Emulsion click microspheres: morphology/shape control by surface cross-linking and a porogen

Abstract

Click chemistry plays a dual role in emulsion microsphere (MS) preparation as both an in situ cross-linking method and a bioconjugation route. The morphology/shape of the MSs can be adjusted by applying surface click cross-linking and a porogen simultaneously. The emulsion click cross-linked MSs have potential application in drug delivery, and the preserved azide groups could be used for further surface conjugation with bioactive molecules.

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Biodegradable polymer microspheres have received a lot of attention since their wide application in drug delivery, biomolecule adsorption/trapping/purification, and endotoxin removal.^1–3 However, in the drug delivery area, the initial burst release and the short release duration of the loaded drugs from the polymer microspheres have always been the two major problems.⁴ To address these problems, cross-linked biodegradable polymer microspheres have been developed.^5,6 In addition, targeting unit or fluorescent probe conjugation onto microspheres has always been needed to realize targeted therapy or bioimaging.^7,8 As one of the most effective surface/interface reactions that are tolerant to water and oxygen, click chemistry (azide–alkyne cycloaddition, AAC), including copper-catalyst azide–alkyne cycloaddition (CuAAC) and copper-free click chemistry (such as strain-promoted azide–alkyne cycloaddition, SPAAC), has been widely used in surface/bulk cross-linking of polymeric elastomers/micells/nanogels/miniemulsions,^9–16 and also surface bioconjugation.^7,17–21 Considering that click chemistry can play versatile roles in designing biomaterials, click chemistry was used as an in situ emulsion surface cross-linking route as well as a surface bioconjugation method to prepare emulsion microspheres in this paper.

First, alkyne and azide functionalized poly(γ-benzyl-L-glutamate) (PBLG), abbreviated as PBALG and PBN₃LG, respectively, were synthesized by ester exchange reactions between PBLG and propargyl alcohol (for PBALG) or 3-azidopropanol (for PBN₃LG) (Scheme 1), according to our previous work.²²

Scheme 1 Synthesis of alkyne and azide functionalized poly(γ-benzyl-L-glutamate): PBALG and PBN₃LG.

Equal weights of PBALG and PBN₃LG were mixed (the molar content of azide was higher than that of alkyne) and dissolved in an organic solvent (mixture of 1,2-dichloroethane and dimethylacetamide (DMAc)) to form an oil phase. An O/W emulsion was prepared under shear force using the oil phase and a polyvinyl alcohol (PVA) solution. After adding a copper catalyst (CuSO₄-L-ascorbic acid sodium salt (NaLAc)) to the water phase, in situ emulsion surface CuAAC was promoted to form in situ emulsion click cross-linked and azide-functionalized poly(L-glutamate) microspheres (iECC-PLG-N₃ MSs) (Scheme 2). The remaining azide groups were used for further surface conjugation of targeting units or fluorescent probes. A porogen was also used, and the co-effect of the porogen and emulsion surface click cross-linking on the morphology/shape of the microspheres was investigated by adjusting the copper catalyst concentration in the water phase and the polymer and porogen concentration in the oil phase (Table 1). The results are shown in Fig. 1.

Scheme 2 Preparation of azide-functionalized poly(L-glutamate) microspheres (iECC-PLG-N₃ MSs) by in situ emulsion click cross-linking of an equal-weight mixture of PBALG and PBN₃LG using CuAAC.

Table 1 Emulsion click microsphere samples

Samples	Polymer in oil phase content %(w/v)	Porogen to polymer ratio (mL g^−1)	Catalyst to alkyne group ratio
1	2	0	0
2	2	1	0
3	5	0	CuSO4 (1.0 eq.), NaLAc (1.0 eq.)
4	2	1	CuSO4 (1.0 eq.), NaLAc (1.0 eq.)
5	5	1	CuSO4 (1.0 eq.), NaLAc (1.0 eq.)
6	2	1	CuSO4 (0.1 eq.), NaLAc (1.0 eq.)

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Fig. 1 Morphology control. Non cross-linked (A and B) and in situ emulsion click cross-linked azide-functionalized poly(L-glutamate) (iECC-PLG-N₃) (C, D, E, and F) microspheres. (A) 2% (w/v) of polymer in the oil phase, no porogen, no catalyst (sample 1); (B) 2% (w/v) of polymer in the oil phase, with a porogen content of 1 mL g⁻¹ polymer, no catalyst (sample 2); (C) polymer: 5% (w/v), no porogen, catalyst: 1.0 eq. of CuSO₄ to alkyne group, 1.0 eq. of NaLAc (sample 3); (D) polymer: 2% (w/v), porogen: 1 mL g⁻¹, catalyst: 1.0 eq. of CuSO₄ to alkyne group, 1.0 eq. of NaLAc (sample 4); (E) polymer: 5% (w/v), porogen: 1 mL g⁻¹, CuSO₄ (1.0 eq.), NaLAc (1.0 eq.) (sample 5); (F) polymer: 2% (w/v), porogen: 1 mL g⁻¹, CuSO₄ (0.1 eq.), NaLAc (1.0 eq.) (sample 6).

It can be seen that without a porogen and surface cross-linking, the obtained microspheres had an ordinary round shape with an average diameter around 10 μm (sample 1, Fig. 1A). After applying the porogen (decahydronaphthalene), but no copper catalyst, the obtained microspheres were also round shaped, but with a bigger diameter (around 20 μm) and possessed a lot of pores (sample 2, Fig. 1B). When no porogen but a copper catalyst was used, the obtained MSs were as nonporous as sample 1 (sample 3, Fig. 1C). Since the solvent was homogeneously dispersed in all microspheres, even after surface cross-linking, it was evaporated easily. When the porogen and the copper catalyst were applied simultaneously, the pores formed by the porogen decreased, some even disappeared. Changing the concentrations of the copper catalyst in the water phase and the polymer in the oil phase had a great effect on the morphology/shape of the obtained microspheres. When the polymer concentration in the oil phase was 2% (w/v), the porogen was 1 mL g⁻¹ polymer, and the copper catalyst to alkyne group ratio was 1/1, the pores were almost sealed by surface click cross-linking. The obtained microspheres were non-porous spheres with a diameter typically of 5–10 μm (sample 4, Fig. 1D). When the polymer concentration in the oil phase was increased from 2% (w/v) to 5% (w/v), the shape of the obtained microspheres changed into a tetrahedron with all surfaces collapsed into the center, and the size of the microparticles also increased to around 15 μm (sample 5, Fig. 1E). It was found that, along with the increase of the polymer content in the oil phase, the size of the microspheres increased accordingly, which resulted in an overall increase of solvent and porogen encapsulated into each microparticle, because the ratio between the porogen and the polymer was kept at 1 mL g⁻¹. Along with the fast evaporation of the organic solvent, followed by the slower evaporation of the gathered big porogen liquid drops, the surfaces of the microparticles collapsed resulting in the tetrahedral shape. Since the surface cross-linking speed was fast enough, nearly all pores were sealed (Fig. 1D). When the polymer concentration in the oil phase was kept at 2% (w/v), but the copper catalyst to alkyne group ratio decreased from 1/1 to 1/10, the obtained microparticles changed into wrinkled spheres with some small holes (sample 6, Fig. 1F). This walnut kernel-like morphology may be caused by the abatement of the surface cross-linking. The slow surface cross-linking could not seal the holes left by the solvent and porogen evaporation, and the thinner cross-linked shells collapsed and shrunk after solvent and porogen evaporation.

Emulsion clicked microspheres can be used as cross-linked drug carriers, which may improve drug loading efficiency and reduce the initial burst release, as reported before.^5,6 The emulsion click method can adjust the morphology/shape²³ of the microspheres or microparticles (Scheme 3A), which has been reported to be an important design parameter in drug delivery.^24,25

Scheme 3 Potential applications of emulsion clicked microspheres: (A) in drug delivery systems to improve drug loading, and to control the MS morphology and drug release; (B) surface conjugation of MSs through click reaction with fluorescent probes or other bioactive molecules.

Since the azide group content is higher than that of the alkyne group in the mixed polymer, some azide groups were preserved after surface cross-linking, which is shown in the FTIR spectrum of the iECC-PLG-N₃ MSs (around 2100 cm⁻¹ in Fig. 2). The residual azide groups could be used for further conjugation (Scheme 3B). As an example, a red fluorescent probe, alkyne-functionalized Rhodamine B,¹⁷ was conjugated onto the surface of the iECC-PLG-N₃ MSs. The fluorescence microscopy image (Fig. 3A, from sample 4) clearly shows the red fluorescence covering the MS surface, which also proved the reactivity of the azide groups on the MS surface, implying the practicability of the surface modification on the iECC-PLG-N₃ MSs by the click reaction. To further prove the pore interconnectivity of the porous MSs (sample 3) and detect the difference between porous MSs and nonporous MSs, fluorescence images of the cross-sections of Rhodamine B labeled porous and nonporous MSs were taken by confocal laser scanning microscopy (CLSM) using Z-section and measurement. The CLSM images show that most of the cross-section of the Rhodamine B labeled porous MS exhibits red fluorescence (Fig. 3B). As for the nonporous MS, only a red ring is observed (Fig. 3C, sample 4). These results further confirmed the interconnectivity of the formed pores, which enabled the permeation of the alkyne functional fluorescent dye, to conduct click reaction with the azide groups on the surface of the MSs in the process of surface conjugation.

Fig. 2 FTIR spectra of (A) non cross-linked microspheres made from an equal-weight mixture of PBALG and PBN₃LG (sample 1), and (B) in situ emulsion click cross-linked azide-functionalized poly(L-glutamate) microspheres (iECC-PLG-N₃ MSs, sample 4) made from an equal-weight mixture of PBALG and PBN₃LG.

Fig. 3 (A) Fluorescence microscope image (left) and white light control image (right) of Rhodamine B labeled iECC-PLG-N₃ MSs (sample 3) (scale bar = 10 μm). (B and C) The fluorescence images of the cross-sections of the porous (B, sample 2) and nonporous (C, sample 4) microspheres were taken by confocal laser scanning microscopy (CLSM) using Z-section and measurement.

To investigate if emulsion surface click cross-linking can improve the drug loading efficiency and reduce the initial burst release, BSA was chosen as a model for the drug loading and release research. The click cross-linked MSs possess a higher drug loading efficiency. The BSA encapsulation percentage was determined to be about 64.5% with a drug loading capacity around 19.6%. BSA loaded non cross-linked MSs (using no copper catalyst) were also prepared as a control, the BSA encapsulation percentage was around 42.4%, and the drug loading capacity was about 13.8%. From Fig. 4, it can be seen that after applying surface click cross-linking, the BSA release speed decreased, and the total release amount of BSA in 7 days was also much lower than that of BSA released from non cross-linked MSs, which agrees with the previous report.⁵

Fig. 4 BSA standard curve (A) and the accumulated release of BSA from non cross-linked and click cross-linked MSs.

Conclusions

In conclusion, copper-catalyzed azide–alkyne cycloaddition (CuAAC, click chemistry) was applied to prepare microspheres using an O/W emulsion method as both surface cross-linking route and surface conjugation method. The co-effect of surface click cross-linking and the porogen on the morphology/shape of the obtained microspheres/microparticles was investigated. By applying surface click cross-linking and the porogen simultaneously, the pores formed by the porogen were sealed to some extent. Microspheres/microparticles with a different morphology/shape were obtained by adjusting the polymer concentration and the copper catalyst ratios. The emulsion surface click cross-linked microspheres can be used as cross-linked drug carriers to improve drug loading efficiency and reduce the initial burst release. Some azide groups were preserved on the surface of the obtained microspheres, which could be further used for further conjugation with a fluorescent probe or targeting unit for applications of bioimaging or targeting. Click chemistry played a dual role as an emulsion surface cross-linking route and as a surface conjugation method, which not only expanded the application areas of click chemistry in the design of biomaterials, but also provide an interesting way to surface cross-link and to adjust the morphology/shape of microspheres made by an emulsion method.

Experimental section

Materials

Alkyne and azide functionalized poly(γ-benzyl-L-glutamate), abbreviated as PBALG and PBN₃LG, respectively, were synthesized by ester exchange reactions between poly(γ-benzyl-L-glutamate) (PBLG) and propargyl alcohol (for PBALG) or 3-azidopropanol (for PBN₃LG) (Scheme 1), according to our previous work.²² The average molecular weights of PBALG and PBN₃LG are 13.3 kDa and 14.3 kDa (by ¹H-NMR), respectively, and the degrees of functional group substitution of PBALG and PBN₃LG used are 19.8% and 37.5%, respectively. L-Ascorbic acid sodium salt (NaLAc) was obtained from Acros Organics. Rhodamine B was obtained from Sigma. Bovine serum albumin fraction V (BSA, 99%) was from Sigma-Aldrich. All other reagents were commercially available and used without further purification.

Instrumentation

Fourier transform infrared spectroscopy (FTIR) spectra were measured with a Bruker Vertex 70 spectrometer using KBr pellets. The morphology of the microspheres was observed by a field emission scanning electron microscope (SEM) (Model XL 30 ESEM FEG from Micro FEI Philips). Fluorescence microscope pictures were taken at an excitation wavelength of 555 nm (for Rhodamine B), and a white light graph was taken for comparison. The fluorescence images of the cross-sections of the Rhodamine B labeled porous and nonporous microspheres were taken by confocal laser scanning microscopy (CLSM, TCS Sp2, Germany) using Z-section and measurement. The BSA concentration was determined by UV absorbance at 280 nm using a UV-2450 spectrometer (Shimadzu, Japan) with a minimum wavelength resolution of 0.2 nm.

Preparation of the microspheres

In situ emulsion click cross-linked azide-functional poly(L-glutamate) microspheres (iECC-PLG-N₃ MSs) were prepared by in situ surface cross-linking the O/W emulsion of an equal-weight mixture of PBALG and PBN₃LG using copper(I)-catalyzed 1,3-dipolar azide–alkyne cycloaddition (CuAAC) (Scheme 2). An equal-weight mixture (0.1 g) of PBALG (0.05 g) and PBN₃LG (0.05 g) was dissolved in 1,2-dichloroethane–DMAc (5 mL, v/v = 4 : 1, the polymer concentration in the oil phase is 2% (w/v)), and 0.1 mL decahydronaphthalene (as porogen) was then added to the solution. The solution was dispersed in 1.0 wt% of a poly(vinyl alcohol) (PVA) solution (100 mL) by a high-shear dispersing emulsifier at a speed of 2000 rpm for 2–3 min. Then the O/W emulsion was poured into another 400 mL of PVA solution (1.0 wt%), which contained CuSO₄ (1.0 eq. to alkyne group). After stirring for 1–2 min, NaLAc (1.0 eq. to alkyne group) was added to the system, and the mixture was stirred at 40 °C for 12–24 h to evaporate the solvent. After that, the microspheres were collected by centrifugation and washed several times with water and hot water, and then freeze-dried. iECC-PLG-N₃ MSs with different polymer concentrations in the oil phase, porogen contents, as well as CuAAC catalyst system (CuSO₄–NaLAc) contents (shown in Fig. 1C–E) were prepared. Non cross-linked microspheres made from an equal-weight mixture of PBALG and PBN₃LG with or without the porogen (A and B in Fig. 1) were also prepared with the same method but without adding the CuAAC catalyst. Cross-linked microspheres without the porogen were also prepared as a control. The microspheres were characterized using a SEM and FTIR.

Rhodamine B labeled iECC-PLG-N₃ microspheres

Rhodamine B was first functionalized with alkyne groups to give alkynyl-Rhodamine B, using the method described before.²² Then, alkynyl-Rhodamine B reacted with iECC-PLG-N₃ MSs through CuAAC (Scheme 3) to get Rhodamine B labeled iECC-PLG-N₃ MSs. The details are described in ref. 22 and the MSs were characterized by fluorescence microscopy with the result shown in Fig. 3A. Alkynyl-Rhodamine B labeled porous microspheres (MSs) and nonporous MSs were also observed by confocal laser scanning microscopy (CLSM) using Z-section and measurement, the images are shown in Fig. 3B and C.

Drug loading and release of iECC-PLG-N₃ microspheres

BSA was chosen as the drug model for the drug loading and release research. Briefly, 1 mL BSA (0.1 g) solution in DI water was dispersed in 5 mL polymer solution (containing 0.125 g PBALG, 0.125 g PBN₃LG, and 0.25 mL decahydronaphthalene) and 1,2-dichloroethane–DMAc (v/v = 4 : 1) to form the W/O emulsion. The W/O emulsion was then dispersed in 100 mL 1.0% PVA solution by a high-shear dispersing emulsifier at a speed of 2000 rpm for 2–3 min to form a W/O/W emulsion, and 400 mL of PVA solution (1.0 wt%) was added under stirring. Then, a calculated amount of CuSO₄ (1.0 eq. to alkyne group) and NaLAc (1.0 eq. to alkyne group) was added. The MS purification process was the same as that of the drug-free MSs. The supernatant was collected and freeze-dried before re-dissolving it in DI water to measure the free BSA amount.

The BSA release study was conducted by putting 100 mg of BSA loaded MSs in a dialysis tube with a molecular weight cut-off of 20 kDa. The dialysis tube was immersed in 20 mL of PBS (pH 7.4) at 37 °C with shaking. At pre-set time points, 1 mL of PBS solution was taken out, and 1 mL of fresh PBS solution was added. The concentration of BSA in the PBS solution was determined by UV-vis, according to the standard curve obtained at the same time (Fig. 4A).

Acknowledgements

The authors would like to thank the financial support from the Ministry of Science and Technology of China (863 Project, no. SS2012AA020603), the National Natural Science Foundation of China (no. 51321062 and 21174143), the “100 Talents Program” of the Chinese Academy of Sciences (no. KGCX2-YW-802), and the Jilin Provincial Science and Technology Department (no. 20100588).

Notes and references

1. S. E. Bae, J. S. Son, K. Park and D. K. Han, J. Controlled Release, 2009, 133, 37.
2. Q. Qian, X. Huang, X. Zhang, Z. Xie and Y. Wang, Angew. Chem., Int. Ed., 2013, 52, 10625.
3. C. Hirayama and M. Sakata, J. Chromatogr. B, 2002, 781, 419.
4. S. D. Allison, Expert Opin. Drug Delivery, 2008, 5, 615.
5. Y. Yuan, B. M. Chesnutt, G. Utturkar, W. O. Haggard, Y. Yang, J. L. Ong and J. D. Bumgardner, Carbohydr. Polym., 2007, 68, 561.
6. Y.-F. Liu, K.-L. Huang, D.-M. Peng, P. Ding and G.-Y. Li, Int. J. Biol. Macromol., 2007, 41, 87.
7. G.-F. Luo, W.-H. Chen, Y. Liu, J. Zhang, S.-X. Cheng, R.-X. Zhuo and X.-Z. Zhang, J. Mater. Chem. B, 2013, 1, 5723.
8. O. Grinberg, A. Gedanken, D. Mukhopadhyay and C. R. Patra, Polym. Adv. Technol., 2013, 24, 294.
9. K. Liang, G. K. Such, Z. Zhu, H. Lomas and F. Caruso, Adv. Mater., 2011, 23, H273.
10. S. M. Garg, X.-B. Xiong, C. Lu and A. Lavasanifar, Macromolecules, 2011, 44, 2058.
11. H. Wang, L. Tang, C. Tu, Z. Song, Q. Yin, L. Yin, Z. Zhang and J. Cheng, Biomacromolecules, 2013, 14, 3706.
12. J. Guo, Z. Xie, R. T. Tran, D. Xie, X. Bai and J. Yang, Adv. Mater., 2014, 26, 1906.
13. Y. Cheng, C. He, C. Xiao, J. Ding, K. Ren, S. Yu, X. Zhuang and X. Chen, Polym. Chem., 2013, 4, 3851.
14. J. Guo, Y. Wei, D. Zhou, P. Cai, X. Jing, X.-S. Chen and Y. Huang, Biomacromolecules, 2011, 12, 737.
15. D. A. Heller, Y. Levi, J. M. Pelet, J. C. Doloff, J. Wallas, G. W. Pratt, S. Jiang, G. Sahay, A. Schroeder, J. E. Schroeder, Y. Chyan, C. Zurenko, W. Querbes, M. Manzano, D. S. Kohane, R. Langer and D. G. Anderson, Adv. Mater., 2013, 25, 1449.
16. J. Zou, Y. Yu, L. Yu, Y. Li, C.-K. Chen and C. Cheng, J. Polym. Sci., Part A: Polym. Chem., 2012, 50, 142.
17. J. Guo, F. Meng, X. Jing and Y. Huang, Adv. Healthcare Mater., 2013, 2, 784.
18. B. Malvi, B. R. Sarkar, D. Pati, R. Mathew, T. G. Ajithkumar and S. S. Gupta, J. Mater. Chem., 2009, 19, 1409.
19. R. Manova, T. A. van Beek and H. Zuilhof, Angew. Chem., Int. Ed., 2011, 50, 5428.
20. J. Guo, F. Meng, X. Li, M. Wang, Y. Wu, X. Jing and Y. Huang, Macromol. Biosci., 2012, 12, 533.
21. X.-Y. Li, T.-H. Li, J.-S. Guo, Y. Wei, X.-B. Jing, X.-S. Chen and Y.-B. Huang, Biotechnol. Prog., 2012, 28, 856.
22. J. Guo, Y. Huang, X. Jing and X. Chen, Polymer, 2009, 50, 2847.
23. M. C. M. van Oers, F. P. J. T. Rutjes and J. C. M. van Hest, J. Am. Chem. Soc., 2013, 135, 16308.
24. J. A. Champion, Y. K. Katare and S. Mitragotri, J. Controlled Release, 2007, 121, 3.
25. J. P. Best, Y. Yan and F. Caruso, Adv. Healthcare Mater., 2012, 1, 35.

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