NANOPARTICLES

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to EP No. 18179257.3 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 poly(lactic-co-glycolic acid) polymer (“PLGA”) and corresponding nanoparticles for theranostic applications. The invention further relates to drug encapsulated fluorinated PLGA 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 ¹⁹ FNMR spectrum ofCF3-PLGA-COOH nanoparticles.

Fig. 2 is a ¹⁹ FNMR spectrum of Methylated (CF3)3-PLGA-OH nanoparticles.

Fig. 3 is a ¹⁹ FNMR spectrum of F ₂₇ -PLGA nanoparticles.

Fig. 4 is a ¹⁹ FNMR spectrum ofCF3-PLGA-OH nanoparticles.

Fig. 5 is a plot showing the pH dependence of ¹⁹ FNMR peak integrals (normalized for the peak of the standard solution) (CF3)3-PLGA-OH nanoparticle. Fig. 6 is a ¹⁹ FNMR spectrum of dexamethasone encapsulated in a (CF ) -PLGA-OH nanoparticle.

Fig. 7 is a ¹⁹ FNMR spectrum of leflunomide encapsulated in a (CF ) -PLGA-OH nanoparticle.

DETAILED DESCRIPTION OF THE INVENTION

Described herein are fluorinated poly(lactic-co-glycolic acid) polymers (“PLGA”). Also described herein are fluorinated PLGA nanoparticles, drug encapsulated fluorinated PLGA nanoparticles, and corresponding dispersions. Relative to corresponding non-fluorinated PLGA nanoparticles, the fluorinated PLGA nanoparticles have desirable ¹⁹ F nuclear magnetic resonance (“NMR”) activities and concomitant relaxation times for use as ¹⁹ F magnetic resonance imaging (“MRI”) probes. It was surprisingly found that, for some fluorinated PLGA nanoparticle embodiments, the ¹⁹ FNMR activity (and therefore the ¹⁹ FMRI activity) could be tuned (e.g. turned on an off) by changing the pH of the nanoparticle dispersion. Similarly, it was also surprisingly discovered that the ¹⁹ FNMR activity could also be tuned by adjusting the relative concentration of the polar organic solvent to water in the formation medium, as described in detail below. Furthermore, because the fluorinated PLGA nanoparticles include, by definition, fluorine, drug loading and encapsulation efficiency is also significantly increased for fluorine containing drugs, relative to corresponding non-fluorinated PLGA.

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 PLGA nanoparticles are unable to serve as an imaging agent.

Described herein are fluorinated PLGA nanoparticles, which can encapsulate drugs. As such, the PLGA nanoparticles can serve as an imaging agent alone or also as a drug delivery vector. The fluorinated PLGA 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. In general, for MRI activity, it is desirable to have small Ti times and large T ₂ times, where Ti times are less than 1 second (“s”) and T ₂ times are larger than 100 ms. However, even in instances where Ti is ls or more, MRI activity can be desirable if where T ₂ is sufficiently long. Additionally, as demonstrated in the examples below, the fluorinated PLGA nanoparticles also have desirable drug loading and encapsulation efficiencies and, therefore, can be suitable for drug delivery.

Of course, the ability of the fluorinated PLGA 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 PLGA nanoparticles described herein have excellent colloidal stability and, therefore, provide stable dispersions suitable for theranostic use.

Fluorinated PLGA Polymers

The fluorinated PLGA polymers of interest herein are represented by the following formula:

where Z ¹ is a hydrogen or a group represented by the following formula:

Z ² is a group selected from the following group of formulae:

-OH; (3)

R ¹ is a hydrogen or a Ci to C12 alkyl group; R; and R _j , at each location, is independently selected from the group consisting of a hydrogen or an alkyl group; R ² to R ¹⁰ are independently selected from the group consisting of a hydrogen, a fluorine,— CF ₃ ,— CHF ₂ ,— CH ₂ F and— CH ₃ ; T is an oxygen or a bond; n and m are independently selected integers from 25 to 200; and ni to n ₄ are independently selected integers from 1 to 12; with the provision that at least one of R ² to R ¹⁰ includes a fluorine and either Z is a hydrogen or Z is an— OH group. As used herein a dashed bond (— ) is used to indicate a bond to an atom outside the denoted structure. Preferably, R ¹ is an alkyl group represented by the following formula:— (CH ₂ ) _n5 -CH ₃ , where n ₅ is an integer from 0 to 12, preferably ns is 0. In some embodiments, R; and R _j , at each location, are hydrogen. Additionally or alternatively, in some embodiments R to R are all the same, preferably fluorine or— CF ₃ . In some embodiments, n and m are independently selected integers from 25 to 150, from 25 to 100, from 35 to 100, or from 35 to 75.

In some embodiments, the fluorinated PLGA polymer is represented by a formula selected from the following group of formulae consisting of:

.(9)

In some embodiments in which the fluorinated PLGA polymer is represented by Formula (6) or (7), R ¹ is a hydrogen or a— CH ₃ . In some embodiments, the fluorinated PLGA polymer has a number average molecular weight of at least 2000 g/mol, at least 3000 g/mol, at least 4000 g/mol. Additionally or alternatively, in some embodiments the fluorinated PLGA polymer has a number average molecular weight of no more than 9000 g/mol, no more than 8000 g/mol, or no more than 7000 g/mol. In some embodiments, the fluorinated PLGA polymer has a number average molecular weight of from 2000 g/mol to 9000 g/mol, from 3000 g/mol to 8000 g/mol or from 4000 g/mol to 7000 g/mol. Number average molecular weight can be determined by Gel Permeation Chromatography (“GPC”) using the polystyrene standard.

In some embodiments, the fluorinated PLGA polymer has a weight average molecular weight of at least 2000 g/mol, at least 3000 g/mol, at least 4000 g/mol. Additionally or alternatively, in some embodiments the fluorinated PLGA polymer has a weight average molecular weight of no more than 20,000 g/mol, no more than 15,000 g/mol, or no more than 10,000 g/mol. In some embodiments, the fluorinated PLGA polymer has a weight average molecular weight of from 2000 g/mol to 20,000 g/mol, from 3000 g/mol to 15,000 g/mol or from 4000 g/mol to 10,000 g/mol. Weight average molecular weight can be determined by Gel Permeation Chromatography (“GPC”) using the polystyrene standard.

Fluorinated PLGA Nanoparticles. Drug Encapsulated PLGA Nanoparticles, and Dispersions

The fluorinated PLGA 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 PLGA 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 PLGA nanoparticles and corresponding encapsulated drugs include, but are not limited to, nanocoprecipitation 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.

In one embodiment, the formation method involves dispersing the fluorinated PLGA 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 PLGA nanoparticles encapsulate a drug, both the fluorinated PLGA nanoparticles and corresponding drug are dispersed in the formation solution.

It was surprisingly discovered that ¹⁹ FNMR signal intensity could be tuned by varying the relative concentration of the polar organic solvent to the water. For clarity, the signal intensity refers to the strongest signal (highest peak) in the ¹⁹ FNMR spectrum. As demonstrated in the examples below, in some embodiments, it was unexpectedly discovered that the ¹⁹ FNMR signal intensity increased with increasing polar organic solvent concentration, relative to the water concentration, in the formation solution. In some embodiments, the volume ratio of polar organic solvent to water in the formation solution is at least 1 :20, at least 1 : 15, at least 1 : 10, or at least 1 :5. Additionally or alternatively, in some embodiments, the volume ratio of the polar organic solvent to water in the formation solution is no more than 2.5:1 , no more than 2:1 , no more than 1.5: 1, no more than 1.2: 1, or no more than 1 :1. In some embodiments, the volume ratio of the polar organic solvent to water in the formation solution is from 1 :20 to 2.5: 1, from 1 :15 to 2:1 , from 1 :10 to 1.5: 1, from 1 :5 to 1.2:1 , or from 1 :5 to 1 :1. Desirable polar organic solvent includes, but are not limited to, acetone, dichloromethane, dimethylsulfoxide, dichloromethane, acetonitrile, and toluene. Preferably, the polar organic solvent is acetone.

In embodiments in which the fluorinated PLGA nanoparticles encapsulate a drug, both the drug and the fluorinated PLGA nanoparticles are dispersed in the formation solution. In such embodiments, the weight ratio of the drug to the fluorinated PLGA 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 PLGA nanoparticles is no more than 1 :l , no more than 1 :5, or no more than 1 :10. In some embodiments, the weight ratio of the drug to the fluorinated PLGA 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 PLGA 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 poloxomers. Poloxomers are nonionic triblock copolymers composed of a central hydrophobic chain of polyoxypropylene flanked by two hydrophilic chains of polyoxyethylene. Poloxomers 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.

It was also surprisingly discovered that ¹⁹ FNMR signal intensity could be tuned by varying the pH of the resulting dispersion. As demonstrated in the examples, in some embodiments, the ¹⁹ FNMR signal intensity of the fluorinated PFGA nanoparticles could be increased by increasing the pH of the dispersion (and correspondingly decreased by decreasing the pH). In some embodiments, for desirable ¹⁹ FNMR signal intensity, the pH of the dispersion is at least 7 or at least 7.5. In some embodiments, fluorinated PFGA nanoparticles in dispersions having a pH of less than 7 generally have insufficient ¹⁹ FNMR signal intensity to be useful for ¹⁹ FMRI imaging.

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. For drug delivery applications, collections of nanoparticles having an average size of from 10 nm to 200 nm and a width of no more than 0.2 nm are desirable, as they provide sufficient colloidal stability.

Particle size distributions can be measured by dynamic light scattering (“DFS”). 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.

The fluorinated PLGA nanoparticles described herein have desirable Z-average particle sizes and PDI values for drug delivery applications. In some embodiments, a collection of fluorinated PLGA nanoparticles have a Z-average particle size of at least 45 nm, at least 50 nm, or at least 55 nm. Additionally or alternatively, in some embodiments the collection of fluorinated PLGA nanoparticles have a Z-average particle size of no more than 90 nm, no more than 85 nm, no more than 80 nm, no more than 75 nm, or no more than 70 nm. In some embodiments, the collection of fluorinated PLGA nanoparticles have a Z-average particle size of from 45 nm to 90 nm, from 50 nm to 90 nm, from 50 nm to 85 nm, from 50 nm to 80 nm, from 50 nm to 80 nm, from 50 nm to 75 nm, from 50 nm to 70 nm or from 55 nm to 70 nm. In some embodiments, a collection of fluorinated PLGA nanoparticles has polydispersity index (“PDI”) of at least 0.05 or at least 0.08. Additionally or alternatively, in some embodiments the collection of fluorinated PLGA nanoparticles has a PDI of no more than 0.2, no more than 0.18, no more than 0.15, or no more than 0.1. In some embodiments, a collection of fluorinated PLGA nanoparticles has a PDI of from 0.05 to 0.2, from 0.05 to 0.18, from 0.05 to 0.15, or from 0.05 to 0.10. PDI is related to dispersion of z-averaged sizes of the particle population according to the following formula: PDI=(o/d) , 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 PLGA nanoparticles as well as the drug encapsulated fluorinated PLGA nanoparticles.

In some embodiments, the fluorinated PLGA nanoparticle dispersions have a fluorinated PLGA 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 PLGA nanoparticle dispersions have a fluorinated PLGA 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 PLGA nanoparticle dispersions have a fluorinated PLGA 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 PLGA dispersions have a drug encapsulated fluorinated PLGA 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 PLGA dispersions have a drug encapsulated fluorinated PLGA 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 PLGA dispersions have a drug encapsulated fluorinated PLGA 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 PLGA 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. Taxol). In some embodiments, the drug encapsulated fluorinated PLGA nanoparticles have a drug loading of at least 3%, at least 4 %, at least 5%, at least 6% or at least 7%. Additionally or alternatively, in some embodiments the drug encapsulated fluorinated PLGA nanoparticles have a drug loading of no more than 20%, no more than 18%, no more than 16%, no more than 15% or no more than 14%. In some embodiments, the drug encapsulated PLGA nanoparitlces have a drug loading of from 3% to 20%, from 4% to 18%, from 5% to 16%, from 6% to 15%, or from 7% to 14%. As described in the Examples, drug loading can be measured using HPLC and determined according to the following formula:

EXAMPLES

Example 1 : Synthesis of Activated PLGA

The present Example demonstrates the synthesis of N-hydroxysuccinimide functionalized PLGA (“activated PLGA”) according to following scheme:

To demonstrate the synthesis, PLGA (0.33 mmol, 1 eq) was dissolved in 10 mL of anhydrous CH2CI2 to form a solution. Solid N-hydroxysuccinimide (“NHS”) (1.32 mmol, 4 eq) was then added to the solution. Subsequently, the mix of reaction was cooled at 0° C and N,N'- dicyclohexylcarbodiimide (“DCC”) (1.38 mmol, 4.2 eq) was added. The reaction mixture was left at room temperature (25° C) for 18 hours (“h”), while being stirred. The resulting urea-DCC solid was filtered and a white powder was precipitated from the remaining CH2CI2 solution with cold ether. The obtained white powder was dried under vacuum. 'HNMR confirmed the white powder was activated PLGA - 'HNMR (400 MHz, CDCI3): d [ppm] = 5.30-5-19 (m, 1H, CH PLGA), 4.89-4.67 (m, 2H, CH ₂ PLGA), 2.84 (m,2H, CH ₂ ), 1.58-1.59 (d, 3H, CH ₃ PLGA).

Example 2: Synthesis of Trifluoroethylamine Functionalized PLGA

The present Example demonstrates the synthesis of 2,2,2 -trifluoroethylamine functionalized PLGA (“CF3-PLGA-OH”) according to following scheme:

To demonstrate the synthesis, activated PLGA (0.33 mmol, leq) was dissolved in 6 mL of anhydrous CH2CI2 to form a solution. Subsequently, 2,2,2-trifluoroethanolamine (1.27 mmol, 3.86 eq) and N,N-diisopropylethylamine (1.43 mmol, 4.34 eq) were added to the solution to form a reaction mixture. The reaction mixture in anhydrous CH2CI2 was left for 18h at room temperature, while being stirred. Subsequently, the mixture was concentrated and CF3-PLGA- OH was precipitated from the mixture with cold ether. In order to increase the purity of the product, it was washed with Brine solution. Finally, CF3-PLGA-OH was dried under vacuum. 1 12 mg of white electrostatic powder were obtained. NMR confirmed the white powder was CF3-PLGA-OH: 'HNMR (400 MHz, CDCl ₃ ): d [ppm] = 6.86-6.55 (brs, OH), 5.26-5-18 (m, 1H, CH PLGA), 4.89-4.67 (m, 2H, CH ₂ PLGA), 3.95 (m, 2H, CH ₂ ), 1.58-1.59 (d, 3H, CH ₃ PLGA); ¹³ CNMR (400 MHz, CDCI3): d [ppm] = 169.49, 166.43, 69.25, 60.88, 40.30, 16.66; and ¹⁹ FNMR (400 MHz, CDCI3): d [ppm] = 72.49 (s, 3F, CF ₃ ). Example 3: Synthesis ofNonafluoro-t-Butoxyethylamine Functionalized PLGA

The present Example demonstrates the synthesis of nonafluoro-t-butoxyethylamine functionalized PLGA (“(CF3)3-PLGA-OH”) according to following scheme:

To demonstrate the synthesis, activated PLGA (0.33 mmol, leq) was dissolved in 6 mL of anhydrous CHCI3 to form a solution. Subsequently, nonafluoro-tbutoxyethylamine HC1 (0.28 mmol, 1 eq) and diisopropylethylamine (“DIPEA”) (1.15 mmol, 4 eq) were added to the solution to create a reaction mixture. The reaction mixture was left for 18 h at room temperature under N2 atmosphere, while being stirred. Subsequently, the reaction mixture was concentrated under vacuum and (CF3)3-PLGA-OH was precipitated from the concentrated reaction mixture with cold ether. The recovered (CF3)3-PLGA-OH was washed with a brine solution and dried under vacuum at 50° C. 700 mg of white electrostatic powder were obtained. NMR confirmed the white powder was (CF3)3-PFGA-OH: 'HNMR (400 MHz, CDCI3): d [ppm] = 6.61 (brs, OH), 5.26-5.19 (m, 1H, CH PEG A), 4.88-4.60 (m, 2H, CH ₂ PEG A), 4.12 (m, 2H, CH ₂ ), 3.59 (m, 2H, CH ₂ ), 1 .57-1.59 (d, 3H, CH ₃ ); ¹³ CNMR (400 MHz, CDCl ₃ ): d [ppm] = 169.52, 166.46, 69.29, 68.54, 60.90, 38.99, 16.69; ¹⁹ FNMR (400 MHz, CDCl ₃ ): d = -70.37 (s, 9F, CF ₃ ); and ¹⁵ NNMR

(400 MHz, CD ₃ CN): d [ppm] = 244.61 (s, 1N, NH).

Example 4: Methylation of Nonafluoro-t-Butoxyethylamine Functionalized PLGA

The present Example demonstrates the methylation of nonafluoro-t-butoxyethylamine functionalized PLGA (“methylated (CF3)3-PLGA-OH”) according to following scheme:

The methylation was performed by modifying the approach described in New journal of Chemistry, 2015, 39, 8720, to Chopra and Panini and incorporated by reference herein. NaH (0.056 mmol, 1.2 eq.) was dispersed in 6 ml of dry THF, under constant stirring, to form a reaction mixture. Subsequently, (CF3)3-PLGA-OH (0.047 mmol, 1 eq.) was added slowly to the reaction mixture, which was refluxed for 2 h at 60° C. After refluxing, the reaction mixture was allowed to cool to room temperature and methyl iodide, (excess amount, 0.6 mL added) was slowly added at 0° C. The reaction mixture was left for 12 h at room temperature, while being stirred. The reaction was quenched with 20 ml of 5% HC1 solution and extracted with ethyl acetate and then washed with brine solution three times. 90 mg of a yellow oil was obtained. NMR confirmed the yellow oil to be methylated (CF3)3-PLGA-OH: 'HNMR (400 MHz, CD ₃ CN): d [ppm] = 5.19-5.03 (m, 1H, CH PLGA), 4.79-4.56 (m, 2H, CH ₂ PLGA), 4.12-4.1 1 (m, 2H, CH ₂ ), 4.07-4.05 (m, 2H, CH ₂ ), 2.30 (brs, OH), 1.48-1.36 (d, 3H, CH ₃ ); ¹³ CNMR (400 MHz, CD ₃ CN): d [ppm] = 170.20, 167.35, 69.54, 59.06, 52.48, 38.67, 29.95, 16.45; ¹⁹ FNMR (400 MHz, CD ₃ CN) : d [ppm] = -71.12 (s, 3F, CF ₃ ); and ¹⁵ NNMR (400 MHz, CD ₃ CN): d [ppm] = 244.61 (s, IN, NH), -199.98 (s, 1N, NCH ₃ ).

Example 5: Synthesis of Trifluoropropylmercaptan Functionalized PLGA

The present Example demonstrates the synthesis of T rifluoropropylmercaptan functionalized PLGA (“CF3-PLGA-COOH”) according to following scheme:

To demonstrate the synthesis, PLGA (0.57 mmol, 1 eq) was dissolved in 100 mL of anhydrous CH ₂ Cl2, under argon. Then, the mix of reaction was cooled at 0°C and Trimethylamine (“TEA”) (1.2 mL, 2 equiv. per hydroxyl group of polymer) was dropwise added. The reaction was left stirred for 15 min. Afterwards, acryloyl chloride (1 mL, 4 equiv.) was added dropwise to the solution and the concomitant reaction proceeded in the dark at room temperature for 24 h. The resulting reaction mixture was filtered to remove triethyl ammonium chloride salt, and purified by precipitation in an excess diethyl ether and dried under vacuum for 24 h. 1.5 gr of a white powder was obtained.

CF3-PLGA-COOH was prepared by mixing 3,3,3 Trifluoropropylmercaptan with acrylate PLGA via Michael-type addition. Tris (2-carboxyethyl)phosphine (“TCEP”) hydrochloride (0.07 mmol, 0.1 eq), was solubilized in 1 mL of phosphate-buffered saline (“PBS”) solution at pH 8 to create a TCEP solution. The acrylate PLGA (0.07 mmol, 1 eq), previously solubilized in 8 mL of acetone, was dropwise added to TCEP solution and, subsequently, 3,3,3 Trifluoropropylmercaptan (0.07 mmol, 1 eq) was added to create a reaction mixture. The reaction mixture was left for 48 h at room temperature under a N ₂ flow, while being stirred. The formation of a yellow gel phase was observed. The isolated solid was dissolved in CH2CI2 and washed with MQ water (Type 1 water according to ISO 3696 - 1987). Then, the organic phase was treated with an excess of cold diethyl ether causing the precipitation of a yellowish gel. The isolated solid was dried under vacuum for 24 h. A yellow oil was finally obtained (50 mg). NMR confirmed the yellow oil was CF3-PLGA-COOH: 'HNMR (400 MHz, CDCI3): d [ppm] = 6.46-6.34 (m, 2H, CH ₂ ), 6.18-6.08 (m, 2H, CH ₂ ), 5.89-5.81 (m, 2H, CH ₂ ), 5.19-5.09 (m, 1H, CH PLGA), 4.83-4.57 (m, 2H, CH ₂ PLGA), 3.45-3.35 (m, 2H, CH ₂ ), 2.81-2.71 (m, 2H, CH ₂ ), 2.67- 2.62 (m, 2H, CH ₂ ), 2.37-2.27 (m, 2H, CH ₂ ), 1.53-1.50 (d, 3H, CH ₃ PLGA); ¹³ CNMR (400 MHz, CDCI3): d [ppm] = 169.50, 166.44, 69.25, 60.89, 16.66; and ¹⁹ FNMR (400 MHz, CDCl ₃ ): d [ppm] = -66.01 (s, 3F, CF ₃ ).

Example 6: Synthesis of Spider-NH _j Functionalized PLGA

The present Example demonstrates the synthesis of 3-(3-((l,l ,l,3,3,3-hexafluoro-2- (trifluoromethyl)propan-2-yl)oxy)-2 ,2-bis(((l , 1 , 1 ,3 ,3 ,3 -hexafluoro-2-(trifluoromethyl)propan-2- yl)oxy)methyl)propoxy)propan-l -amine (“Spider-N ’) functionalized PLGA (“F ₂₇ -PLGA”) according to following scheme:

To demonstrate the synthesis, PLGA (0.12 mmol, leq) was dissolved in 6 mL of anhydrous CHCl ₃ , under N ₂ flow. Spidcr-NFE (0.12 mmol, 1 eq) was solubilized in a mix solution of CHsOHiDMSO (7: 1) and added to the reaction solution. Subsequently, DIPEA (0.47 mmol, 4 eq) was added at 0° C. The reaction was left stirring at room temperature for 4 days, under N ₂ atmosphere. The solution was concentrated and was washed with Brine solution. Finally, it was dried under vacuum. 492 mg of a gel were obtained. NMR analysis confirmed the gel was F ₂₇ -PLGA: *HNMR (400 MHz, CDC13): d [ppm] = 5.23-5.09 (m, 1H, CH, PEG A), 4.83- 4.52 (m, 2H, CH ₂ , PEG A), 3.98 (s, 6H, CH ₂ ), 3.61-3.55 (m, 2H, CH ₂ ), 3.31 (s, 2H, CH ₂ ), 3.06- 2.99 (m, 2H, CH ₂ ), 1.65-1.75 (m, 2H, CH ₂ ) 1.52-1.50 (d, 3H, CH ₃ , PLGA); ¹³ CNMR (400 MHz, CDCI ₃ ): d [ppm] = 169.30, 166.34, 68.96, 60.93, 53.41 , 16.63; ¹⁹ FNMR (400 MHz, CDC13, d) : d [ppm] = -70.38 (s, 27F, CF ₃ ). Example 7: Formation. Size Characterization and ¹⁹ FNMR Activity of Fluorinated PLGA Nanoparticles

The present Examples demonstrates the formation, size characterization and NMR activity of fluorinated PFGA nanoparticles.

Nanoparticles of PFGA (as a comparative), CF ₃ -PFGA-OH, (CF ₃ ) ₃ -PFGA-OH, CF ₃ - PFGA-COOH, methylated CF ₃ -PFGA-COOH and F ₂₇ -PFGA were formed as described in Journal of Transplantation, vol. 212, p. 1 -9, to Tang et al. and incorporated by reference herein. More specifically, 10 mg of PFGA or fluorinated PFGA were added to an acetone solution to form a 10 mg/mF polymer solution. The polymer solutions were dropwise added into 1 mF, 5, mF, 10 mF or 20 mF of either PBS (at a pH of either 7.2 or 8) or MQ water (at a pH 6.5). To evaporate the organic solvent (acetone), the resulting nanoparticle suspension was left for 18 h at room temperature, while being stirred. The final concentration of the fluorinated PFGA nanoparticles in the dispersion was 0.5 mg/mF, 1 mg/mF, 2 mg/mF or 10 mg/mF.

Z-average particle size and PDI were obtained as described above, using DFS. More particularly, multiangle-DFS measurements were performed on an ALV/CGS-3 Platform-based Goniometer System equipped with an ALV-7004 correlator and an AFV / CGS-3 goniometer. The signal was detected by an ALV-Static and Dynamic Enhancer detection unit. The light source was the second harmonic of a diode -pumped Coherent Innova Nd:YAG laser (l = 633 nm), linearly polarized in the vertical direction. Measurements were performed at 25 °C. Approximately 1 mF of sample solution was transferred into the cylindrical Hellma scattering cell. The dynamic information on nanoparticles dispersions were derived from the normalized autocorrelation function g ₂ (q, t) of the scattered intensity, which is measured according to the following formula:

where q is the scattering vector, t is the relaxation time and I is the scattered intensity. Data analysis was performed with the cumulant method. For each sample, at least three measurements were performed at four different angles (70°, 90°, 1 10°, 130°), corresponding to four different scattering vectors (q): q = (4 ph/l) sin(6/2), where n is the refractive index of the medium and Q is the scattering angle. Table 1 displays sample parameters and the results of particle size distribution measurements:

TABLE 1

Referring to Table 1 , the fluorinated PLGA nanoparticles all had desirable Z-average size distributions for drug delivery applications. The Z-average particles sizes ranged from about 37 to about 87 and the PDI was no more than about 0.2.

To further demonstrate MRI activity, ¹⁹ FNMR of samples of the fluorinated nanoparticles were obtained. Figs. 1 to 3 display the ¹⁹ FNMR spectra of samples 4, 7 and 8, respectively. The 1 ⁹ FNMR spectra demonstrate fluorine signal around -48 ppm, -70 ppm and -72 ppm for samples 4, 7 and 8, respectively. Comparison of samples 2 with sample 5 and sample 3 with samples 6 and 6A, demonstrates the tunability of ¹⁹ FNMR activity with pH. No ¹⁹ FNMR signals were observed with sample 2 (prepared with water at a pH of 6.5). However, for sample 5 (prepared with a PBS with a pH of 8), ¹⁹ FNMR signals were observed at about -72 ppm, as shown in Fig. 4. Similarly, samples 3, 6 and 6A also demonstrate the tenability of the ¹⁹ FNMR signal. Fig. 5 is a plot showing the integral value of the ¹⁹ FNMR peak (normalized for the peak of the standard solution - trifluoroethanol) of samples 3, 6 and 6A. Referring to the figure, the integral increases with increasing pH, with sample 3 having the lowest value and sample 6 having the highest value. The comparison demonstrates that ¹⁹ FNMR activity can selectively triggered with a change in pH. In conjunction with the ¹⁹ FNMR analysis, Ti and T ₂ times were determined. Fluorine Tl measurements were performed with Inversion recovery sequence, with a pw=90°, relaxation delay=about 5*Ti, acquisition time=ls, number of transient=4 (or varied on the basis of S/N ratio), variable delay was exponentially increased usually varying from 0.0025s, to 20.5s with 14 steps. Fluorine T ₂ measurements were performed with CPMG sequence, with a pw=90°, relaxation delay= about 5*Ti, acquisition time=l .5s, number of transients 6 (or varied on the basis of S/N ratio), variable delay was exponentially increased, usually varying from 0.075s, to l9.2s with 9 steps. Samples 9 and 1 1 were prepared as described above for samples 6 and 4 (polymer concentration in dispersion was 0.5 mg/mL). Samples 10 and 12 were also prepared as described above for samples 6 and 4, respectively, however the polymer concentration in the dispersion was 2 mg/mL. The T i and T ₂ times are displayed in Table 2.

TABLE 2

Referring to Table 2, samples 9 to 12 demonstrate suitable relaxation times for MRI activity.

Example 8: Formation. Size Characterization. Loading Performance and ¹⁹ FNMR Activity of Drug Encapsulated Fluorinated PLGA Nanoparticles

The present example demonstrates the formation, size characterization, encapsulation performance and ¹⁹ FNMR activity of dexamethasone and leflunomide encapsulated fluorinated PLGA nanoparticles.

To demonstrate formation 6 sample dispersions were formed (samples 13 to 18). To form each sample, dexamethasone or leflunomide was solubilized in acetone solution to obtain a concentration of 1 mg/mL or 2 mg/mL drug solution. 10 mg or 20 mg of polymer (either PLGA, CF ₃ -PLGA-OH, or (CF ₃ ) ₃ -PLGA-OH) was subsequently were solubilized in 1 ml of the drug solution to create a mixture. The mixture was added dropwise to 1 mL of MQ water and the suspension was stirred at room temperature from 15 min to 12 h. After stirring, the acetone was removed with a rotovap.

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 from which two different dilutions, 1 :10 and 1 : 100, to form HPLC analysis samples. Each analysis sample was injected (10 pL) in a Cl 8 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 and the LEF peak after aboutlO min. The detection wavelength was set at 254 nm. Calibration curves were previously obtained with different DEX and LEF 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

Dexamethasone Encapsulated Fluorinated PLGA Nanoparticles

Results of the Z-average particle distributions are displayed in Table 3, and demonstrate that dexamethasone encapsulated (CF ) -PLGA-OH have excellent stability for drug delivery applications.

TABLE 3

Referring to Table 3, at a drug:polymer weight ratio (“DP ratio”) of 1 :10, the Z-average particle sizes and PDI of both samples 13 and 15 are comparable to those obtained for the corresponding fluorinated PLGA nanoparticles without drug encapsulation (samples 1 and 3, respectively). The result demonstrates the stability of DEX encapsulated PLGA and (CF ) - PLGA-OH at DP ratio of 1 :10. However, at a DP ratio of 2:20 (samples 14 and 16), the DEX encapsulated PLGA had a PDI that was significantly larger than 0.20 and the DEX encapsulated (CF ) -PLGA-OH had a PDI that was significantly less than 0.20. The results demonstrate that at a DP ratio of 2:20, while the DEX encapsulated PLGA was not suitably stable, the DEX encapsulated (CF ) -PLGA-OH had excellent stability for drug delivery applications.

Results of the encapsulation performance measurements are displayed in Table 4, and demonstrate that dexamethasone encapsulated (CF ) -PLGA-OH had significantly increased drug loading and encapsulation efficiency values relative to the corresponding dexamethasone encapsulated PLGA.

TABLE 4

Referring to Table 4, for the DP ratios tested, the (CF3)3-PFGA-OH nanoparticles had up to twice the drug loading capability and significantly increased encapsulation efficiency, relative to the PFGA nanoparticles. For example, comparison of samples 13 with sample 15 demonstrate that at a DP ratio of 1 :10, (CF3)3-PFGA-OH nanoparticles encapsulated twice the amount dexamethasone, and had significantly increase encapsulation efficiency, relative PGA. Similar results were seen at a DP ratio of 2:20.

Fig. 6 is a ¹⁹ FNMR spectrum of sample 15 and demonstrates the ¹⁹ FNMR activity of the (CF3)3-PFGA-OH encapsulated dexamethasone nanoparticles. The spectrum shows a strong signal at about -70 ppm.

Leflunomide Encapsulated Fluorinated PLGA Nanoparticles

Results of the Z-average particle distributions are displayed in Table 5, and demonstrate that (CF3)3-PFGA-OH encapsulated leflunomide nanoparticles have excellent stability for drug delivery applications.

TABLE 5

Referring to Table 5, sample 18 ((CF3)3-PLGA-OH) had significantly reduced Z-average particle size and PDI relative to sample 17 (PLGA). Furthermore, while sample 17 is not suitable for drug delivery applications ( e.g . PDI > 2.0), based at least in part upon the Z-average particle size and PDI, sample 18 had excellent colloidal stability for drug delivery applications. Results of the encapsulation performance measurements are displayed in Table 6, and demonstrate that leflunomide encapsulated (CF3)3-PLGA-OH had significantly increased drug loading and encapsulation efficiency values relative to the corresponding leflunomide encapsulated PLGA.

TABLE 6

Referring to Table 6, the (CF3)3-PLGA-OH nanoparticles had increased drug loading and encapsulation efficiency, relative to the PLGA nanoparticles.

Fig. 7 is a ¹⁹ FNMR spectrum of sample 18, which demonstrates the ¹⁹ FNMR activity of

(CF3)3-PLGA-OH encapsulated leflunomide nanoparticles. The spectrum shows a distinct F ¹⁹ signals at about -62 ppm and -70 ppm.

Example 9: Dependence of ¹⁹ FNMR Activity on Nanoparticle Formation Method

The following example demonstrates the dependence of ¹⁹ FNMR Activity on the formation method.

To demonstrate the dependence, 3 samples were formed. The samples included (CF3)3- PLGA-OH nanoparticles formed as described above in Example 5, using 3 different volume ratios of acetone to water: 1 :1 , 1 :5 and 1 :10 (samples 19 to 21 , respectively). ¹⁹ FNMR spectra were obtained for each of the samples and the peak integrals, normalized for the peak of the standard solution (trifluoroethanol), were obtained.

Fig. 8 is a plot showing the polar organic solvent to water ratio dependence of ¹⁹ FNMR peak integrals (normalized for the peak of the standard solution) (CFA-PLGA-OH nanoparticle. Referring to the figure, the integral (and therefore the intensity) of the ¹⁹ FNMR peak integrals decrease with increasing amounts of water.

Further Inventive Concepts

1. A fluorinated poly(lactic-co-glycolic acid) polymer (“PLGA”) represented by the following formula:

, (1)

wherein

Z ¹ is a hydrogen or a group represented by the following formula:

Z ² is a group represented by a formula selected from the following group of formulae:

-OH; (3)

i ( t I ^{^} 313

; (5) wherein

R ¹ is a hydrogen or a Ci to C12 alkyl group;

Ri and R _j , at each location, is independently selected from the group consisting of a hydrogen or an alkyl group;

R ² to R ¹⁰ are independently selected from the group consisting of a hydrogen, a fluorine,— CF ₃ ,— CHF ₂ ,— CH ₂ F and— CH ₃ ;

T is an oxygen or a bond;

n and m are independently selected integers from 25 to 200; and

- ni to n ₄ are independently selected integers from 1 to 12 with the provisio that at least one of R ² to R ¹⁰ includes a fluorine and either Z ¹ is a hydrogen or Z is an—OH group.

2. The fluorinated PLGA of inventive concept 1 , wherein the fluorinated PLGA is represented by a formula selected from the following group of formulae:

.(9) 3. The fluorinated PLGA of either inventive concept 1 or 2, wherein the fluorinated PLGA is represented by Formula (4) or Formula (5).

4. The fluorinated PLGA of either inventive concept 1 or 2, wherein the fluorinated PLGA is represented by Formula (9).

5. The fluorinated PLGA of either inventive concept 2 or 3, wherein R ¹ is a hydrogen or an alkyl group represented by the following formula:— (OH ₂ ) ₃ -OH ₃ , where n ₃ is an integer from 0 to 12, preferably n ₃ is 0.

6. A fluorinated PLGA nanoparticle dispersion comprising:

fluorinated PLGA nanoparticles comprising the fluorinated PLGA of any one of inventive concepts 1 to 5; and

a solvent comprising water.

7. The dispersion of inventive concept 6, wherein the fluorinated PLGA nanoparticles have a Z-average particle size of at least 45 nm, at least 50 nm, or at least 55 nm, as determined by dynamic light scattering (“DLS”).

8. The dispersion of either inventive concept 6 or 7, wherein the fluorinated PLGA nanoparticles have a Z-average particle size of no more than 90 nm, no more than 85 nm, no more than 80 nm, no more than 75 nm, or no more than 70 nm, as determined by DLS.

9. The dispersion of any one of inventive concepts 6 to 8, wherein the dispersion has a PDI of at least 0.05 or at least 0.08, as determined by DLS

10. The dispersion of any one of inventive concepts 6 to 9, wherein the dispersion has a PDI of no more than 0.2 or no more than 1.18, as determined by DLS.

1 1. The dispersion of any one of inventive concepts 6 to 10, wherein the solvent has pH of greater than 7, preferably greater than 7.5. 12. The dispersion of any one of inventive concepts 5 to 11 , wherein the fluorinated PLGA nanoparticles further comprise a hydrophobic drug.

13. A method for forming the dispersion of any one of inventive concepts 5 to 1 1, the method comprising dispersing the fluorinated PLGA nanoparticles in a solution including a polar organic solvent and water, and, subsequently, removing the polar organic solvent.

14. The method of inventive concept 13, wherein the solution further includes a DMARD or a steroidal anti-inflammatory drug.

15. The method of either inventive concept 13 or 14, wherein the solvent is selected from the group of solvents consisting of acetone, dichloromethane, dimethylsulfoxide, dichloromethane, acetonitrile, and toluene, preferably acetone.

16. The dispersion of inventive concept 12, wherein the hydrophobic drug is a disease modifying antirheumatic drug (“DMARD”), preferably leflunomide, or a steroidal anti inflammatory drug, preferably dexamethasone.

17. The dispersion of either inventive concept 12 or 16, wherein the drug loading of the hydrophobic drug in the fluorinated PLGA nanoparticles is at least 3%, at least 4 %, at least 5%, at least 6% or at least 7%.

18. The dispersion of either one of inventive concepts 12, 16 or 17, wherein the drug loading of the hydrophobic drug in the fluorinated PLGA nanoparticles is no more than 20%, no more than 18%, no more than 16%, no more than 15% or no more than 14%.

20. A method of increasing the ¹⁹ FNMR signal intensity of a fluorinated PLGA nanoparticle, the method comprising:

increasing the pH of the solvent of the dispersion of any one of claims 6 to 12. 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.