Egg components composited inverse opal particles for synergistic

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Biological and Medical Applications of Materials and Interfaces

Egg components composited inverse opal particles for synergistic drug delivery Yuxiao Liu, Changmin Shao, Feika Bian, Yunru Yu, Huan Wang, and Yuanjin Zhao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b03483 • Publication Date (Web): 27 Apr 2018 Downloaded from http://pubs.acs.org on April 28, 2018

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ACS Applied Materials & Interfaces

Egg components composited inverse opal particles for synergistic drug delivery †

†

Yuxiao Liu, Changmin Shao, Feika Bian, Yunru Yu, Huan Wang, Yuanjin Zhao*

State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China Keywords: microfluidics; particle; inverse opal; egg; drug delivery

Abstract

Microparticles have a demonstrated value in drug delivery systems. The attempts to develop this technology focus on the generation of functional microparticles by using innovative but accessible materials. Here, we present egg components composited microparticles with hybrid inverse opal structure for synergistic drug delivery. The egg component inverse opal particles were achieved by using egg yolk to negatively replicate colloid crystal bead templates. Due to their huge specific surface areas, abundant nanopores and complex nanochannels of the inverse opal structure, the resultant egg yolk particles could load with different kinds of drugs, such as hydrophobic camptothecin (CPT), by simply immersing them into the corresponding drug solutions. Attractively, additional drugs, such as the hydrophilic doxorubicin (DOX), could also be encapsulated into the particles through the secondary filling of the drugs-doped egg white

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hydrogel into the egg yolk inverse opal scaffolds, which realized the synergistic drug delivery for the particles. It was demonstrated that the egg-derived inverse opal particles were with large quantity and lasting releasing for the CPT and DOX co-delivery, and thus could significantly reduce cell viability, and enhance therapeutic efficacy in treating cancer cells. These features of the egg components composited inverse opal microparticles indicated that they were ideal microcarriers for drug delivery.

1. Introduction Microparticle-based drug delivery systems have attracted increasing interests because of their distinctive advantages in practical applications.1-4 Compared with traditional drug delivery forms like systemic, local or oral delivery, which are often difficult to control and have great side effects or large fluctuations of the drug concentration in the blood, the microparticle-based drug delivery systems are with high controllable as they can load sufficient amounts of drugs, prevent the drug failures during the lifetime of release, and monitor the process of the release over the course of days to years.5-7 Thus, the microparticle-based drug delivery systems are effective and stable, which have lower side effects and can greatly reduce the pain of patients.8,9 To generate microparticles of the drug systems, many kinds of biocompatible and biodegradable polymer components, such as collagen, gelatin, sodium alginate, poly(D/L-lactide-co-glycolide), polycaprolactone, and so on, were employed for housing therapeutic agents.10-15 The release of the loaded drugs from the microparticles can be adjusted via choosing different polymers with suitable degradation kinetics, changing the physical characteristics of the particles or controlling the size distribution of the particles.6,16 Although these microparticles have found important values for drugs delivery, their polymer monomers are usually not directly available, which


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require complex chemical synthesis or biological extraction. In addition, during the formation of the microparticles, some of the polymer components need to be dissolved in organic solvents or be polymerized under extreme conditions that might cause a debatable biocompatibility of the materials.17,18 Furthermore, because of the simple structure and content of the microparticles generated from the common single-emulsion templates, synergistic delivery of two or more drugs, in particular the hydrophobic and hydrophilic agents, is relatively difficult.19 Therefore, the generation of functional microparticles with accessible materials and synergistic delivery capabilities is still anticipated for biomedicine. In this paper, we present novel egg components composited inverse opal microparticles for the drug delivery with the desired features. Eggs are a kind of common food of human beings, which are extremely easy to obtain at low cost due to the increasing popularity of factory breeding.20 As the main chemical composition of eggs like proteins, lecithin, total fat and cholesterol, have exhibited significant biocompatibility, eggs have also found wide application in biomedical fields, such as in the preparation of vaccines and in combination with inorganic template.21-23 On the other hand, inverse opals are a kind of spatially ordered porous structure, which can be formed by negatively replicating the structure of colloidal crystal templates.24-28 The homogeneous pore structure endows the inverse opal materials with huge specific surface areas, abundant nanopores and complex nanochannels for loading of different biological actives.29-34 Thus, it is conceivable that the combination of egg components and inverse opal structures would be conducive to the generation of functional microparticles for drug delivery, as indicated in Figure 1. For this purpose, the egg component inverse opal particles were achieved by using egg yolk to negatively replicate colloid crystal beads. Different kinds of drugs, such as hydrophobic camptothecin (CPT), could be loaded into the resultant egg yolk inverse opal


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particles by simply immersing the particles in the corresponding drug solutions. More attractively, additional drugs, such as the hydrophilic doxorubicin (DOX), could also be encapsulated into the particles through the secondary filling of the DOX-doped egg white hydrogel into the egg yolk inverse opal scaffolds, which contributed to the realization of the synergistic drug delivery. It was demonstrated that the egg-derived particles were with large quantity and lasting releasing for the CPT and DOX co-delivery, and thus the particles could significantly reduce cell viability, and enhance therapeutic efficacy in treating cancer cells. These features make the egg components composited inverse opal microparticles ideal for synergistic and sustained drug delivery applications.

Figure 1 Schematic illustration of the fabrication and synergistic drug delivery application of the egg-derived inverse opal miroparticles.

2. Results and Discussion


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In a typical experiment, the egg yolk inverse opal particles were generated by the negatively template replication of silica colloidal crystal beads (SCCBs), which were derived from the selfassembly of silica nanoparticles in microfluidic droplets. During the process of the water evaporation from the droplets, the silica nanoparticles became closely arranged and formed orderly hexagonal packed microstructures. This arranging of the silica nanoparticles also generated interconnected nanopores throughout the SCCBs, which could provide void spaces for the infiltration of the pregel solutions. To generate the egg yolk inverse opal particles, it was crucial to choose the appropriate concentration of the egg yolk solution, since the low concentration of the solutions could not provide enough mechanical strength, while the excessively high concentration would reduce the infiltration capacity of the solutions. Thus, the mechanical strengths and viscosities of egg yolk solutions with different concentrations have been investigated, from which an optimized egg yolk concentration of 95% was chosen for the following experiment, as shown in Figure S1. Besides the egg yolk inverse opal scaffolds, the appropriate concentration of the egg white pregel solution is also needed in order to achieve the secondary filling of hydrogel into the inverse opal particles. Since we require stripping out the egg white filled egg yolk inverse opal microparticles from the egg white gel, the concentration of the egg white pregel solution should be at a relatively lower level, which should also ensure that the egg white is in a solidified condition. Therefore, the solidification states of the egg white under different concentrations were also investigated, as shown in Figure S1. An optimized concentration of the egg white pregel solution at 40% was employed for inverse opal scaffolds filling as it was not only soft enough to peel off the microspheres, but also could form stable hydrogel state in the void space of the inverse opal scaffolds.


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Based on the above investigating results, the desired egg components composited microparticles have been fabricated through the negatively replicating, hydrofluoric acid or sodium hydroxide corrosion, and secondary hydrogel filling in process. The microstructures of the template SCCBs, the inverse opal scaffold and the final resultant hybrid microparticles were characterized through a scanning electron microscope (SEM), as shown in Figure 2 and Figure S2. It could be observed that there were obvious hexagonal alignments on the surface and inside the template SCCBs, as shown in Figure 2b-c. From the image of the egg yolk hydrogel hybrid SCCBs in Figure 2d, it was found that the egg yolk hydrogel had efficiently filled the nanovoids in the template SCCBs. Ascribe to the highly ordered structure of the templates, the egg yolk inverse opal microparticles replicated from the template SCCBs were imparted with the similar ordered and interconnected nanopores, which could be beneficial to the drug encapsulating and delivering (Figure 2e). After filling the egg white hydrogel into the nanopores of the inverse opal particles, most of the interconnected porous structures were filled by the egg white, as shown in Figure 2f.


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Figure 2 The SEM microscopes of different kinds of microparticles: (a) the whole view of the template SCCBs. (b, c) the microstructures of the surface (b) and inside (c) of the template SCCBs. (d) the inside microstructures of the egg yolk hydrogel hybrid SCCBs. (e, f) the inside microstructures of the egg yolk inverse opal microparticles (e) and the egg white hydrogel filled inverse opal microparticles (f). The scale bars are 100µm、1µm、2µm、1µm、1µm、1µm, respectively.

Due to their orderly arranged nanostructure, the SCCB templates and its derived microparticles were all imparted with unique photonic band gaps (PBGs). These PBGs lead light with certain wavelengths or frequencies to be located in and reflected instead of propagating through the materials. As a result, the SCCB templates, the inverse opal particles, together with their derived hybrid microparticles all showed vivid colors and possessed characteristic reflection peaks, as shown in Figure 3 and Figure S3. Under normal incidence, the main reflection peak position λ of these microparticles can be estimated by Bragg’s equation, λ=1.633dnaverage, where d is the center-to-center distance between nearest nanoparticles or nanopores, naverage refers to the average refractive index of the materials. It was obvious that in comparable with the SCCB templates, the structural color of the egg yolk inverse opal particles changed to blue remarkably and the characteristic reflection peak blue shifted simultaneously due to the decrease of the average refractive index and egg yolk hydrogel shrinking during the silica etching (Figure 3c and Figure S3). However, the reflection peak of the egg components composited microparticles had a slight red shift due to the increase of the average refractive index when the egg white hydrogel was secondary filled into the inverse opal scaffolds, as shown


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in Figure 3d and Figure S3. These results also indicated that the orderly arranged nanostructures in each kind of microparticles were well maintained during their fabrications. Moreover, since the size of SCCB templates could be easily adjusted from several to hundreds of microns through changing the flow rates of inner and outer phases during the generating process, the inverse opal microparticles with injectable sizes about several to hundreds of microns could be well obtained, which could meet the requirements of different drug delivery methods and made the microparticles more versatile (Figure S4).

Figure 3 (a-d) The reflection images of the template SCCBs (a), the egg yolk hydrogel hybrid SCCBs (b), the egg yolk inverse opal microparticles (c), and the egg white hydrogel filled inverse opal microparticles (d). The scale bar is 200 µm.


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Since the egg yolk contains oil-soluble substances like cholesterol, lecithin, total fat, etc., it was expected to have the capacity of loading the hydrophobic drugs. In order to verify the hypothesis, the egg yolk solution was employed for encapsulating hydrophobic drugs. The results indicated that the hydrophobic drugs could be loaded in the solid egg yolk, and the drug could maintain activity in the egg yolk for several days (Figure S5). It was worth to mention that the contact angle of the solid 95% egg yolk membrane to bean oil was about 21.29°, which indicated an oleophilic surface feature of the materials, as shown in Figure S6. Thus, the hydrophobic drugs could also be simply loaded in the egg yolk inverse opal scaffolds by physically adsorbing. For the hydrophilic drugs, as the egg white pregel filling solution contained nearly 90% of water, they could be easily dissolved in the solution and loaded into the microparticles during the egg white filling process, as shown in Figure S7. Thus, the microparticles with the egg yolk inverse opal scaffolds and egg white filling were imparted with the ability to load drugs with different solubility, which contributed to the synergistic drug delivery. To implement the concept of synergistic drugs delivery by the egg components composited microparticles, the egg yolk inverse opal scaffolds were immersed in the hydrophobic CPT solution, so as to load the drug. During this process, the CPT powders were firstly dissolved in an organic solvent, which would volatilize later and had no side effects in future applications. The CPT were loaded into the inverse opal particles through the porous adsorption and physically adsorbing by the egg yolk scaffolds. After the process of drug loading, the intrinsic blue fluorescent of CPT could be observed throughout the microparticles via the confocal laser scanning, as shown in Figure 4. Besides the CPT, the hydrophilic DOX was chosen as the codelivery drug. To achieve this, the DOX powders were dissolved in the egg white dilute solution,


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which was then used to immerse the CPT loaded microparticles and fulfill the inverse opal structure of the microparticles. After solidifying the egg white solution and collecting the hybrid microparticles, it was observed that both of the intrinsic fluorescents of CPT and DOX were existed in the delivery system, as shown in Figure 4.

Figure 4 (a-l) The layer by layer confocal laser scanning photographs of DOX (a-d), CPT (e-h), and CPT and DOX (i-l) loaded microparticles. The scale bar is 200 µm.

To investigate the performance of the CPT and DOX co-loaded microparticles as drug delivery systems, the release kinetics of the two-different kind of drugs in the PBS buffer were recorded. The relative drug release amounts were calculated via the fluorescent changes of the microparticles. The intrinsic fluorescent intensity of CPT and DOX in the microparticles reduced gradually (Figure S8), and the release curves recorded in Figure 5. It was obvious that the release of both the two kinds of drugs were with long-term processes, which indicated a


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sustainable release effects. However, as the DOX was encapsulated in the egg white hydrogel and the CPT was simply immersed into the egg yolk scaffolds, the CPT was relatively easy to release from the microparticles. Thus, the relative release percentage of CPT was faster than the relative amount of DOX. It was worth to mention that the total fluorescent intensity of CPT was obviously lower than DOX by using the same concentrations of drugs for the loading. This should be ascribed to the different main loading position of CPT and DOX, and the slight loss of the loaded CPT during the process of secondary hydrogel filling. In addition, the drug-loaded microparticles could remain in a stable morphology during the release process, which obviously ensured the successful sustained drug release. Moreover, since the microparticles with different sizes (several to hundreds of microns) could be generated, they are appropriate for hypodermic or intratumor injection. Besides, when they act as oral delivery microcarriers, the loaded drug molecules could be released from them in gastrointestinal area and cross the gut barrier, while the egg-composited microparticles could be digested and broken down eventually.

Figure 5 (a) the curves of the OD values of DOX and CPT during the release process. (b) the release percentage curves of CPT and DOX from the microparticles.


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To confirm the effects of the synergistic drug delivery system, CPT and DOX co-loaded microparticles were used for oncotherapy. CPT is a cytotoxic quinolone alkaloid that can inhibit the DNA enzyme topoisomerase I, prevent DNA re-ligation, and cause DNA damage, which results in apoptosis;35 while DOX is the inhibitor of DNA enzyme topoisomerase II, which can hinder DNA replication and transcription via embedding in the DNA base fragments, and thus inhibiting the tumor cell growth.36,37 Moreover, since the CPT and DOX act on the different DNA strands and cell cycles, they have obvious synergistic anti-tumor effects. Here, the human liver cancer cell line (HepG2 cells) were treated for 24 h with the unloaded, single DOX loaded, single CPT loaded, and DOX and CPT co-loaded microparticles, respectively. Representative optical and fluorescence microscopy of the HepG2 cell culture results were presented in Figure 6a-d. It was observed that the HepG2 cells grew well in the culture plate with the presence of microparticles unloaded with the drugs. While less than 20% HepG2 cells could survive with the treatment of the CPT and DOX synergistic drugs delivery microparticles after 3 days culture, as confirmed by the cell MTT assay, which is the most common method of quantitative study of cell viability (Figure 6e). Thus, the co-delivery of DOX and CPT in the egg components composited microparticles could significantly reduce cell viability and enhance therapeutic efficacy in treating cancer cells. In addition, during the experiment process, the microparticles could remain a stable condition in the culture media, which was conducive to the successful sustained drug release. Compared with most of double-emulsion drug delivery systems, these egg composited microparticles were much more economical and easier to fabricate due to the broad source of eggs and single-emulsion derived particle templates (the original SCCBs). Since the biocompatibility-debatable elements (e.g. hydrofluoric acid) only existed during the inverse opal


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microparticle formation process, those elements would not affect the applications of the final microparticles in subsequent drug delivery and oncotherapy processes. In addition, novel microfluidic devices could be employed to further simplify the generation process and improve the throughput of particles.38 It is worth to mention that to demonstrate the practical values and real applications of the drug delivery system, such as the immune rejection of egg proteins in specific groups, the in vivo experiments and clinical trial are still anticipated in the future research.


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Figure 6 (a-d) The fluorescent images of the HepG2 cells after 24 hours cultured in the control group (a), treated with DOX loaded microparticles (b), treated with CPT loaded microparticles (c), and treated with DOX and CPT co-loaded microparticles (d), respectively. The scale bar is 50 µm. (e) The results of the MTT assay of the HepG2 cells cultured in the different groups for 3 days.

3. Conclusion In summary, we have developed egg derived hybrid microparticles for synergistic drug delivery. The hybrid microparticles were composed of the egg yolk inverse opal scaffolds and egg white hydrogel filler. Based on the huge specific surface areas, abundant nanopores and complex nanochannels of the inverse opal structure, the egg yolk inverse opal scaffolds could load with different kinds of drug like hydrophobic CPT by simply immersing them into the corresponding drug solutions; while additional hydrophilic drugs (such as DOX) could also be encapsulated into the particles through the secondary filling of the drugs-doped egg white hydrogel into the egg yolk inverse opal scaffolds. Thus, synergistic delivery of two drugs, in particular the hydrophobic and hydrophilic agents, could be realized in the microparticles. It was demonstrated that the egg-derived inverse opal particles with DOX and CPT co-delivery could significantly reduce cell viability and enhance therapeutic efficacy in treating liver cancer cells. In addition, as the co-loaded drugs were with large quantity and lasting releasing, the microparticles were also imparted with a long-term synergistic effect for the tumor treatments. These features indicated that the egg components composited inverse opal microparticles were idea for constructing synergistic drugs delivery systems.


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4. Experimental Section Materials: SiO2 nanoparticles with the size of 255nm were purchased from NanJing DongJian Biological Technology Co., Ltd. The Egg white and Egg yolk pre-gel solutions were prepared in the laboratory. Doxorubicin hydrochloride (DOX) and Camptothecine (CPT) were all purchased from Sigma Aldrich Co. The HepG2 cells were obtained from the Cell Bank of the Chinese Academy of Sciences, Shanghai, China. The Calcein-AM was purchased from Molecular Probes Co. The MTT powders were purchased from J&K Scientific Ltd., Shanghai, and the MTT solution was prepared at a concentration of 5mg/mL in PBS. The dimethyl sulfoxide (DMSO) was purchased from Sigma, USA. The PBS buffer was prepared in the laboratory. Deionized water with a resistivity of 18.2 MΩ·cm-1 was obtained from a Millipore Milli-Q system. All other chemical reagents were of the best grade available and used as received and the deionized water was used in all experiments. Preparation of egg white and egg yolk pre-gel solutions: Chicken eggs were purchased less than 2 days after laying. The eggs were thoroughly washed by the 70% ethyl alcohol, and then they were broken manually and the egg white was separated from egg yolk by egg separator. The egg white was filtered with gauze and the resulting dilute solution was used in the following experiments. Comparison of egg white and egg yolk with different concentrations: Egg white and egg yolk were prepared into solutions at different concentrations respectively. The solutions were heated in 80℃ water bath, and the coagulation conditions of each group were explored after that. In addition, the viscosity of different concentrations of egg yolk were compared via the NDJ-1 rotational viscometer. Generation of template colloidal crystal beads: The silica colloidal crystal beads (SCCBs) were


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generated by the microfluidic droplet template method. The silicon oil and the aqueous suspension were pumped into the single emulsion microfluidic device. The concentration of the used silica nanoparticles was 20% (w/v). The injection speeds of the oil and dispersed phase were 0.4mL/h and 8mL/h, respectively. Due to the fluid shear force, the aqueous suspension was sheared into droplets by the oil phase in the channel. The resultant droplets were collected in a plastic container filled with silicon oil. Then the silica nanoparticles in the droplets selfassembled into ordered lattices during the evaporation process at 75℃ in an oven, and this process should last for a night. Later, the silicon oil should be gently and thoroughly washed out from the silica colloidal crystal beads. Finally, the silica colloidal crystal beads should be calcined at 800℃ for 3 hours so as to improve their mechanical strength. The size distribution of the SCCBs was also characterized in Figure S9. Fabrication of the egg yolk inverse opal microparticles: The SCCBs were immersed in the 95% egg yolk pre-gel solution for 6 hours, so as to the solution thoroughly filled the nanovoids in the SCCBs. Then, the egg yolk pre-gel was thermosetted by the constant temperature water bath at 80℃. Next, the egg yolk gel was exposed under the UV light and immersed in the 75% ethyl alcohol for further disinfection. Later, the egg yolk hydrogel hybrid SCCBs were stripped out from the egg yolk hydrogel in the deionized water. Finally, the egg yolk inverse opal particles were obtained after removing the silica template by immersing in hydrofluoric acid (4%, v/v) for 6 hours and immersed in 10% CaCl2 solution for 24 hours to wipe off the residue of the hydrofluoric acid, or the inverse opal particles could be obtained through immersing the templates in the 5mol/L sodium hydroxide solution for 24 hours to remove the templates. Secondary filling of the egg white into the inverse opal microparticles: The resultant egg yolk inverse opal microparticles were immersed in the 40% egg white pre-gel solution for a night.


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Then, the solution was put in the constant temperature water bath at 80℃ for 3 minutes, so as to solidify the egg white pre-gel. Later, the secondary filled inverse opal microparticles were stripped out from the egg white hydrogel in the deionized water. Finally, the resultant microparticles had been obtained. Characterization: Photographs of the SCCBs, egg yolk hydrogel hybrid SCCBs, egg yolk inverse opal microparticles, and the secondary filled microparticles were taken with a light microscope (OLYMPUS BX51) equipped with a color CCD camera (Media Cybernetics Evolution MP 5.0). Reflection spectra of the particles were recorded by the microscope (OLYMPUS BX51) equipped with a fiber optic spectrometer (Ocean Optics, QE65000).The microstructures of the microparticles were characterized by a scanning electron microscopy (SEM, Hitachi, S-300N). Drug loading: The egg yolk inverse opal microparticles were immersed in the CPT/CH2Cl2 solution to load the first kind of drug, CPT. The DOX powders were dissolved in the egg white solution and loaded into the inverse opal scaffold through the secondary filling process. To verify whether the drugs had been loaded successfully, the fluorescent images were captured through the Laser Scanning Confocal Microscope (FV10i, Olympus, Tokyo, Japan). Drug release in vitro: We studied the release kinetics of the drug-loaded microparticles through the changes of the fluorescent intensity of CPT and DOX in the PBS buffer solution as the release media (pH 7.4). The red (for DOX) and blue (for CPT) fluorescent images of the microparticles were captured via the Nikon inverted microscope (Nikon Eclipse TE200) every hour for 3 weeks and 20 microparticles were captured at least. Then the OD values of DOX and CPT of the microparticles were calculated by the ImageJ via the fluorescent images. Finally, we could obtain the release curves through the changes of the OD values.


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The effects of drug-loaded microparticles on tumor cells: To realize the specific experiments, the HepG2 cells were divided into four groups in the 24-well plate (Thermo, USA), the control group, the group treated with DOX loaded microparticles, the group treated with CPT loaded microparticles, and the group treated with DOX and CPT loaded microparticles, and each group had 12 holes for observation and MTT assays. The 1mg drug-loaded microparticles were added into the holes of the experimental group, and the HepG2 cells were homogeneously dispersed in the culture media with a concentration of 4×105cells/mL in each hole. During the culturing process, the cells were stained by Calcein-AM for observing the cells growth condition and the images were obtained from the confocal laser scanning microscope. The MTT assays were carried out after cells seeding for 0 days, 1 days, 2 days, and 3 days. To carry out the MTT assays, we first sucked out the culture medium and the microparticles, and then added culture medium with 10% MTT solution and incubated at 37 °C for 4 h. After incubation, the medium was removed and 400 µL of dimethyl sulfoxide was added to dissolve the formazan crystals in the cells. Then the OD values were detected by a microplate reader (SYNERGY|HTX). After the OD values were obtained, we set the value of the control group at the first day as the standard, and marked it as 100%, and then calculated the percentage of other groups based on it, and finally the statistical histogram for MTT assay was obtained.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI:


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The viscosities and solidification states of egg yolk and egg white; The whole views of the inverse opal particle and the final particle under SEM; The reflection peaks; SCCBs with different sizes; The fluorescent images of drug loaded microparticles; The contact angles of egg yolk membranes; Size distribution image. (PDF)

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]. Author Contributions † Y.X. L. and C.M.S. contributed equally to this work. Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT This work was supported by the National Science Foundation of China (Grant Nos. 21473029 and 51522302), the NSAF Foundation of China (Grant No.U1530260), the National Science Foundation of Jiangsu (Grant No.BK20140028), the Program for New Century Excellent Talents in University, and the Scientific Research Foundation of Southeast University.

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Egg components composited inverse opal particles for synergistic

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