Polymeric micelle, methods of production and uses thereof

Abstract

The present disclosure relates to enzymatic and redox responsive polymeric micelle, its method of production as well as its uses. Specifically, use of the enzymatic- and redox-responsive polymeric micelle for drug delivery.

Claims

1. A polymeric micelle comprising a micelle core comprising a palmitic acid that encapsulates a hydrophobic active ingredient; and a micelle branch comprising glutathione and methoxypolyethylene glycol, wherein the methoxypolyethylene glycol is bonded to glutathione the micelle core and micelle branch forming a methoxypolyethylene glycol amine-glutathione-palmitic acid; wherein the methoxypolyethylene glycol amine-glutathione-palmitic acid forms a copolymer with a hydrophobic active ingredient encapsulated.

2. (canceled)

3. The polymeric micelle of claim 1 wherein the polymeric micelle is enzymatic and redox responsive.

4. A pharmaceutical composition comprising an effective amount of the polymeric micelle of claim 1.

5. The polymeric micelle of claim 1 wherein the polymeric micelle is a vehicle for drug delivery.

6. A method of treating inflammatory diseases in a subject, the method comprising administering the polymeric micelle of claim 1 to the subject.

7. A method of treating osteoarthritis or rheumatoid arthritis in a subject, the method comprising administering the polymeric micelle of claim 1 to the subject.

8. The polymeric micelle of claim 1 wherein the active ingredient is a hydrophobic drug.

9. The polymeric of claim 1 wherein the hydrophobic drug is dexamethasone, prednisolone, betamethasone, or combinations thereof.

10. The polymeric micelle of claim 1 wherein the size of the micelle is at least 100 nm.

11. The polymeric micelle of claim 1 wherein the micelle has an encapsulation efficiency from 30% to 70%.

12. The polymeric micelle of claim 1 wherein the micelle to drug ratio feed weight is in the range of from 1:0.2 to 1:0.8 for a polymer concentration of 1 mg/mL.

13. The polymeric micelle of claim 1 wherein the amount of hydrophobic active ingredient is from 0.2 mg to 6.0 mg.

14. The polymeric micelle of claim 1 wherein the hydrophobic active ingredient is dexamethasone and wherein the polymeric micelle releases the dexamethasone when the glutathione reductase concentration is at least 50 mU.

15. The polymeric micelle of claim 1 wherein the hydrophobic active ingredient is dexamethasone and wherein the polymeric micelle releases the dexamethasone when the glutathione concentration is at least 10 μM.

16. A pharmaceutical composition comprising the polymeric micelles of claim 1 and a suitable pharmaceutical vehicle.

17. The pharmaceutical composition of claim 16 wherein the concentration of the polymeric micelles is less than or equal to 50 μg/mL.

18. The pharmaceutical composition of claim 16 wherein the composition is a suspension for systemic administration.

19. A method of producing the polymeric micelles of claim 1 comprising: covalently linking methoxypolyethylene glycol amine and glutathione using coupling agents to form a first copolymer; adding the first copolymer to palmitic acid in tetrahydrofuran to form a second copolymer; nano-precipitating the second copolymer to form the polymeric micelles.

20. The method of claim 19 wherein the coupling agents are 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide and N-Hydroxysuccinimide.

21. The method of claim 19 further comprising: adding 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (TBTU) and triethylamine (TEA) as catalysers when forming the second copolymer.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0045] The following figures provide preferred embodiments for illustrating the disclosure and should not be seen as limiting the scope of invention.

[0046] FIG. 1A shows the results of FTIR analyses of the methoxypolyethylene glycol (mPEG), Glutathione (GSH), Palmitic acid (PA) and the polymeric micelles (Mic). FIG. 1B shows the size distribution of the polymeric micelles. FIG. 1C shows the results of stability evaluation of polymeric micelles kept in water for 6 months, at 4° C. FIG. 1D shows the SEM micrographs of the polymeric micelles.

[0047] FIG. 2 shows the release profile of dexamethasone (Dex) from the polymeric micelles under different artificial milieu: PBS, 10 mM of Glutathione (GSH), and 50 mU of Glutathione Reductase (GR). The analysis was performed at 37° C. and the results captured over a period of 96 hours.

[0048] FIG. 3 shows the biological performance of (A) hACs, (B) EA cell line, and (C) THP-1 cell line cultured with different concentrations of micelles: (I) cell viability, (II) cell proliferation and (Ill) total protein synthesis after 1, 3, and 7 days of culture. Asterisk (*) denotes significant differences (p<0.01) compared to the control (0 μg/mL).

[0049] FIG. 4 shows the SEM micrographs of the polymeric micelles cultured with (A) hACs, (B) EA cell line and (C) THP-1 cell line in the (I) absence (control) and in the presence of the micelles at different concentrations: (II) 50 and (III) 100 μg/mL. Scale bar 10 μm.

[0050] FIG. 5 shows the biochemical performance of (A) hACs and (B) THP-1 cultured in monolayers and hACs co-cultured with activated M1 macrophages after different treatments: control (Ctr, no treatment), micelles encapsulating Dexamethasone (Mic+Dex) and dexamethasone (Dex). The samples were analyzed for (i) cell viability, (ii) cell proliferation, (C) TNF-α concentration, and (D) IL-6 concentration. The alphabet “a” denotes significant difference as compared to hACs Ctr, “b” denotes significant difference as compared to THP-1 Ctr, and “c” denotes significant difference as compared to co-culture Ctr, where p<0.01.

[0051] FIG. 6 shows SEM micrographs of (A) hACs cultured in monolayer and (B) co-cultured with activated M1 macrophages after 14 days of treatment under different conditions: (i) no treatment, (ii) micelles encapsulating Dexamethasone (Dex) and (iii) Dex. Scale bars: 10 μm.

DETAILED DESCRIPTION

[0052] The present disclosure relates to an enzymatic and redox responsive polymeric micelle, its method of production as well as its uses. Specifically, use of the enzymatic and redox responsive polymeric micelle for drug delivery.

[0053] An aspect of the present disclosure relates to the use of the disclosed polymeric micelles to: a) increase the therapeutic index of a hydrophobic drug through encapsulating the drug in the polymeric micelles; b) reduce systemic side effects through the controlled release profiles of the drug and consequently reducing unnecessary exposure to healthy tissues; and c) increase the therapeutic efficacy of currently used drugs, including anti-inflammatory, anti-cancer and many other therapeutic agents.

[0054] In an embodiment, polymeric micelles comprising methoxypolyethylene glycol amine-glutathione-palmitic acid (mPEG-GSH-PA) copolymer were produced. The copolymer was synthetized through 2-step reactions. Firstly, mPEG was covalently linked to GSH using 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-Hydroxysuccinimide (NHS) as coupling agents. Secondly, mPEG-GSH was allowed to react with PA in tetrahydrofuran (THF) with 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (TBTU) and triethylamine (TEA) acting as catalysers. After copolymer synthesis, polymeric micelles were prepared through nanoprecipitation.

[0055] In an embodiment, after physicochemical characterization of the polymeric micelles, including Fourier-transform infrared spectroscopy (FTIR), particle size, Polydispersity Index (PDI), zeta-potential and morphology analyses, Dex was encapsulated into the polymeric micelles. The Dex content in the polymeric micelles was determined using different micelle:Dex feed weight ratios. The micelle:Dex feed weight ratios varies from 1:0.2 to 1:0.8 for a polymer concentration of 1 mg/mL.

[0056] In an embodiment, in vitro drug release profiles under different external stimulations were evaluated using PBS, 10 mM GSH, and 50 mU GR.

[0057] In an embodiment, micelles cytocompatibility was assessed in the presence of endothelial cell line (EA.hy926), human monocyte-like cell line (THP-1), and human articular chondrocytes (hACs). A co-culture model of inflammation was also established by culturing hACs and stimulated M1 macrophages.

[0058] In an embodiment, the polymeric micelles were characterized.

[0059] In an embodiment, tri-block amphiphilic copolymer was synthesized via a two-steps polymerization reaction. Firstly, mPEG reacts with the carboxylic groups of GSH, and then the free amine groups of GSH reacts with the PA. FTIR analysis of the micelles (FIG. 1A) showed a shift of the amine group from the mPEG and GSH (two N—H stretch absorptions at 3300-3000 cm.sup.−1) to amide in the polymeric micelles (one N—H stretch absorption at 3300 cm.sup.−1 and a C═O peak at 1680-1630 cm.sup.−1). Moreover, while the GSH present a weak thiol (S—H) peak at 2550-2620 cm.sup.−1, the polymeric micelles presented a weak disulphide (S—S) peak at 700-550 cm.sup.−1. The size of the polymeric micelles (FIG. 1B) was 101.3±3.4 nm with a zeta potential of −19.7±3.42 mV and PDI values of 0.092±0.011 (FIG. 1B). Given that the PDI value was lower than 0.2, the micelles population can be considered homogeneous. The assessment of the polymeric micelles' storage stability (FIG. 1C) shows that the micelles are stable over time, without a significant increase in size and PDI for at least 6 months. Based on the SEM analyses (FIG. 1D), the polymeric micelles appear to be largely spherical in shape and has an average diameter of 100 nm. This result is in agreement with the dynamic light scattering (DLS) measurements.

[0060] FIG. 1A shows the results of the FTIR analysis of the methoxypolyethylene glycol (mPEG), Glutathione (GSH), Palmitic acid (PA), as well as the polymeric micelles (Mic). FIG. 1B shows the size distribution of the polymeric micelles. FIG. 1C shows the results of the stability evaluation of the polymeric micelles kept in water for 6 months, at 4° C. FIG. 1D shows the SEM micrographs of the polymeric micelles.

[0061] In an embodiment, polymeric micelles efficiency as delivery device was assessed using Dex as a model drug. Being a hydrophobic drug, Dex was dissolved in the organic phase of the micelles using different micelle:Dex feed weight ratios. The micelle:Dex feed weight ratios varies from 1:0.2 to 1:0.8 at a polymer concentration of 1 mg/mL (Table 1). The results show that the Dex loading content and entrapment efficiency increases with the feed weight ratio. The maximum entrapment efficiency observed was for micelle:Dex feed weight ratio of 1:0.8 which has an efficiency of about 64%. After entrapment of the drug, the polymeric micelles' size is about 118.8±0.2 nm, with 0.105±0.009 of PDI value and, a zeta potential of −17.4±2.7 mV.

[0062] In an embodiment, after quantification of encapsulated Dex into the polymeric micelles, micelle:Dex feed weight ratio of 1:0.8 ratio was chosen for in vitro release profile evaluation (FIG. 2). The in vitro release profile evaluation was performed using the dialysis method in the presence of different artificial milieu. The polymeric micelles showed almost no release during the first 24 hours in the presence of PBS at 37° C. Maximum release of 20% was observed after 5 days. In contrast, the addition of GSH at an intracellular level (10 mM) increased Dex release, maximum release of about 50% was observed after 5 days. The addition of GR at 50 mU was able to induce a burst release of the drug of about 80% during the first 12-24 hours. These results show that the controlled release profiles of the polymeric micelles.

TABLE-US-00001 TABLE 1 Dexamethasone (Dex) loading content (mg) and encapsulation (or entrapment) efficiency (%) into the polymeric micelles, using different micelle:Dex feed weight ratios at a polymer concentration of 1 mg/mL. Micelles:Dex Dex loading Entrapment feed weight ratio content [mg] efficiency [%] 1:0.2 0.57 ± 0.03 35.3 ± 1.9 1:0.4 1.38 ± 0.08 39.8 ± 1.3 1:0.6 2.50 ± 0.10 51.9 ± 2.1 1:0.8 4.65 ± 0.11 64.6 ± 1.6

[0063] FIG. 2 shows the release profile of dexamethasone (Dex) from the polymeric micelles under different artificial milieu: PBS, 10 mM of Glutathione (GSH), and 50 mU of Glutathione Reductase (GR) at 37° C.

[0064] In an embodiment, polymeric micelles' cytocompatibility was analysed. In vitro cellular studies were carried out to assess the viability of different relevant cells that can be affected. The cells used for in vitro cellular analysis are: hACs from diseased knee arthroplasties (phenotype associated with arthritis disease), endothelial cells (main cells of the blood vessels), and macrophages (immune system). After 1, 3 and 7 days of culture, different biological assays were performed to assess cell viability (Alamar Blue—AB—assay), cell proliferation (DNA quantification), total protein synthesis, and cell morphology (SEM). For all the cell types analysed, the results as shown in FIG. 3 revealed that the micelles are cytocompatible until the concentration of the micelle reaches 50 μg/mL. Beyond 50 μg/mL, the polymeric micelles reduced cell viability and cell proliferation as compared with the control. This is especially prominent for polymeric micelles concentration of 200 μg/mL. SEM analyses show that the cell morphology was not affected by the polymeric micelles (FIG. 4). Thus, the preferable concentration of micelles is not more than 50 μg/mL.

[0065] FIG. 3 shows the biological performance of (A) hACs, (B) EA cell line, and (C) THP-1 cell line cultured with different concentrations of micelles: (I) cell viability, (II) cell proliferation and (111) total protein synthesis after 1, 3, and 7 days of culture. Asterisk (*) denotes significant differences (p<0.01) as compared to the control (0 μg/mL).

[0066] FIG. 4 shows SEM micrographs of the polymeric micelles cultured with (A) hACs, (B) EA cell line and (C) THP-1 cell line in the (I) absence (control) and in the presence of the micelles at (II) 50 μg/mL and (111) 100 μg/mL. Scale bar 10 μm.

[0067] In an embodiment, the biological effects of dexamethasone (Dex) in monocultures and co-culture of hACs and THP-1 were analysed. To compare the biological effects of free Dex and the polymeric micelles encapsulating Dex, monoculture and co-culture systems of hACs and stimulated M1 macrophages were used. Three different conditions were tested: (i) no treatment (Ctr), (ii) treatment with micelles encapsulating Dex (Mic+Dex), and (iii) treatment with free Dex (Dex). The concentration of Dex was 100 WI, and in the co-culture system, 50 mU of GR was added.

[0068] In an embodiment, hACs' viability and proliferation was significantly reduced with the free Dex treatment (FIG. 5A). Interestingly, the encapsulation of Dex into the micelles were able to block Dex's nefarious effects on hACs, as no differences between the Ctr and Mic+Dex groups was observed. In addition, morphological analysis of the hACs (FIG. 6A) shows that the encapsulation of the drug did not affect cell density and morphology as observed in the Dex group. This effect was also observed in the THP-1 cell line (FIG. 5B), where treatment with polymeric micelles showed higher cell viability and proliferation as compared to treatment with free Dex.

[0069] In an embodiment, co-culture of hACs with activated M1 macrophages significantly decreased cell viability and proliferation as compared to the hACs control. While treatment with Mic+Dex was able to reduce this harmful effect on chondrocytes, the treatment with free Dex was not. Mic+Dex treatment significantly increases cell viability as compared to co-culture without treatment. These results were also corroborated with morphological analysis of hACs (FIG. 6B). After 14 days of co-culture, hACs showed altered morphology with cell shrinkage and a reduction of cell density. Treatment with polymeric micelles encapsulating Dex was able to prevent cell shrinkage and reduction of cell density to a larger degree as compared to treatment with free Dex. Additionally, the co-culture system not shows any nefarious effects on the THP-1 cells. However, the addition of the Mic+Dex and Dex significantly reduced the quantity of DNA, especially after 1 day of treatment.

[0070] In an embodiment, the co-culture of hACs and activated macrophages shows a significant reduction in the amount of pro-inflammatory cytokines produced by those cells. TNF-α and IL-6 cytokines (FIGS. 5C and D, respectively) were quantified in the medium. While the hACs almost do not produce TNF-α, activated M1 macrophages produce around 1.3 ng/mL after 1 day, which was reduced to 0.1 ng/mL after 14 days of treatment. In this case, all conditions (Ctr, Mic+Dex and Dex in THP-1) have a similar reduction of TNF-α in the medium. The polymeric micelles encapsulating Dex were able to reduce more TNF-α in the co-culture system than free Dex, especially after 3 days of treatment. This may be explained due to the controlled release of Dex from the polymeric micelles over the time. With regard to IL-6, the establishment of the co-culture system increased IL-6 amount to a maximum of about 1.2 μg/mL. In this case, both the polymeric micelles with encapsulated Dex and free Dex were able to effectively reduce the amount of IL-6 in the medium to about 0.1 μg/mL.

[0071] In an embodiment, the polymeric micelles are able to protect chondrocytes from nefarious effects, such as cell shrinkage and density, of Dex. The encapsulation of Dex in polymeric micelles not compromises the biological action of the drug in inflammation. Additionally, they are able to protect the chondrocytes during inflammation by reducing pro-inflammatory cytokines (TNF-α and IL-6) amount in the media. Therefore, the overall results show that polymeric micelles encapsulating Dex are able to prolong and extend the Dex half-life, as well as increase the efficacy and to reduce some side effects of free Dex.

[0072] FIG. 5 shows the biochemical performance of (A) hACs and (B) THP-1 cultured in monolayers and hACs co-cultured with activated M1 macrophages after treatment with different conditions: control (Ctr, no treatment), micelles encapsulating dexamethasone (Mic+Dex) and dexamethasone (Dex). The samples were analyzed for (i) cell viability, (ii) cell proliferation, (C) TNF-α concentration, and (D) IL-6 concentration. The alphabet “a” denotes significant difference compared to the hACs Ctr, “b” denotes significant difference compared to the THP-1 Ctr, and “c” denotes significant difference as compared to the co-culture Ctr where p<0.01.

[0073] FIG. 6 shows SEM micrographs of (A) hACs cultured in monolayer and (B) co-cultured with activated M1 macrophages after 14 days of treatment under different conditions: (i) no treatment, (ii) polymeric micelles encapsulating dexamethasone (Dex) and (iii) free Dex. Scale bars: 10 μm.

[0074] The term “comprising” whenever used in this document is intended to indicate the presence of stated features, integers, steps, components, but not to preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

[0075] The disclosure should not be seen in any way restricted to the embodiments described and a person with ordinary skill in the art will foresee many possibilities to modifications thereof.

[0076] The above described embodiments are combinable.

REFERENCES

[0077] 1. Petros R A, DeSimone J M. Strategies in the design of nanoparticles for therapeutic applications. Nat Rev Drug Discov. 2010; 9(8):615-627. [0078] 2. Jhaveri A M, Torchilin V P. Multifunctional polymeric micelles for delivery of drugs and siRNA. Frontiers in pharmacology. 2014; 5:77. [0079] 3. Nicolas J, Mura 5, Brambilla D, et al. Design, functionalization strategies and biomedical applications of targeted biodegradable/biocompatible polymer-based nanocarriers for drug delivery. Chem Soc Rev. 2013; 42(3):1147-235. [0080] 4. Kamaly N, Yameen B, Wu J, et al. Degradable Controlled-Release Polymers and Polymeric Nanoparticles: Mechanisms of Controlling Drug Release. Chem Rev. 2016; 116(4):2602-63. [0081] 5. Pu H L, Chiang W L, Maiti B, et al. Nanoparticles with Dual Responses to Oxidative Stress and Reduced pH for Drug Release and Anti-inflammatory Applications. Acs Nano. 2014; 8(2):1213-1221. [0082] 6. Li Y, Xiao K, Luo J, et al. Well-defined, reversible disulfide cross-linked micelles for on-demand paclitaxel delivery. Biomaterials. 2011; 32(27):6633-45. [0083] 7. Lu S C. Regulation of glutathione synthesis. Molecular aspects of medicine. 2009; 30(1-2):42-59. [0084] 8. Kim H C, Kim E, Ha T L, et al. Highly stable and reduction responsive micelles from a novel polymeric surfactant with a repeating disulfide-based gemini structure for efficient drug delivery. Polymer. 2017433:102-109. [0085] 9. Chiang Y T, Yen Y W, Lo C L. Reactive oxygen species and glutathione dual redox-responsive micelles for selective cytotoxicity of cancer. Biomaterials. 2015; 61:150-161. [0086] 10. Sredzinska K, Galicka A, Porowska H, et al. Glutathione reductase activity correlates with concentration of extracellular matrix degradation products in synovial fluid from patients with joint diseases. Acta Biochim Pol. 2009; 56(4):635-40. [0087] 11. Zhu Z, Du S, Du Y, et al. Glutathione reductase mediates drug resistance in glioblastoma cells by regulating redox homeostasis. Journal of neurochemistry. 2018; 144(1):93-104.