CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to EP No. 18179265.6 filed on June 22, 2018, the whole content of this application being incorporated herein by reference for all purposes.

FIELD OF THE INVENTION

The invention relates to fluorinated hyper-branched polyglycerol (“HPG”) polymers and corresponding nanoparticles for theranostic applications. The invention further relates to drug encapsulated HPG nanoparticles.

BACKGROUND OF THE INVENTION

There is a significant interest in theranostic nanoparticles, due to their ability to provide therapeutic delivery and medical imaging ( e.g . diagnostic) information on a single platform. In some instances, the theranostic nanoparticles are used to probe selective drug efficacy on different patients. For example, in some cases, a drug may work for one patient and not another. In such instances drug delivery can be monitored using the imaging activity of the theranostic nanoparticle. As such, theranostic nanoparticles can diagnosis disease, provide a drug delivery vector and monitor therapeutic response, all on a single platform. Accordingly, there is significant interest in developing increasingly effective theranostic nanoparticles.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a schematic depiction of a fluorinated HPG polymer, where F refers to a fluorinated group.

Fig. 2 is an ¹ HNMR spectrum of a purified final solution containing perfluoro-tert- butanol functionalized glycidol and DIAD.

Fig. 3 is an 'HNMR spectrum of 2-[(2,2,2-Trifluoroethoxy)methyl]oxirane.

DETAILED DESCRIPTION OF THE INVENTION Described herein are fluorinated hyper-branched polyglycerol (“HPG”) polymers and corresponding synthesis methods. Also described herein are fluorinated HPG nanoparticles, drug encapsulated fluorinated HPG nanoparticles, and corresponding dispersions. Relative to corresponding non-fluorinated HPG polymers, the fluorinated HPG have have desirable ¹⁹ F nuclear magnetic resonance (“NMR”) activities for use as ¹⁹ F magnetic resonance imaging (“MRI”) probes. Furthermore, because the fluorinated HPG nanoparticles include, by definition, fluorine, drug loading and encapsulation efficiency is also significantly increased for fluorine containing drugs, relative to corresponding non-fluorinated HPG nanoparticles.

Theranostic systems include, among other things, nanoparticles that provide both diagnostic and therapeutic agents on a single platform. More particularly, theranostic systems aim to combine therapeutic agents ( e.g . drugs) and imaging agents (e.g. contrast) on a single platform (e.g. a nanoparticle). While polyesters nanoparticles are widely used due to their biocompatibility and biodegradability, as well as their desirable in vitro release behavior of drugs encapsulated within the polyester nanoparticles, they lack ¹⁹ FMRI imaging activity, at least in part because of the lack of a fluorine. While the lack of fluorine is not problematic for ^! HMRI, ¹⁹ FMRI has gained much attention as a more useful tool in diagnostic and therapeutic imaging. In contrast to 'HMRI, ¹⁹ FMRI allows direct detection of labeled agents or cells for unambiguous identification and quantification, unlike many metal-based contrast agents. Accordingly, bare HPG nanoparticles are unable to serve as an imaging agent.

Described herein are fluorinated HPG nanoparticles, which can encapsulate drugs. As such, the HPG nanoparticles can serve as an imaging agent alone or also as a drug delivery vector. The fluorinated HPG nanoparticles have desirable ¹⁹ FNMR activities as well as T i and T ₂ relaxation times, which make the nanoparticles desirable for ¹⁹ FMRI imaging. Ti relaxation time refer to the time it takes for the longitudinal component of the net magnetization vector to reach (1 - l/e) of its maximum value. T ₂ relaxation time refers to the time required for the transverse components of the magnetization to fall to l/e of its initial value. The Ti and T ₂ relaxation times influence the response efficiency of fluorinated compounds in MRI. Additionally, as demonstrated in the examples below, the fluorinated HPG nanoparticles also have desirable drug loading and encapsulation efficiencies and, therefore, can be suitable for drug delivery. Of course, the ability of the fluorinated HPG nanoparticles to act as theranostic nanoparticles is intimately link to their stability in dispersions. That is, to the extent that nanoparticles cannot be suitably dispersed, they cannot provide theranostic capabilities in vivo. The fluorinated HPG nanoparticles described herein have excellent colloidal stability and, therefore, provide stable dispersions suitable for theranostic use.

Fluorinated HPG Polymers

The fluorinated HPG polymers comprise recurring units formed from the polymerization of an hydroxyalkyl oxirane monomer and a fluorinated glycidyl ether monomer. The hydroxyalkyl oxirane monomer and fluorinated glycidyl ether monomer are represented by the following formulae, respectively:

where R;, R _j , R _k , Ri, R _q and R _s , at each location, is selected from the group consisting of a hydrogen or an alkyl group; T is represented by a formula selected from the group of formula consisting of -CH ₂ F, -CHF ₂ and -CF ₃ ; and ni to n ₃ are independently selected integers from 1 to 12. In some embodiments, Ri, R _j , R _k , Ri, R _q and R _s at each location is a hydrogen. Additionally or alternatively, in some embodiments, ni to n ₃ are all 1. In some embodiments, T is either -CFFF or -CF ₃ . In some embodiments, the hydroxyalkyl oxirane monomer is glycidol, the fluorinated glycidyl ether monomer is trifluoroethoxymethyl oxirane, or both. Fig. 1 is a schematic representation of one embodiment of a fluorinated HPG polymer. For ease of reference, monomers according to formula (2) are referred to as fluorinated glycidyl ether monomers, even though ni can be greater than 1 and R _k , Ri, R _q and R _s can be other than H.

For the sake of clarity, the fluorinated HPG polymers are sometimes referred to using the following designation: Fx,yHPGz. X refers to the number of fluorine atoms in T (if X=l , the T is -CH ₂ F; if X=2, then T=-CHF ₂ ;and if X=3, then T is -CF ₃ ); Y refers to the degree of polymerization with the respect to the fluorinated glycidyl ether monomer, and Z refers to the degree of polymerization with respect to the hydroxyalkyl oxirane monomer. The degree of polymerization (“DP”), defined as the number of monomeric units in a macromolecule, is given by:

DP can be measured using 'HNMR, as described in detail in the examples below.

In some embodiments, the fluorinated HPG polymers have a degree of polymerization with respect to the hydroxyalkyl oxirane monomer (“DP _OH ”) of at least 15, at least 20, or at least 25. Additionally or alternatively, in some embodiments the fluorinated HPG polymers have a DP _OH of no more than 80, no more than 70, or no more than 65. In some embodiments, the fluorinated HPG polymers have a DP _OH of from 15 to 80, from 20 to 70, or more 25 to 65. In some embodiments, the fluorinated HPG polymers have a degree of polymerization with respect to the fluorinated glycidyl ether monomer (“DPp”) of at least 5, at least 10, or at least 15. Additionally or alternatively, in some embodiments, the fluorinated HPG polymers have a DPp of no more than 50, no more than 40 or no more than 35. In some embodiments, the fluorinated HPG polymers have a DP _F of from 5 to 50, from 10 to 40, from 15 to 40, or from 15 to 35.

In some embodiments, the fluorinated HPG polymer has a number average molecular weight (“Mn”) of at least 1,000 g/mol, at least 2,000 g/mol, or at least 3,000 g/mol. Additionally or alternatively, in some embodiments the fluorinated HPG polymer has an Mn of no more than 12,000 g/mol, no more than 1 1,000 g/mol, 10,000 g/mol, no more than 9,000 g/mol or no more than 8,000 g/mol. In some embodiments, the fluorinated HPG polymer has an Mn of from 1,000 g/mol to 12,000 g/mol, from 2,000 g/mol to 12,000 g/mol, from 3,000 g/mol to 12,000 g/mol, from 3,000 g/mol to 1 1,000 g/mol, from 3,000 g/mol to 10,000 g/mol, from 3,000 g/mol to 9,000 g/mol, or from 3,000 g/mol to 8,000 g/mol. Number average molecular weight can be calculated as described in the Examples.

Synthesis of Fluorinated HPG Polymers

The fluorinated HPG polymers are synthesized using a ring opening multi-branching polymerization. The polymerization includes reacting a first hydroxyalkyl oxirane monomer and fluorinated glycidyl ether monomer in the presence of an initiator to form the fluorinated HPG. While reacting a fluorinated glycidol monomer ( e.g . epifluorohydrin) with a hydroxyalkyl oxirane monomer is a more direct route to fluorinated HPG polymer synthesis, it was surprisingly discover that by using a fluorinated glycidyl ether monomer in place of the fluorinated glycidol monomer, desirable ¹⁹ FNMR activity could be obtained, as demonstrated in the examples below.

The first hydroxyalkyl oxirane monomer and fluorinated glycidyl ether monomer are represented by Formulae (1) and (2), respectively. The initiator is a polyhydric alcohol, which is deprotonated at least one hydroxyl group to form the nucleophile that initiates the polymerization. The polyhydric alcohol is represented by the following formula: R-(OH) _n , where n is an integer from 1 to 4 and R is any molecule stable under the conditions of the polymerization reaction. In some embodiments, R is a Ci to Ci ₂ substituted or unsubstituted, straight chain or branched alkyl group. In some embodiments, the initiator is a Suitable initiators include, but are not limited to, tris- (n=3) or tetrafunctional (n=4) initiator, including, but not limited to, 1 , 1 , 1 ,-tris(hydroxymethyl)propane (“TMP”) or 1 ,1 ,1 - tris(hydroxymethyl)ethane (“TME”), Benzene-l ,3,5-triol (“THB”), pentaerythrol (“PE”).

In some embodiments, the first hydroxyalkyl oxirane monomer and the fluorinated glycidyl ether monomer can be polymerized at a temperature of from 60° C to 150° C, from 70° C to 120° C, from 80° C to 1 10° C, or from 85° C to 1 10° C.

In some embodiments, the polymerization reaction further includes reacting the fluorinated HPG polymer with a second hydroxyalkyl oxirane monomer. The second hydroxyalkyl oxirane is also represented by Formula (1), and is the same or different than the first hydroxyalkyl oxirane monomer. Preferable, the second hydroxyalky oxirane is the same as the first hydroxyalkyl oxirane. In such embodiments, fluorinated HPG is grown by addition of hydroxyalky oxirane monomer units. The fluorinated ends of the HPG, formed by initial polymerization between the first hydroxyalkyl oxirane monomer and the fluorinated glycidyl ether monomer, are, therefore, generally disposed interior to the HPG. The surface chemistry of the fluorinated HPG in such embodiments is dominated by the hydroxyl ends formed from polymerization with the first and second hydroxyalkyl oxirane monomers. The resulting internalization of the fluorine atoms increases the stability of the fluorinated HPG nanoparticles in aqueous solvents ( e.g . water). In some embodiments, the second hydroxyalkyl oxirane monomer is reacted with the fluorinated HPG polymer in the same temperature range as described above with respect to the polymerization of the first hydroxyalkyl oxirane monomer and the fluorinated glycidyl ether monomer.

It was also surprisingly discovered that the fluorinated glycidyl ether monomers could be synthesized by reacting a haloalkyl oxirane with a haloalkyl alcohol in the presence of a base according to the following nucleophilic substitution reaction scheme:

where X is a halogen, preferably Cl. Suitable bases include, but are not limited to, metal hydroxides. Examples of metal hydroxide bases include, but are not limited to, sodium hydroxide and potassium hydroxide. In some embodiments, the reacting can be done at room temperature. Alternative approaches to synthesis of fluorinated glycidyl ether monomers use a Mitsunobu reaction scheme. In such a reaction scheme, a glycidol is reacted with haloalkyl alcohol in the presence of triphenylphosphine and a dialkyl azodicarboxylate to convert the haloalkyl alcohol in to an ether. More particularly, the triphenylpho spine combines with the dialkyl azodicarboxylate to generate a phosphonium intermediate that binds to the alcohol oxygen, activating it as a leaving group. However, it was discovered that when using primary and tertiary haloalkyl alcohols, the dialkyl azodicarboxylate could not be desirably separated from the final glycidol ether monomer product or that the reaction did not proceed, as demonstrated in the example below. On the other hand, it was surprisingly discovered that synthesis of the fluorinated glycidyl ether monomers using nucleophilic substation as described above allowed for desirable yields and purity.

Fluorinated HPG Nanoparticles. Drug Encapsulated HPG Nanoparticles, and Dispersions

The fluorinated HPG nanoparticles and corresponding encapsulated drugs are formed in solution ( e.g . as a dispersion), using methods well known in the art. The dispersions generally include a solvent and the fluorinated HPG nanoparticles. Optionally, the dispersions can further include other components that can aid in increasing the colloidal stability of the nanoparticles. Methods for making the fluorinated HPG nanoparticles and corresponding encapsulated drugs include, but are not limited to, nanoprecipitation methods, emulsion-diffusion methods, double emulsification methods, emulsion-coacervation methods, polymer-coating methods and layer-by- layer methods are described in detail in Int. J. Pharm 385 (2010) 1 13 - 142, to Mora-Huertas et al. and incorporated by reference herein. Preferably, a nanoprecipitation method is used.

In one embodiment, the formation method involves dispersing the fluorinated HPG nanoparticles in a formation solution including a polar organic solvent and water and, subsequently, removing the polar organic solvent to form the dispersion. Optionally, the formation solution can further include a dispersant. In embodiments in which the fluorinated HPG nanoparticles encapsulate a drug, both the fluorinated HPG nanoparticles and corresponding drug are dispersed in the formation solution. Desirable polar organic solvent includes, but are not limited to, acetone, dimethylsulfoxide, dimethylformamide and acetonitrile. Preferably, the polar organic solvent is acetone.

In embodiments in which the fluorinated HPG nanoparticles encapsulate a drug, both the drug and the fluorinated HPG nanoparticles are dispersed in the formation solution. In some such embodiments, the weight ratio of the drug to the fluorinated HPG nanoparticles is at least 1 :30, at least 1 :25, or at least 1 :20. Additionally or alternatively, in some embodiments the weight ratio of the drug to the fluorinated HPG nanoparticles is no more than 1 :1 , no more than 1 :5, or no more than 1 :10. In some embodiments, the weight ratio of the drug to the fluorinated HPG nanoparticles is from 1 :30 to 1 :1 , from 1 :25 to 1 :5, or from 1 :20 to 1 :10.

In some embodiments, the formation solution includes a dispersant, to aid in the dispersion of the fluorinated HPG nanoparticles (or corresponding drug encapsulating nanoparticles). In some embodiments, the dispersant is a surfactant. Desirable surfactants include, but are not limited to, poly(vinyl alcohol) and poloxamers. Poloxamers are nonionic triblock copolymers composed of a central hydrophobic chain of polyoxypropylene flanked by two hydrophilic chains of polyoxyethylene. Poloxamers are commercially available, for example, from Sigma-Aldrich® under the trade name Pluronic® (e.g. F-127, F-68).

Subsequent to dispersion, the polar organic solvent is removed. The polar organic solvent can be removed using techniques well known in the art. Examples include, but are not limited to, evaporation under vacuum or at ambient pressure.

The resulting dispersions (subsequent to removal of polar organic solvent) have desirable stability for theranostic applications. As explained above, the suitability of nanoparticles for theranostic use depends, in part, on their colloidal stability (the ability of the nanoparticles to form stable dispersion). In turn, for a given nanoparticle composition and solvent, the colloidal stability is dependent, in part, on particle size. Generally, the relationship between nanoparticle size and colloidal stability is not linear. In some instances, colloidal stability increases with nanoparticle size to a maximum, and thereafter decreases as nanoparticle size continues to increase. Of course, in any given collection of nanoparticles, there is a distribution of sizes, so that the collection of nanoparticles is characterized by an average size as well a width.

Particle size distributions can be measured by dynamic light scattering (“DLS”). With particular reference to colloidal stability, the z-average size is of particular interest as it provides sizing information of a particle in a fluid ( e.g . dispersions of nanoparticles) based on their Brownian motions. The Z-average size is extracted from the analysis of the scattering intensity weighted auto-correlation function of a dispersion of particles. Evaluation of this size distribution is prescribed in ISO International Standard 13321 , Methods for Determination of Particle Size Distribution Part 8: Photon Correlation Spectroscopy, 1996, incorporated herein by reference. The Z-average particle size as well as polydispersity index (PDI), can be obtained from the fits to the time correlation functions. For clarity, the Z-average sizes reflect secondary particle sizes (i.e. the size of the particles in a dispersion). The primary particle size is roughly the lower limit of the secondary particle size for a particular collection of particles, so that the average secondary particle size can be approximately the average primary particle size if the primary particles are effectively completely dispersed in the medium. In general, fluorinated HPG nanoparticles having a z-average particle size of less than 400 nm and a PDI of less than 0.4 are most desirable with respect to colloidal stability.

The fluorinated HPG nanoparticles described herein have desirable Z-average particle sizes and PDI values for drug delivery applications. In some embodiments, a collection of fluorinated HPG nanoparticles have a Z-average particle size of at least 30 nm, at least 50 nm, or at least 60 nm. Additionally or alternatively, in some embodiments, the collection for fluorinated HPG nanoparticles has a Z-average particle size of no more than 350 nm, no more than 250 nm, or no more than 200 nm. In some embodiments, the collection of fluorinated HPG nanoparticles has a Z-average particle size of from 30 nm to 350 nm, from 30 nm to 250 nm, from 30 nm to 200 nm, from 50 nm, to 200 nm, or from 60 nm to 200 nm. In some embodiments, a collection of fluorinated HPG nanoparticles has polydispersity index (“PDI”) of no more than 0.8, no more than 0.7, no more than 0.6, no more than 0.5, no more than 0.4, no more than 0.3, or no more than 0.2. PDI is related to dispersion of z-averaged sizes of the particle population according to the following formula: PDI=(o/d) ² , where s is the standard deviation of the Z-average size distribution and d is the z-average particle size. For clarity, the Z-average particle size ranges above apply to both the fluorinated HPG nanoparticles as well as the drug encapsulated fluorinated HPG nanoparticles.

In some embodiments, the fluorinated HPG nanoparticle dispersions have a fluorinated HPG concentration of at least 0.01 wt.%, at least 0.05 wt.%, or at least 0.08 wt.%. Additionally or alternatively, in some embodiments, the fluorinated HPG nanoparticle dispersions have a fluorinated HPG concentration of no more than 1 wt.%, no more than 0.2 wt.% or no more than 0.15 wt.%. In some embodiments, the fluorinated HPG nanoparticle dispersions have a fluorinated HPG concentration of from 0.01 wt.% to 1 wt.%, from 0.05 wt.% to 0.2 wt.% or from 0.08 wt.% to 0.15 wt.%. In some embodiments, the drug encapsulated fluorinated HPG dispersions have a drug encapsulated fluorinated HPG nanoparticle concentration of at least 0.01 wt.%, at least 0.05 wt.%, at least 0.1 wt.%, at least 0.15 wt.% or at least 0.18 wt.%. Additionally or alternatively, in some embodiments the drug encapsulated fluorinated HPG dispersions have a drug encapsulated fluorinated HPG nanoparticle concentration of no more than 2 wt.%, no more than 1.5 wt.%, no more than 1 wt.%, no more than 0.7 wt.%, or no more than 0.5 wt.%. In some embodiments, the drug encapsulated fluorinated HPG dispersions have a drug encapsulated fluorinated HPG nanoparticle concentration of from 0.01 wt.% to 2 wt.%, from 0.05 wt.% to 1.5 wt.%, from 0.1 wt.% to 1 wt.%, from 0.15 wt.% to 0.7 wt.%, or from 0.18 wt.% to 0.5 wt.%. As used herein, wt.% is relative to the total weight of the dispersion.

While there is no limit on the type of drug that is encapsulated within the fluorinated HPG nanoparticles, hydrophobic drugs are particularly desirable. Examples of hydrophobic drugs include, but are not limited to, disease-modifying antirheumatic drugs (“DMARD”) (e.g. Leflunomide), steroidal anti-inflammatory drugs (e.g. Dexamethasone), and anti-cancer drugs (e.g. Paclitaxel, commercially available under the trade name Taxol®). In some embodiments, the drug encapsulated fluorinated HPG nanoparticles have a drug loading of at least 1.5%, at least 2.0%, or at least 2.5%. Additionally or alternatively, in some embodiments the drug encapsulated fluorinated HPG nanoparticles have a drug loading of no more than 10%, no more than 9%, no more than 8%, no more than 7%, no more than 6%, or no more than 5%. In some embodiments, the drug encapsulated HPG nanoparitlces have a drug loading of from 1.5% to 10%, from 2.0% to 10%, from 2.5% to 10%, from 2.5% to 9%, from 2.5% to 8%, from 2.5% to 7%, from 2.5% to 6%, or from 2.5% to 5%. As described in the Examples, drug loading can be measured using HPLC and determined according to the following formula:

EXAMPLES

In the following Examples, the DP is determined as follows. For the HPG polymers, the DP was determined by ^! HNMR spectroscopy, in CD3OD, as follows:

( A ^~ 6)/5

DP_ OH C/3

where A is the area of the peak at 4.10-3.47 -ppm, corresponding to the 5 protons of glycidol unit (1 CH and 2 CFE) and the 6 protons of the initiator TMP (3 CFE) which do not belong to the monomeric unit; and C is the area of the peak at 0.88 ppm corresponding to the 3 protons of the initiator TMP (1 CH3).

For the fluorinated HPG polymers, two different DP values were determined, by ¹ HNMR spectroscopy, in (CD3) ₂ SO. The DP_ _F , (the DP of the fluorinated glycidyl ether monomer) was determined as follows:

B/2

DP. _f Ί

Where B is the area of the peak at 4.04 ppm corresponding to the - CH ₂ - protons next to the - CF3 group of the fluorinated monomer unit; and C is the area of the peak at 0.79 ppm corresponding to the 3 protons of the initiator TMP (1 CH3). DP_ _0H (the DP of the hydroxyalkyl oxirane monomer) was determined as follows:

Where A is the area of the peak at 3.69-3.22 ppm corresponding to 5 protons of hydroxyalkyl oxirane monomer (1 CH and 2 CH ₂ ), 6 protons of TMP (3 CH ₂ ), and 5 protons of the fluorinated monomer unit; B is the peak at 4.04 ppm corresponding to the - CH ₂ - protons next to the - CF ₃ group of the fluorinated monomer unit; and C is the area of the peak at 0.79 ppm corresponding to 3 protons of initiator TMP (1 CH ₃ ).

Number average molecular weight is obtained from the following formula: Mn = DP _OH M _OH + DP _F *M _F + Mi _mtiator , where M _OH is the molar mass of the hydroxyalkyl oxirane monomer, Mp is the molar mass of the fluorinated glycidyl ether monomer and M _utator is the molar mass of the initiator.

Example 1 : Synthesis and Characterization ofHPG Polymers

The following example demonstrates the synthesis and characterization ofHPG polymers having different number average molecular weights.

The HPG polymers were synthesized using a ring opening multibranching polymerization method as described in “Synthesis, Characterization, and Viscoelastic Properties of High Molecular Weight Hyperbranched Polyglycerols,” Macromolecules 2006, 39, 7708-7717, to Kumar et al. and incorporated herein by reference. Specifically, l,l,l-Tris(hydroxymethyl)propane (“TMP”) was added to a flask under nitrogen atmosphere and partially deprotonated (10%) with potassium methoxide solution in methanol (25% in Methanol). The resulting mixture was stirred using a magnetic stirrer bar for 15 min at room temperature (25° C), after which excess methanol was removed in a vacuum for 1 hour, until the bubbling stopped. The reaction flask was kept in an oil bath at 95° C, and glycidol was added dropwise over a period of 12 hours (“h”) using a syringe pump. After completion of monomer addition, the mixture was stirred for an additional 5 h. The reaction was monitored through ¹ HNMR spectra. The amounts of TMP, potassium methylate solution and glycidol for used for the synthesis of each of the three HPG polymers is displayed in Table 1. In Table 1 , as well as other references to HPGx in the Examples, the subscript X refers to the number average molecular weight. TABLE 1

The product obtained from the synthesis described above was dissolved in methanol, neutralized by passing three times through a column containing cation-exchange resin (Dowex MAC-3 ion exchange resin). The polymer was then precipitated in acetone and dried under vacuum. The glycidol conversion was greater than 95% and the final yield was greater than 90%. The product was confirmed to be HPG polymer by NMR: ¹ HNMR (400 MHz, Methanol-d4), 5[ppm] = 4.10-3.47 (m, 5H x DP + 6H of TMP), 1.68 - 1.1 1 (m, 2H), 0.88 (t, J = 7.5 Hz, 3H), ¹³ CNMR (400 MHz, Methanol-d4), 5[ppm] = 80.15 (d, J = 24.1 Hz, C _LI3 ), 78.58 (d, J = 21.4 Hz, C _D ), 72.55 (s, 2C _LH ) , 71.99 - 70.49 (m, 2C _T , 2C _D ), 69.43 (d, J = 27.7 Hz,

C _LIS , C _LM ), 63.07 (d, J = 9.2 Hz, C _T ), 61.44 (s, Cus) .

The HPG polymers were characterized by degree of polymerization (“DP”) number average molecular weight (“M _n ”), Z-average particle size and PDI. Multiple methods were used to determine the aforementioned values. Mn was obtained from 'HNMR spectra as follows: M _n = DP * M _giyCidoi + M _TMP , where M _giyCid0i and M _TMP are the molar mass of glycidol and TMP, respectively. Z-average particle size and PDI were obtained by DLS. Specifically, 10 mg of the HPG polymer were dispersed in 1 mL of dionized (“DI”) water and measured with DLS at 25° C with a backscattering angle of 173°. The results of the characterization are provided in Table 2.

TABLE 2

Example 2: Comparative Synthesis and Characterization of Perfluoro-Tert-Butyl Alcohol functionalized HPG Polymers by Direct Functionalization

The following example demonstrates the synthesis and characterization of a perfluoro- tert-butyl alcohol functionalized HPG polymer by direct functionalization according to the following reaction scheme:

Hypertonanched Polyglycerol

f

HourinaHil Hyperbrimslied Polygtycml

In the above reaction scheme, the person of ordinary skill in the art will recognize that the depictions of the starting HPG and fluorinated HPG are schematic representations of the polymers, not exact chemical structures.

To demonstrate the synthesis, triphenylphosphine (2.95 g, 1 1.25 mmol, 84 Eq.) was placed in an oven-dried 50 mL flask, under nitrogen flow for 30 minutes. HPG polymer (prepared as sample 1 , above) (500 mg, 0.134 mmol, 1 Eq.) and anhydrous DMF (4.82 mL) were placed in a flask under nitrogen flow for 30 minutes under stirring to create a polymer solution. The polymer solution and anhydrous THF (9.79 mL) were added in the reaction flask to form a reaction mixture. The reaction mixture was cooled to 0 °C. Diisopropylazodicarboxylate (“DIAD”) (2.22 mL, 1 1.25 mmol , 84 Eq.) was added dropwise to the reaction solution under stirring. The resulting solution was warmed up to room temperature and stirred for additional 20 minutes. Subsequently, perfluoro-tert-butyl alcohol (1 ,6 mL, 1 1.25 mmol, 84 Eq.) was added, in one lot, to the reaction mixture. The final clear, yellow solution was stirred at 45 °C for 72 hours under static nitrogen atmosphere. The reaction was monitored using ¹ HNMR and

¹⁹ FNMR spectroscopy. After cooling the solution down to room temperature, the solution was concentrated under vacuum. Example 3: Comparative Functionalization of Glycidol with Either Perfluoro-Tert-Butanol or Trifluoroethanol

The present example demonstrates a comparative functionalization of glycidol with either perfluoro-tert-butanol or trifluoroethanol using a Mitsunobu reaction according the following schemes, respectively:

To demonstrate synthesis of the perfluoro-tert-butanol functionalized glycidol, triphenylphosphine (4 g, 14.85 mmol) was placed in an oven-dried 50 mL flask, under nitrogen flow for 30 minutes. Glycidol (1 g, 13.5 mmol) and anhydrous THF (19.3 mL) were placed in aflask under nitrogen flow for 30 minutes under stirring. Afterwards, triphenylphosphine was added to the reaction mixture, which was cooled to 0 °C.. DIAD (2.92 mL, 14.85 mmol) was added dropwise to the reaction mixture under stirring. The resulting solution was warmed up to room temperature and stirred for additional 20 minutes, before adding perfluoro-tert-butyl alcohol (2.07 mL, 14.85 mmol) in one portion. The final clear, yellow solution was stirred at room temperature for 22 hours under static nitrogen atmosphere. The reaction was monitored thought ¹⁹ FNMR spectra.

The final solution was purified using flash chromatography as well vacuum distillation, however, desirable separation between the final product and the remaining DIAD was not obtained. Fig. 2 is a ¹ HNMR spectrum of the purified final solution containing the perfluoro- tert-butanol functionalized glycidol and DIAD. Referring to Fig. 2, a significant amount of DIAD was still present in the final solution subsequent to purification.

To demonstrate synthesis of the trifluoroethanol functionalized glycidol, triphenylphosphine (3.56 g, 13.5 mmol) was placed in an oven-dried 50 mL flask, under nitrogen flow for 30 minutes. Glycidol (1 g, 0.895 mL, 13.5 mmol) and anhydrous THF (19.3 mL) were placed in a flask under nitrogen flow for 30 minutes under stirring. Afterwards, triphenylphosphine was added to the reaction mixture, which was cooled to 0 °C. DIAD (2.66 mL, 2.73g, 13.5 mmol) was added dropwise under stirring. The resulting solution was warmed up to room temperature and stirred for additional 20 minutes, before adding trifluoroethanol (0.85 mL, 1.13g, 1 1.25 mmol) in one portion. The finally clear (yellow) solution was stirred at room temperaturefor 24 hours under static nitrogen atmosphere. The reaction was monitored thought ¹⁹ FNMR spectra. However, the reaction failed to proceed and the glycidol was not significantly functionalized. Example 4: Synthesis of Fluorinated Glycidyl Ether Monomers

The present example demonstrates the synthesis of a fluorinated glycidyl ether monomer according to the following nucleophilic substitution reaction:

Epichlorohydrin Trifluoroethyl alcohol

1 eq. 1 eq.

NaOH O

roam temperature

overnight

2-[(2,2.2-Trifluoroeyhoxy)melhyl]oxirane static N;

To demonstrate synthesis, trifluoroethanol (10 g, 0.1 mol) and epichlorohydrin (9.2 g, 0.1 mol) were added to a cooled solution of 5 g (0.125 mol) of sodium hydroxide in 60 mL of water (2.08 M). The reactants were mixed thoroughly and the mixture was allowed to stand at room temperature overnight. Then the organic layer was separated, washed twice with water, and dried with Na ₂ S0 ₄ . The reaction product was subjected to vacuum distillation. The final yield = 1 1%. The final product was confirmed to be 2-[(2,2,2-Trifluoroethoxy)methyl]oxirane by NMR:

1H NMR (400 MHz, Methanol-d4): 5[ppm] = 4.07 - 3.88 (m, 2H _D ,lH _c ), 3.47 (m, J = 11.9, 6.3 Hz, lHc), 3.16 (m, J = 6.8, 2.6 Hz, lH _B ), 2.85 - 2.73 (m, lH _A ), 2.61-259 (m, lH _A ). Fig. 3 is an ¹ HNMR spectrum of the 2-[(2,2,2-Trifluoroethoxy)methyl]oxirane. The 'HNMR spectrum demonstrates the purity of the fluorinated glycidyl ether monomer.

Example 5: Comparative Synthesis and Characterization of a Fluorinated HPG Polymer. F | . _S.2- H PG ₅₂

The present example demonstrates the synthesis and characterization of a fluorinated HPG polymer by a ring opening multibranching polymerization with Epifluorohydrin (“F _I ,5 _.2- HPG5 ₂ ”) according to the following reaction scheme:

CH ₃ O K ⁺

Potassium metiundd

l.l. Epifluaroh^dni

pic pace 20 eg. 20 eq.

1 -q

In the above reaction scheme, the person of ordinary skill in the art will recognize that the depictions of the fluorinated HPG are schematic representations of the polymers, not exact chemical structures.

To demonstrate synthesis, l,l ,l-Tris(hydroxymethyl)propane (GMR) (90 mg, 0.67 mmol) was added to a flask under nitrogen atmosphere followed by potassium methoxide solution in methanol (53 pL, 0.72 mmol, 0.05 lg, 1.08 eq.) (25% in Methanol). The mixture was stirred using a magnetic stirrer bar for 15 minutes at room temperature. Afterwards, the excess of methanol was removed under vacuum for 1 hour, until the bubbling stopped. The reaction flask was kept in an oil bath at 80 °C, and 0.895 mL of glycidol (13 mmol, 1 g, 0.895 mL, 20 eq.) and 0.927 mL of epifluorohydrin (13 mmol, 0.927 mL, 0.989 g, 20 eq.) were added dropwise over a period of 12 h using a syringe pump. After completion of monomers addition, the mixture was stirred for an additional 7 h. Finally a second amount of glycidol (13 mmol, 1 g, 0.895 mL, 20 eq.) was added dropwise over a period of 12 h using a syringe pump, at 95°C. After completion of monomer addition, the mixture was stirred for an additional 9 h. The reaction was monitored through ¹ HNMR spectra. The product was dissolved in methanol, neutralized by passing through a column containing cation-exchange resin (Dowex MAC-3 ion exchange resin). The polymer was then dried for 2 h under vacuum. Conversion(glycidol) > 90% Conversion(epifluorohydrin) < 50%. The final yield of the reaction was > 97%. The products was confirmed by NMR: 'PINMR^OO MHz, Methanol-d4): 5[ppm] = 4.66 (m, J = 120.6, 59.8 Hz, -OH), 4.61-4.41 (m, 2H near -F), 3.90-3.58 (m, 5H x DPgi _yC idoi + 5H x DP _fluori nated monomer + 6H of TMP), 1.40 (m, 2H of TMP), 0.90 (s, 3H of TMP). However, ¹⁹ FNMR spectra revealed very weak fluorine in both CH ₃ OD and D ₂ 0. The ¹⁹ FNMR results demonstrate that the F _{| .2O} -H PG _6O was unsuitable for theranostic use, at least because of expected lack of ¹⁹ FNMR activity.

Values of Mn, DP, Z-average particle and PDI were obtained, as described above. Table 3 displays the results of the measurements.

Example 6: Synthesis of a Fluorinated HPG Polymer. Fi 70-HPG77. and Characterization

The present example demonstrates the synthesis of a fluorinated HPG polymer (F3 _, 2 _O -HPG27) by ring opening multi-branching polymerization with 2-[(2,2,2,- trifluoroethoxy)methyl] oxirane according to the following reaction scheme:

l,l,l-Tris( hydroxyme thy 1 Ipropa lie

1 eq.

Potassium methoxide

solution

Glycidol 2- [{2,2 ,2,-Tri fluoroethoxy) *re.irton ⁼ ' F

20 eq. methyl] oxirane

20 eq.

In the above reaction scheme, the person of ordinary skill in the art will recognize that the depictions of the fluorinated HPG is a schematic representation of the polymer, not an exact chemical structure.

To demonstrate the synthesis, l,l,l-Tris(hydroxymethyl)propane (“TMP”) (61 mg, 0.45 mmol) was added to a flask under nitrogen atmosphere followed by 36 pL of potassium methoxide solution in methanol (25% in Methanol). The mixture was stirred using a magnetic stirrer bar for 15 minutes at room temperature. Afterwards, excess of methanol was removed under vacuum for 1 hour, until the bubbling stopped. The reaction flask was kept in an oil bath at 95 °C, and 0.67 mL of glycidol (9.05 mmol, 20 eq.) and 1.41 mL of 2-[(2,2,2,-

Trifluoroethoxy)methyl] oxirane (9.05 mmol, 20 eq.) were added dropwise over a period of 12 h using a syringe pump. After completion of monomers addition, the mixture was stirred for an additional 7 h. The reaction was monitored through ¹ HNMR spectra. The crude was dissolved in methanol, neutralized by passing through a column containing cation-exchange resin (Dowex MAC-3 ion exchange resin). The polymer was then dried for 2 h under vacuum. Conversion (for both glycidol and 2-[(2,2,2,-trifluoroethoxy)methyl] oxirane)> 95 %. The final yield was > 90%. The structure of the fluorinated HPG was confirmed with NMR: 'HNMR (400 MHz, DMSO-d6): 5[ppm] = 4.64 (m, J = 120.1 , 71.3 Hz, -OH), 4.04 (s, 2H near CF ₃ ), 3.69-3.22(m, J =

89.5, 49.5 Hz, 5H x DP _giyCidoi + 5H x DP _fluori „ated monomer + 6H of TMP), 1.28 (m, 2H of TMP), 0.79 (s, 3H of TMP); ¹⁹ FNMR (400 MHz, DMSO-d6): 5[ppm] = -73.18 (s).

Values of Mn, DP, Z-average particle and PDI were obtained, as described above. The values are displayed in Table 4. TABLE 4

Example 7: Synthesis of a Fluorinated HPG Polymer. F3.7 _. 8-HPG37. and Characterization

The present example demonstrates the synthesis of a fluorinated HPG polymer

(F3 _7. 8-HPG32) by ring opening multi-branching polymerization with 2 -[(2,2,2, -

Trifluoroethoxy)methyl] oxirane according to the following reaction scheme:

1,L 1-Trii( liydroxyme thyl ipropa lie

1 eg.

OH , O K ⁺

Potassium methoxide

solution

32 eq. inethyljoxiraiie

5 eq.

In the above reaction scheme, the person of ordinary skill in the art will recognize that the depictions of the fluorinated HPG is a schematic representation of the polymer, not an exact chemical structure.

To demonstrate synthesis, l ,l ,l -Tris(hydroxymethyl)propane (TMP) (63 mg, 0.477 mmol) was added to a flask under nitrogen atmosphere followed by 37 pL of potassium methoxide solution in methanol (25% in Methanol). The mixture was stirred using a magnetic stirrer bar for 15 minutes at room temperature, and the excess of methanol was removed under vacuum for 1 hour, until the bubbling stopped. The reaction flask was kept in an oil bath at 95 °C, and 1 mL of glycidol (15.08 mmol, 32 eq.) and 0.46 mL of 2-[(2,2,2,- trifluoroethoxy)methyl] oxirane (9.05 mmol, 20 eq.) were added dropwise over a period of 12 h using a syringe pump. After completion of monomers addition, the mixture was stirred for an additional 7 h. The reaction was monitored through ¹ HNMR spectra. The crude was dissolved in methanol, neutralized by passing through a column containing cation-exchange resin (Dowex MAC-3 ion exchange resin). The polymer was then dried for 2 h under vacuum. Conversion (for both glycidol and 2-[(2,2,2,-trifluoroethoxy)methyl] oxirane) > 95 %. The final yield was > 90%. The structure was confirmed with NMR: 1HNMR (400 MHz, DMSO-d6): 0[ppm] = 4.65 (m, J = 124.2, 61.8 Hz, -OH), 4.04 (s, 2H near CF ₃ ), 3.7-3. l7(m, J = 89.5, 49.5 Hz, 5H x DPgiycidol + 5H x DPfluorinated monomer + 6H of TMP), 1.25 (m,2H of TMP), 0.79 (m, 3H of TMP); ¹⁹ FNMR (400 MHz, DMSO-de): 5[ppm] = -73.02.

Values of Mn, DP, Z-average particle and PDI were obtained, as described above. The values are displayed in Table 5. TABLE 5

Example 8: Synthesis of a Fluorinated HPG Polymer. F ₃ .i9- d Characterization

The present example demonstrates the synthesis of a fluorinated HPG polymer (F3 _,i 9_HPG6i) by ring opening multi-branching polymerization with 2-[(2,2,2,- trifluoroethoxy)methyl] oxirane according to the following reaction scheme:

In the above reaction scheme, the person of ordinary skill in the art will recognize that the depictions of the fluorinated HPG is a schematic representation of the polymer, not an exact chemical structure.

To demonstrate synthesis, l,l ,l-Tris(hydroxymethyl)propane (GMR) (61 mg, 0.45 mmol) was added to a flask under nitrogen atmosphere followed by 36 pL of potassium methylate solution in methanol (25% in Methanol). The mixture was stirred using a magnetic stirrer bar for 15 minutes at room temperature, and the excess of methanol was removed under vacuum for 1 hour, until the bubbling stopped. The reaction flask was kept in an oil bath at 95 °C, and

0.67 mL of glycidol (9.05 mmol, 20 eq.) and 1.41 mL of 2-[(2,2,2,-

Trifluoroethoxy)methyl]oxirane (9.05 mmol, 20 eq.) were added dropwise over a period of 12 h using a syringe pump. After completion of monomers addition, the mixture was stirred for an additional 7 h. Afterwards, a second amount of glycidol (1.2 mL, 18.01 mmol, 40 eq.) was added dropwise over a period of 12 h using a syringe pump; after completion of monomer addition, the mixture was stirred for an additional 5 h. The reaction was monitored through ¹ HNMR spectra. The product was dissolved in methanol, neutralized by passing it through a column containing cation-exchange resin (Dowex MAC-3 ion exchange resin). The polymer was then dried for 2 h under vacuum. The conversion rate (for both glycidol and 2-[(2,2,2,-trifluoroethoxy)methyl] oxirane) was > 97 % and the final yield was also > 97%. The structure of the fluorinated HPG was confirmed with NMR: 'HNMR (400 MHz, DMSO-d6): 5[ppm] = 4.66 (m, J = 120.6, 59.8 Hz, -OH), 4.04 (s, 2H near CF ₃ ), 3.78-3.19 (m, 5H x DP _giycidoi + 5H x DP _fluori „ated monomer + 6H of TMP), 1.38-1.24 (m, 2H of TMP), 0.79 (s, 3H of TMP); ¹⁹ FNMR (400 MHz, DMSO-d6): 5[ppm] = -73.18 (s).

Values of Mn, DP, Z-average particle and PDI were obtained, as described above. The values are displayed in Table 6.

TABLE 6

Example 9: Formation. Size Characterization, and Loading Performance and ¹⁹ FNMR Activity of Drug Encapsulated Fluorinated HPG Nanoparticles

The present example demonstrates the formation, size characterization, and encapsulation performance and dexamethasone encapsulated in fluorinated HPG nanoparticles.

To demonstrate formation 2 sample dispersions (fluorinated HPG nanoparticles) and 2 comparative dispersions (HPG nanoparticles) were formed. The drug encapsulated particles were formed using a nanoprecipitation method as described in: C.E. Mora-Huertas, H. Fessi, and A. Elaissari, “Polymer-based nanocapsules for drug delivery” International Journal of Pharmaceutics, 385(1): 113, 142, 2010; Michael Chorny, Ilia Fishbein, Haim D Danenberg and Federica Lince, Daniele L. Marchisio, and Antonello A. Barresi,“Strategies to control the particle size distribution of poly-epsilon-caprolactone nanoparticles for pharmaceutical applications”, Journal of Colloid and Interface Science, 322(2):505, 515, 2008; all of which are incorporated herein by reference.

To form each sample, 10 mg of polymer (either HPG or fluorinated HPG) were dissolved with 1 mL of deionized water and the solution was stirred for 15 minutes. A stock solution of Dexamethasone in Acetone (with a concentration of 1 mg/mL) was prepared and stirred for 15 minutes. 1 mL of obtained solution was added dropwise in the polymer solution under stirring. Evaporation of acetone was performed under reduced pressure at 40°C. To demonstrate size characterization, Z-average particle distributions were obtained as described above.

To demonstrate encapsulation performance, drug loading and encapsulation efficiency were determined by HPLC. For each sample was lyophilized to remove aqueous solvent. The lyophilized polymer was dissolved in 1 mL of acetonitrile/water (1 : 1 , by volume) from which two different dilutions, 1 :10 and 1 :100, to form HPLC analysis samples. Each analysis sample was injected (10 pL) in a C18 reversed-phase chromatography column at 30 °C with a flow rate of 1 mL/min in a solution of acetonitrile :water (1 :1 by volume). The DEX peak was detected after about 3 min. The detection wavelength was set at 254 nm. Calibration curves were previously obtained with different DEX concentration (1.0 mg/mL, 0.5 mg/mL, 0.1 mg/mL, 0.01 mg/mL, and 0.001 mg/mL). The drug loading (“DL”) and encapsulation efficiency (“EE”) values were determined according to the following equations:

Weight of Drug Encapsulated

DL% = 100 *

Weight of Drug Encapsulated + Weight of Polymer Nanoparticles

Weight of Drug Encapsulated In Polymer Nanoparticles

EE% = 100 *

Weight of Drug Used in Encapsulation Method

Results of the drug loading measurements are displayed in Table 7.

TABLE 7

Referring to Table 7, the fluorinated HPG nanoparticles had desirable drug loading and encapsulation efficiencies. Moreover, relative toHPG _37i9 , F _{3 X8} -HPG ₃₂ had increased drug loading and encapsulation efficiency, while having a smaller a Z-average particle size. Similar results were seen for F 720-H PG27 relative to HPG7555, though the Z-average particle size was larger in the former.

The embodiments above are intended to be illustrative and not limiting. Additional embodiments are within the inventive concepts. In addition, although the present invention is described with reference to particular embodiments, those skilled in the art will recognized that changes can be made in form and detail without departing from the spirit and scope of the invention. Any incorporation by reference of documents above is limited such that no subject matter is incorporated that is contrary to the explicit disclosure herein.