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Facile fabrication of drug-loaded PEGDA microcapsules for drug evaluation using droplet-based microchip
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Xindi Sun1, Qirui Wu1, Wei Li, Xiuqing Gong, Jun-Yi Ge, Jinbo Wu*, Xinghua Gao*
Chinese Chemical Letters | 2022, 33(5) : 2697 - 2700
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Chinese Chemical Letters | 2022, 33(5): 2697-2700
• Communication •
Facile fabrication of drug-loaded PEGDA microcapsules for drug evaluation using droplet-based microchip
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Xindi Sun1, Qirui Wu1, Wei Li, Xiuqing Gong, Jun-Yi Ge, Jinbo Wu*, Xinghua Gao*
Affiliations
  • Materials Genome Institute, Shanghai University, Shanghai 200444, China
Published: 2022-05-15 doi: 10.1016/j.cclet.2021.08.122
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Droplet-based microfluidic technology can be utilized as a microreactor to prepare novel functional monodisperse microcapsules. In this study, a droplet-based microfluidic chip with surface modification, which allowed the one-step preparation of double emulsion microcapsules. An O/W/O double emulsion using polyethylene (glycol) diacrylate (PEGDA) solution as the intermediate water phase was prepared by regulating the hydrophilicity and hydrophobicity of the chip surface, with PEGDA microcapsules prepared using UV polymerization. And then anti-tumor drug paclitaxel and neurotoxin 6-OHDA were encapsulated in microcapsules for drug and toxicology evaluation, respectively. Compared to controls, drug-loaded microcapsules caused a significant increase in the death rate of PC12 cells. This indicates that the obtained drug-loaded microcapsules could be used in drug evaluation and potentially in drug screening and delivery.

PEGDA microcapsule  /  Droplet-based microfluidic  /  PC12 cells  /  Drug evaluation
Xindi Sun, Qirui Wu, Wei Li, Xiuqing Gong, Jun-Yi Ge, Jinbo Wu, Xinghua Gao. Facile fabrication of drug-loaded PEGDA microcapsules for drug evaluation using droplet-based microchip[J]. Chinese Chemical Letters, 2022 , 33 (5) : 2697 -2700 . DOI: 10.1016/j.cclet.2021.08.122
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Microcapsules enclose or adsorb solid, liquid, or gaseous material in a microscopic hermetically-sealed capsule through the use of inert natural or synthetic polymers. With the rapid advance of biotechnology, microcapsule technology has been used widely within biomedicine, with applications including drug delivery, tissue engineering, and biosensor, amongst others [1-4]. However, shortcomings such as inadequate mono-dispersity and function, limited repeatability, and poorly controllable morphology have limited the development and application of microcapsules in biomedical science. Droplet-based microfluidic technology provides precise control of fluid flow, and shows great potential in the controlled generation of monodisperse emulsion droplets [5-6]. Such characteristics, combined with appropriate materials and manufacturing methods, allow us to create functional microcapsules with a variety of complex structures and morphologies; in turn, these enable active substances to be directly encapsulated in microcapsules with well-defined functions [7-9]. Here, droplet-based microfluidic chip allowing the one-step preparation of double emulsion microcapsules was established. An O/W/O double emulsion using PEGDA solution as the intermediate water phase was prepared by regulating the hydrophilicity and hydrophobicity of the chip surface, with PEGDA microcapsules prepared using UV polymerization (Scheme 1). The microchip could be used repeatedly by hydrophilic and hydrophobic modification method. We have also probed the application of drug-loaded PEGDA microcapsules in drug and toxicological evaluation.
In this study, droplet-based microfluidic chip was used to prepare O/W/O double emulsion in one step and then obtain microcapsules. A PDMS microchip was firstly prepared using soft lithography, as shown in Fig. 1A. The microchip mainly comprises two convergence channels, with a height of 120 µm. Channel width on both sides of the first convergence decreases from 150 µm to 85 µm, while the channel width on both sides of the second convergence decreases from 250 µm to 150 µm. This design reduces the channel's cross-sectional area at the point of convergence, speeding flow of liquid in the continuous phase on both sides, thus the continuous phases on both sides exert an enhanced pressure on the dispersed liquid in the middle channel, which stretches and fractures the dispersed phase to form droplets. We used 20% PEGDA as the water phase; this contains the photoinitiator 2-hydroxy-2-methylpropiophenone (2 wt%) and the surfactant Tween 20 (4 wt%). The corn oil and PEGDA/water phases together form O/W droplets at the first convergence and O/W/O droplets at the second convergence. After 405 nm ultraviolet light irradiation, the middle PEGDA/water phase of the O/W/O double emulsion solidified into a shell to form PEGDA microcapsules.
As far as droplet-based microfluidic technology is concerned, O/W droplets are easy to achieve, but O/W/O droplets have certain requirements for the hydrophilicity and hydrophobicity of the microchannel surface. We modified the microchannel surface to generate O/W/O droplets. Modification methods were evaluated, including plasma and chemical coating methods for hydrophilic modifications, and epoxy resin glue for hydrophobic modification. Generally, PDMS is hydrophobic under normal conditions, and the water contact angle is greater than 90° [10]. First, plasma treatment was undertaken for 2 min on the PDMS surface, generating many surface active groups. Analysis using FTIR spectroscopy of the chip surface indicated the presence of hydroxyl and carboxyl functional groups (Fig. 1B) [11], transforming the chip surface from hydrophobic to hydrophilic. The water contact angle simultaneously fell to almost zero. Fig. 1C shows how the contact angle changed after different storage periods. We found that the PDMS surface could return quickly to its original hydrophobicity. This greatly limits the available time of the chip. To improve the effect of hydrophilic modification, we also modified the PDMS surface with chemical coating method: Polyvinyl alcohol (2 wt%) and glycerol (5 wt%) (PVA-GLY). Similarly, to plasma treatment, FTIR spectroscopy analysis indicated the presence of hydrophilic functional groups (Fig. 1B). Fig. 1D indicates that prolonged storage at room temperature maintains the water contact angle of the modified PDMS surface at approximately 24°, and that the PDMS surface was more hydrophilic. In addition, the hydrophilicity of the chip could be maintained for longer compared with plasma treatment. To modulate the relative hydrophobicity of the chip surface, epoxy glue was applied to the required parts of the chip and substrate before they were sealed. The contact angle between the part covered by epoxy resin and water was approximately 111° (Fig. 1E). The pattern covering the epoxy resin on the chip can maintain the hydrophobic properties of the PDMS. Our experimental protocol can control the surface wettability of the PDMS chip, allowing different wettability on different areas of one chip. The microchip could be used repeatedly by hydrophilic and hydrophobic modification method.
After PDMS chip modification, fluorescence characterization of the hydrophilic and hydrophobic areas can be seen in Fig. 2A. We injected a 0.026 mmol/L Rhodamin-123 (RH-123) solution into the microchannel and then remove it. No RH-123 solution remained on the hydrophobic regions (Fig. 2B). However, RH-123 solution did remain on the hydrophilic areas (Fig. 2C), which was indicated by the green fluorescence seen on the inner wall of the hydrophilic areas. After treating the chip surface, the innermost oil phases no longer adhered to the middle channel's inner wall. Figs. 2D and E show droplet formation before and after modifying the chip. No droplets can be produced on an unmodified chip. However, after modification, droplets can be produced smoothly and evenly. On this basis, we tried to generate one-core and two-core microcapsules, as shown in Figs. 2F and G.
In addition, during formation of double emulsion microcapsules, due to the interfacial tension between liquids, careful selection of a suitable outermost oil phase is needed to form a stable double emulsion. We experimented using mineral oil, FC40, and olive oil, finding that only olive oil generates stable double emulsions without adhesion (Fig. 3A). Therefore, we used olive oil as the outermost oil phase. We also experimented with several inner oils and found that the type of inner oil had little effect on the system. Considering the biocompatibility, heat resistance and stability of corn oil, we finally used corn oil as the inner phase oil [12]. An injection pump was used to adjust the flow rate of each phase, allowing us to adjust core and shell size and the number of cores. Results are shown in Figs. 3B and C. For the first channel converges to form core droplets, while keeping the innermost phase flow rate (QI) constant, the core size decreases as the intermediate phase flow rate (QM) increases. For the second channel converges to form a double emulsion, while keeping the flow rate of the innermost phase (QI) and the middle phase (QM) constant, the shell size will increase as the flow rate of the outermost oil phase (QO) decreases. Moreover, an appropriate flow rate can be selected to generate the required core-shell size. By changing the flow rate, multi-core microcapsules can be prepared. If the outermost oil phase flow rate continues to decrease, multiple oil cores will accumulate before shearing, forming microcapsules with two cores (Fig. 3D). PEGDA microcapsules were prepared using the double emulsion template method. Fig. 3E shows dried PEGDA microcapsules observed using a scanning electron microscope. Fig. 3F shows a microcapsule rupture image. Our results indicate we can prepare PEGDA microcapsules with a core-shell structure. We can successfully prepare O/W/O droplets after controllable modification of the chip surface. In addition, to characterize the release of small-molecule drugs in microcapsules, we use coumarin 6, which has a molecular weight close to that of paclitaxel and 6-OHDA, as a dye for the drug to verify drug release. We encapsulated coumarin 6 in microcapsules, and the fluorescence intensity of the released coumarin 6 was measured every day, as shown in Fig. 3G. The results showed that the normalized fluorescence intensity (Ii/Ib) increased gradually with time and reached balance in 7-10 days. In addition, for the stability of microcapsules, it is reported that PEGDA had good biocompatibility and stability [13]. Our experimental results also showed that PEGDA microcapsules could stably release drugs.
To verify the utility of preparing PEGDA microcapsules in drug evaluation and toxicology evaluation, we encapsulated the anticancer compound paclitaxel at a concentration of 16 µmol/L and the neurotoxin 6-OHDA at a concentration of 40 µmol/L in PEGDA microcapsules respectively. According to the size of the microcapsules, we estimated that the drug loadings of paclitaxel and 6-OHDA in the microcapsules were about 0.014 µg/mg and 0.013 µg/mg, respectively. PC12 cells are rat adrenal pheochromocytoma cells. Anti-tumor drugs are known to decrease the viability of PC12 cells. Neurotoxins can also damage PC12 cells [14-15]. As shown in Fig. 4A, PC12 cells were treated with pure PEGDA microcapsules, with approximately 1 × 103 mL−1 PEGDA microcapsules encapsulating paclitaxel, and with 2 × 103 mL−1 PEGDA microcapsules encapsulating paclitaxel. The rate of cell death after adding pure PEGDA microcapsules was below 5%, indicating that pure PEGDA has little effect on cells, and it can be used as a control group for cell experiments. Compared to control group, paclitaxel-loaded microcapsule gave rise to significant PC12 cell death (P < 0.001). The 2 × 103 mL−1 paclitaxel-loaded microcapsule group had higher cell mortality (P < 0.001). In addition, the effect of the drug is also related to time, and the death rate on the third day was significantly higher than that on the first day. The similar results also appeared in the 6-OHDA-loaded microcapsule experiment, the results were shown in Fig. 4B. The cell death rate was proportional to the concentration and time of the neurotoxin 6-OHDA loaded PEGDA microcapsules. This indicates that PEGDA microcapsules can be successfully used as drug carriers.
In summary, a droplet-based microfluidic chip with a modified surface was established, which allowed the one-step preparation of double emulsion microcapsules. We used this approach to prepare O/W/O double emulsions, and then obtained PEGDA microcapsules with a core-shell structure by UV polymerization. Two kinds of drug-loaded microcapsules were prepared for drug and toxicology evaluation respectively, including anti-tumor drug paclitaxel microcapsules and neurotoxin 6-OHDA microcapsules. Compared to controls, drug-loaded microcapsules can cause a significant increase in the death rate of PC12 cells; resulting cell death was proportional to the concentration of the encapsulated drug, and at the same drug concentration, cell death was proportional to time. This indicated that the obtained drug-loaded microcapsules could be used in drug evaluation, and have potential to be used in biomedical related fields such as drug screening and delivery.
Declaration of competing interest
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The authors declared that they have no conflicts of interest to this work.
Acknowledgment
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This work was supported by the National Natural Science Foundation of China (Nos. 31800848 and 21775101).
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Year 2022 volume 33 Issue 5
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Article Info
doi: 10.1016/j.cclet.2021.08.122
  • Receive Date：2021-06-25
  • Online Date：2025-12-19
  • Published：2022-05-15
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History
  • Received：2021-06-25
  • Revised：2021-08-16
  • Accepted：2021-08-29
Affiliations
    Materials Genome Institute, Shanghai University, Shanghai 200444, China
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表12种不同金属材料的力学参数
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Family		 属数
Number of
genus		 种数
Number of
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Percentage of
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鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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