Polymeric micelles stabilized by polyethylenimine–copper (C₂H₅N–Cu) coordination for sustained drug release

Abstract

In order to improve the release properties of water-insoluble drugs from polymeric micelles, we develop polymeric micelles stabilized by polyethylenimine–copper (C₂H₅N–Cu) coordination between the amino groups in ABC triblock copolymer poly(ethylene glycol)-block-linear polyethylenimine-block-poly(ε-caprolactone) (PEG-PEI-PCL) and divalent copper cations.

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Polymeric micelles are typically prepared by self-assembly of amphiphilic polymers once the concentration of amphiphilic polymers exceeds the critical micelle concentration (CMC).¹ Depending on the polymer structure and concentration, polymeric micelles have different forms such as spherical,² worm-like,³ vesicle,⁴ rod,⁵ flower-like,⁶ and so on.⁷ In general, core–shell structure, with a particle size in the range of tens to hundreds of nano-meters, is typical in polymeric micelles as drug carriers.⁸ In the application of drug release, polymeric micelles can achieve controlled and targeted release of the loaded drug, thus affecting the distribution of the drug in vivo and delivering the drug to the target site, to improve drug efficacy and reduce its side effects.⁹

The self-assembly of amphiphilic polymers is mainly derived from non-covalent interactions including hydrophobic interactions, hydrogen bonding, host–guest interactions, electrostatic interactions, etc.¹⁰ Typically, polymeric micelles are not very stable when used in vivo because the concentration of polymeric micelles will be diluted by blood to below CMC.¹¹ In order to enhance the stability of polymeric micelles, crosslinking,¹² complexation,¹³ coordination,¹⁴ and structure modification¹⁵ are developed. Among them, metal–ligand coordination is particularly attractive because of its high specificity and directionality. The coordination polymers have been widely studied in the stimuli-responsive systems and bio-related materials.

It is well-known that AB diblock and ABA triblock copolymers with two different polymer chains have been widely studied for polymeric micelles.¹⁶ At present, ABC triblock copolymers with three different polymer chains are attracting more and more attention due to their unique features, which do not exist in AB diblock and ABA triblock copolymers.¹⁷ For example, temperature, molecular weight, ratio of each block, preparation method, block sequence in the polymer chains, and the modification of a block can affect the morphology of the self-assembly of ABC triblock copolymers.^18–20 So far, ABC triblock copolymers have been widely used in conventional drug delivery systems^21–23 and the preparation of hollow inorganic nanoparticles.^24,25 ABC triblock copolymers are mainly synthesized by controlled/living radical polymerization (e.g., nitroxide-mediated radical polymerization (NMP),²⁶ atom transfer radical polymerization (ATRP),²⁷ and reversible addition-fragmentation chain transfer (RAFT) polymerization²⁸).

In our previous report, ABC triblock copolymer PEG-PEI-PCL was synthesized through the condensation reaction of diblock copolymer poly(ethylene glycol)-block-linear polyethylenimine (PEG-PEI) and monocarboxy-capped poly(ε-caprolactone) (PCL-COOH).²⁹ Compared with the above common methods, the synthesis process of ABC triblock copolymer PEG-PEI-PCL is very simple and operable. We then prepared highly dispersed AuNPs using PEG-PEI-PCL as the stabilizer due to the electrostatic interactions between the N atoms in the polycation block and metal precursor ions.²⁹ Here we describe polymeric micelles stabilized by polyethylenimine–copper (C₂H₅N–Cu) coordination between the amino groups in PEG-PEI-PCL and divalent copper cations (Scheme 1). As the ability of branched PEI to bind to Cu²⁺, linear PEI can also coordinate Cu²⁺.³⁰ The formation of the C₂H₅N–Cu complexes in the micelles will enhance the stability of the micelles.³¹ The formation of the C₂H₅N–Cu complexes was explored by the solution color change in vision and UV-Vis absorption spectra. The micelle properties (morphology, particle size, and drug-loading capacity) and in vitro drug release behaviour of stabilized micelles were investigated.

Scheme 1 Schematic illustration of the formation of polymeric micelles stabilized by polyethylenimine–copper (C₂H₅N–Cu) coordination.

ABC triblock copolymer PEG-PEI-PCL with a hydrophilic–polycation–hydrophobic structure could form micelles by self-assembly in water. In this study, polymeric micelles were prepared by adding deionized water dropwise to a DMF solution of PEG-PEI-PCL under vigorous stirring, followed by dialysis against deionized water and lyophilization. The lyophilized powder was re-dissolved in deionized water and filtered through a syringe filter (pore size: 0.22 μm). Fig. 1 shows the images of CuSO₄ aqueous solution, non-stabilized micelle solution and stabilized micelle solution. The CuSO₄ aqueous solution and non-stabilized micelle solution were both clear and transparent. Upon mixing, the solution color changed to blue, which confirmed the formation of the C₂H₅N–Cu complexes.³² Pure CuSO₄ aqueous solution and non-stabilized micelle solution had no absorption bands in UV-Vis region. The absorption bands at 276 nm and 623 nm in the UV-Vis spectrum of stabilized micelle solution further confirmed the formation of the C₂H₅N–Cu complexes.³²

Fig. 1 Pictures of (A) 1.0 mg mL⁻¹ of CuSO₄ aqueous solution, (B) non-stabilized micelle solution at the polymer concentration of 1.0 mg mL⁻¹, and (C) stabilized micelle solution at the polymer concentration of 1.0 mg mL⁻¹ ([C₂H₅N] : [Cu²⁺] = 1 : 1). (D) UV-Vis absorption spectra of CuSO₄ aqueous solution, non-stabilized micelle solution, and stabilized micelle solution in a scanning range of 200–700 nm.

Fig. 2 displays the size and zeta potential obtained from dynamic light scattering (DLS) measurements for samples containing different [C₂H₅N] : [Cu²⁺] ratios prepared by adding the designed amount of copper(II) sulfate pentahydrate to the micelle solution. In the absence of the Cu²⁺ ions, PEG-PEI-PCL self-assembled to form sun-40 nm polymeric micelles with zeta potential of 28.5 mV. The addition of the Cu²⁺ ions induced the formation of the C₂H₅N–Cu complexes, as evident from the change of the size and zeta potential in Fig. 2. When Cu²⁺ ions were added at ratios of 1, small micelles with average hydrodynamic diameter of 47.7 nm and zeta potential of 19.2 mV were observed. Remarkably, when the amounts of Cu²⁺ ions introduced exceeded the stoichiometric ratio of 1, the particle size increased and zeta potential did not change significantly. These larger particles are probably due to the aggregation of the C₂H₅N–Cu complexes.³³ Therefore, stabilized micelles with [C₂H₅N] : [Cu²⁺] ratio of 1 were studied for the following morphology and the drug release. In fact, the morphology (TEM image) of stabilized micelles containing different [C₂H₅N] : [Cu²⁺] ratios did not have obvious difference.

Fig. 2 Size and zeta potential determined by DLS for samples containing different [C₂H₅N] : [Cu²⁺] ratios.

The morphology of polymeric micelles was characterized by TEM as shown in Fig. 3. The particles for polymeric micelles were nano-sized in spherical shape. The average diameters of non-stabilized micelles and stabilized micelles were 31.2 ± 4.5 nm and 39.8 ± 2.6 nm from the TEM images, respectively. The size and size distribution of polymeric micelles were determined by DLS with the distribution profile shown in Fig. 3. The non-stabilized micelles had a mean diameter of 39.2 ± 2.8 nm (PDI: 0.044) based on the intensity-averaged values by DLS. The stabilized micelles had a mean diameter of 47.7 ± 4.3 nm (PDI: 0.054). The diameters of the non-stabilized micelles and the stabilized micelles had significant difference, indicating that the addition of the Cu²⁺ ions affected the micelle size. The main reason for slightly higher diameter than that of non-stabilized micelles may be that the addition of CuSO₄ enlarges the volume of the micelles when it goes into the interior of the micelles and forms the C₂H₅N–Cu complexes at the polycation segment. The change in PDI upon complexation with the Cu²⁺ ions should be that the different number of the Cu²⁺ ions entering the interior of the micelles results in the different volume change of the micelles. Methotrexate, a hydrophobic anticancer drug, was encapsulated by addition to the polymer solution before the self-assembly formation. The methotrexate loading and the complex formation caused an increase in micelle size to 56.2 ± 3.7 nm (PDI: 0.059) and 65.3 ± 4.8 nm (PDI: 0.065), respectively. The PDI increased after loading the drug. The reason for the PDI increase is probably that the different number of the drug entering the interior of the micelles results in the different volume change of the micelles.

Fig. 3 Morphology images characterized by TEM of (A) non-stabilized micelles and (B) stabilized micelles ([C₂H₅N] : [Cu²⁺] = 1 : 1). Size distribution profile determined by DLS of (C) non-stabilized micelles and (D) stabilized micelles ([C₂H₅N] : [Cu²⁺] = 1 : 1). Scale bar: 50 nm.

The structure of methotrexate consists of a pteridine ring and dimethyl-p-aminobenzoic acid residue linked with glutamic acid (Scheme S1†). The dissociation constants of the α-carboxyl (pK_a 4.7), γ-carboxyl (pK_a 3.4), and N(1) of the pteridine-ring (pK_a 5.7) lead to the overall dissociation status in the pH range from 5.8 to 7.6.³⁴ Although methotrexate contains several amino groups, it is a weak dicarboxylic acid, and thus mostly ionized at physiologic pH. In the presence of the Cu²⁺ ions, methotrexate simultaneously loses two protons of the α-carboxyl and γ-carboxyl. The Cu²⁺ ions bind to the oxygen atoms of the α-carboxyl and γ-carboxyl. The forced dissociation of amide moiety then causes the binding of N_amide to metal ions. The model of coordination (α-COO–, γ-COO–, and N_amide) is referred in the literature.³⁵ So, the amino groups in methotrexate do not have significant interaction with the Cu²⁺ ions. In addition, linear PEI as used in ABC triblock copolymer PEG-PEI-PCL has pK_a of 8.44.³⁶ The cationic species of ethylene diamine derivatives exist stronger coordination ability with the Cu²⁺ ions than the carboxyl groups.³⁷ It should be also mentioned that methotrexate is mainly encapsulated in the hydrophobic core of polymeric micelles. The Cu²⁺ ions are more likely to be in a hydrophilic environment. Therefore, it is suggested that methotrexate do not exist considerable coordination with the Cu²⁺ ions.

The methotrexate loading contents into non-stabilized micelles and stabilized micelles were 2.32% and 3.52%, respectively. The methotrexate loading efficiencies into non-stabilized micelles and stabilized micelles were 11.3% and 17.0%, respectively. The result indicated that stabilized micelles had a higher drug loading capacity than non-stabilized micelles. The C₂H₅N–Cu complexes can enhance the stability of the micelles and improve the drug loading property. The drug release behaviour was investigated in PBS (pH 7.4) by monitoring the drug amounts released from the drug-loaded micelle solution that was placed in a dialysis bag. The initial polymer concentration of the micelle solution in the drug release experiment is 0.1 mg mL⁻¹. As shown in Fig. 4, rapid releases of approximately 64% of methotrexate-encapsulated by non-stabilized micelles and approximately 35% from stabilized micelles were observed in the first day. Too much released drug at the beginning will be wasted. In addition, high concentration of the released drug will cause toxicity. Thus, stabilized micelles probably control the effective concentration of the drug in low-level and high-efficiency without toxic and side effects. After two days, the released drug from non-stabilized micelles in each day is less than 2%, while the release of the drug from stabilized micelles is sustained and slow over a prolonged period up to 2 weeks. The sustained release of stabilized micelles was probably due to the presence of stabilized layers between the inner core and outer shell caused by polyethylenimine–copper (C₂H₅N–Cu) coordination.³⁸ Due to the burst release of the drug from non-stabilized micelles in the first day and invalid concentration of the drug after two days, the patients need to take the drug by injection or oral every day and suffer from potential toxic effects. If using stabilized micelles to release the drug, the patients probably take the drug each week or every two weeks (which saves labor cost) and the drug is used efficiently at the level of safety (which reduces the cost of pay).

Fig. 4 In vitro methotrexate release profile from non-stabilized micelles (□) and stabilized micelles (●) ([C₂H₅N] : [Cu²⁺] = 1 : 1) in aqueous medium at 37 °C.

The cytotoxicity is very important when considering polymeric micelles including the metal ions to be applied in the fields of biomedicine. Here, in vitro cytotoxicity of CuSO₄, the as-prepared copolymer PEG-PEI-PCL, and the as-prepared stabilized micelles containing the C₂H₅N–Cu complexes on COS7 cells were investigated by the commonly used MTT assay. The initial concentration of CuSO₄ or PEG-PEI-PCL was 1.0 mg mL⁻¹. The stabilized micelles consist of the polymer of 1.0 mg mL⁻¹ and equal mass of CuSO₄. The results as shown in Fig. S1† indicated that CuSO₄ was toxic to COS7 cells at higher than 0.01 mg mL⁻¹ concentrations³⁹ and PEG-PEI-PCL could be thought of the non-toxic polymer at lower than 0.01 mg mL⁻¹ concentrations. The stabilized micelles exhibited the closed cytotoxicity of PEG-PEI-PCL. The Cu²⁺ ions in the stabilized micelles did not seem to have a toxic effect on cells. As discussed in above part about particle size and zeta potential, [C₂H₅N] : [Cu²⁺] ratio of 1 was the most appropriate proportion for the formation of the stabilized micelles. The formation of the C₂H₅N–Cu complexes decreased the zeta potential of polymeric micelles. In addition, the Cu²⁺ ions were mainly coordinated with the PEI unit in the interlayer between the core and the shell of polymeric micelles. Therefore, the as-prepared stabilized micelles containing the C₂H₅N–Cu complexes did not have significant increase in cytotoxicity. Typically, the concentration of polymeric micelles will be diluted by tissue fluid when injected in vivo. The stabilized micelles were worthy of application in the fields of biomedicine such as cancer treatment. Certainly, the non-toxic metal ions such as cerium cation⁴⁰ should also be considered and used in our next research, in particular, in vivo studies.

Conclusions

In summary, we describe polymeric micelles stabilized by polyethylenimine–copper (C₂H₅N–Cu) coordination between the amino groups in ABC triblock copolymer PEG-PEI-PCL and divalent copper cations. The formation of stabilized polymeric micelles was confirmed by the solution color change and UV-Vis absorption spectra. The polymeric micelles were nano-sized in spherical shape characterized by TEM. The stabilized micelles had a higher drug loading capacity and more sustained drug release than non-stabilized micelles. Therefore, metal–ligand coordination provides a way of preparing stabilized polymeric micelles for sustained drug release.

Acknowledgements

This work is supported by the Natural Science Foundation of Hubei Province (2015CFB697) and State Key Laboratory of Advanced Technology for Materials Synthesis and Processing (Wuhan University of Technology).

Notes and references

1. N. Lefevre, C. A. Fustin and J. F. Gohy, Macromol. Rapid Commun., 2009, 30, 1871–1888.
2. I. Larue, M. Adam, E. B. Zhulina, M. Rubinstein, M. Pitsikalis, N. Hadjichristidis, D. A. Ivanov, R. I. Gearba, D. V. Anokhin and S. S. Sheiko, Macromolecules, 2008, 41, 6555–6563.
3. V. Preziosi, G. Tarabella, P. D'Angelo, A. Romeo, M. Barra, S. Guido, A. Cassinese and S. Iannotta, RSC Adv., 2015, 5, 16554–16561.
4. L. J. Li, X. R. Zheng, B. R. Yu, L. P. He, J. Zhang, H. M. Liu, Y. Cong and W. F. Bu, Polym. Chem., 2016, 7, 287–291.
5. D. Li, Z. M. Tang, Y. Q. Gao, H. L. Sun and S. B. Zhou, Adv. Funct. Mater., 2016, 26, 66–79.
6. B. Liu, H. Y. Chen, X. Li, C. N. Zhao, Y. K. Liu, L. J. Zhu, H. P. Deng, J. C. Li, G. L. Li, F. L. Guo and X. Y. Zhu, RSC Adv., 2014, 4, 48943–48951.
7. S. J. Holder and N. Sommerdijk, Polym. Chem., 2011, 2, 1018–1028.
8. K. Kataoka, A. Harada and Y. Nagasaki, Adv. Drug Delivery Rev., 2012, 64, 37–48.
9. Z. Ahmad, A. Shah, M. Siddiq and H. B. Kraatz, RSC Adv., 2014, 4, 17028–17038.
10. A. Blanazs, S. P. Armes and A. J. Ryan, Macromol. Rapid Commun., 2009, 30, 267–277.
11. S. C. Owen, D. P. Y. Chan and M. S. Shoichet, Nano Today, 2012, 7, 53–65.
12. H. S. Han, K. Y. Choi, H. Ko, J. Jeon, G. Saravanakumar, Y. D. Suh, D. S. Lee and J. H. Park, J. Controlled Release, 2015, 200, 158–166.
13. M. Naito, T. Ishii, A. Matsumoto, K. Miyata, Y. Miyahara and K. Kataoka, Angew. Chem., Int. Ed., 2012, 51, 10751–10755.
14. W. Li, Y. Kim, J. F. Li and M. Lee, Soft Matter, 2014, 10, 5231–5242.
15. C. Porsch, Y. N. Zhang, C. Ducani, F. Vilaplana, L. Nordstierna, A. M. Nystrom and E. Malmstrom, Biomacromolecules, 2014, 15, 2235–2245.
16. A. Rosler, G. W. M. Vandermeulen and H. A. Klok, Adv. Drug Delivery Rev., 2012, 64, 270–279.
17. I. W. Wyman and G. J. Liu, Polymer, 2013, 54, 1950–1978.
18. N. Ghasdian, D. M. A. Buzza, P. D. I. Fletcher and T. K. Georgiou, Macromol. Rapid Commun., 2015, 36, 528–532.
19. T. Higuchi, H. Sugimori, X. Jiang, S. Hong, K. Matsunaga, T. Kaneko, V. Abetz, A. Takahara and H. Jinnai, Macromolecules, 2013, 46, 6991–6997.
20. Y. Zhou, X. P. Long, X. G. Xue, W. Qian and C. Y. Zhang, RSC Adv., 2015, 5, 7661–7664.
21. Q. Jin, T. J. Cai, H. J. Han, H. B. Wang, Y. Wang and J. Ji, Macromol. Rapid Commun., 2014, 35, 1372–1378.
22. X. B. Zhao and P. Liu, ACS Appl. Mater. Interfaces, 2015, 7, 166–174.
23. P. Zhou, Y. Y. Liu, L. Y. Niu and J. Zhu, Polym. Chem., 2015, 6, 2934–2944.
24. T. Kjellman and V. Alfredsson, Chem. Soc. Rev., 2013, 42, 3777–3791.
25. M. Sasidharan and K. Nakashima, Acc. Chem. Res., 2014, 47, 157–167.
26. E. Poggi, C. Guerlain, A. Debuigne, C. Detrembleur, D. Gigmes, S. Hoeppener, U. S. Schubert, C. A. Fustin and J. F. Gohy, Eur. Polym. J., 2015, 62, 418–425.
27. X. H. He, W. Q. Sun, D. Y. Yan, M. R. Xie and Y. Q. Zhang, J. Polym. Sci., Part A: Polym. Chem., 2008, 46, 4442–4450.
28. Q. L. Li, F. Huo, Y. L. Cui, C. Q. Gao, S. T. Li and W. Q. Zhang, J. Polym. Sci., Part A: Polym. Chem., 2014, 52, 2266–2278.
29. Y. Dai, Y. Li and S. P. Wang, J. Catal., 2015, 329, 425–430.
30. Y. Wang and E. J. Goethals, Macromolecules, 2000, 33, 808–813.
31. A. Movahedi, A. Lundin, N. Kann, M. Nyden and K. Moth-Poulsen, Phys. Chem. Chem. Phys., 2015, 17, 18327–18336.
32. Z. H. Wang and M. W. Urban, Polym. Chem., 2013, 4, 4897–4901.
33. J. Y. Wang, R. H. M. de Kool and A. H. Veders, Langmuir, 2015, 31, 12251–12259.
34. P. Breedveld, D. Pluim, G. Cipriani, F. Dahlhaus, M. A. J. van Eijndhoven, C. J. F. de Wolf, A. Kuil, J. H. Beijnen, G. L. Scheffer, G. Jansen, P. Borst and J. H. M. Schellens, Mol. Pharmacol., 2007, 71, 240–249.
35. J. Nagaj, P. Kolkowska, A. Bykowska, U. K. Komarnicka, A. Kyziol and M. Jezowska-Bojczuk, Med. Chem. Res., 2015, 24, 115–123.
36. D. Sprouse and T. M. Reineke, Biomacromolecules, 2014, 15, 2616–2628.
37. P. S. Donnelly, J. M. Harrowfield, B. W. Skelton and A. H. White, Inorg. Chem., 2001, 40, 5645–5652.
38. E. J. Cha, J. E. Kim and C. H. Ahn, Eur. J. Pharm. Sci., 2009, 38, 341–346.
39. S. Shaligram and A. Campbell, Toxicol. In Vitro, 2013, 27, 844–851.
40. Y. Kapilov-Buchman, E. Lellouche, S. Michaeli and J. P. Lellouche, Bioconjugate Chem., 2015, 26, 880–889.

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Footnote

† Electronic supplementary information (ESI) available: General experimental details. See DOI: 10.1039/c6ra02300b

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