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Responsive drug-delivery microcarriers based on the silk fibroin inverse opal scaffolds for controllable drug release
Applied Materials Today 19 (2020) 100540
Contents lists available at ScienceDirect
Applied Materials Today journal homepage: www.elsevier.com/locate/apmt
Responsive drug-delivery microcarriers based on the silk fibroin inverse opal scaffolds for controllable drug release Hui Zhang a,b,c , Yuxiao Liu a , Canwen Chen c,e , Wenguo Cui a , Chunwu Zhang a,∗ , Fangfu Ye b,d,∗∗ , Yuanjin Zhao a,b,c,∗ a
Department of Orthopaedic Traumatology, The First Affiliated Hospital of Wen Zhou Medical University, Wenzhou, 325035, China Wenzhou Institute, University of Chinese Academy of Sciences, Wenzhou, Zhejiang 325001, China c State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing, 210096, China d Beijing National Laboratory for Condensed Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing, 100190, China e Department of General Surgery, Jinling Hospital, Medical School of Nanjing University, Nanjing, 210002, China b
a r t i c l e
i n f o
Article history: Received 27 October 2019 Received in revised form 11 December 2019 Accepted 17 December 2019 Keywords: Drug delivery Silk fibroin Inverse opal Hydrogel Biomaterial Responsive
a b s t r a c t Tumor has always been a major threat to human health due to its high morbidity and mortality. Among many cancer treatments, drug delivery microcarriers as a newly emerging method has attracted much attention. Here, we present a novel kind of multifunctional drug-delivery microcarriers formed from silk fibroin inverse opal scaffold and temperature-responsive poly (N-isopropylacrylamide) (PNIPAM) hydrogel for controllable and sustained drug release. Because of their uniform porous microstructure and interconnected nanopores, the PNIPAM hydrogel loaded with therapeutic drug can fill into the inverse opal scaffolds. It was demonstrated that these microcarriers had the ability to release drugs controllably and sustainably with temperature changes, which could not only reduce the drug waste but improve the effect of oncotherapy as well. In addition, due to the excellent biocompatibility of the silk fibroin, the inverse opal scaffolds could also support the adhesion and growth of normal cells, which was contributed to the tissue regeneration in the lesions. These features indicate that the designed drug-delivery microcarriers are ideal for oncotherapy and tissue engineering. © 2019 Elsevier Ltd. All rights reserved.
1. Introduction Tumors seriously threaten human life and health due to their high morbidity and mortality [1–3]. With the continuous expansion and aging of the world population, the global burden of tumors is also increasing. Therefore, it is particularly urgent to find better treatments for oncotherapy. In recent years, compared with traditional treatments which are often difficult to maintain a certain drug concentration and have great side effects due to the potential overdose of drugs, microcarriers have shown their great potential as drug carriers since their advantages of non-toxicity, excellent biocompatibility, good targeting and high drug loading efficiency [4–16]. Thus, the methods of tumor therapy based on microcarriers have attracted increasing attentions. Various materials have
∗ Corresponding authors at: Department of Orthopaedic Traumatology, The First Affiliated Hospital of Wen Zhou Medical University, Wenzhou, 325035, China. ∗∗ Corresponding author at: Beijing National Laboratory for Condensed Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing, 100190, China. E-mail addresses: [email protected] (C. Zhang), [email protected] (F. Ye), [email protected] (Y. Zhao). https://doi.org/10.1016/j.apmt.2019.100540 2352-9407/© 2019 Elsevier Ltd. All rights reserved.
been used for developing functional microcarriers, such as chitosan, cellulose, calcium phosphate, etc [17–20]. These microcarriers have improved the drug administration and therapeutic effects of cancer treatment, which significantly contributed to the field of oncotherapy. However, most of them worked solely, each kind of microcarriers could only achieve a single function, and their static way of drug release would lead to drug waste, which greatly limited their therapeutic effects. Hence, the multi-functional microcarriers with controllable release are still anticipated for better oncotherapy. In this paper, we presented a controllable multifunctional drug-delivery microcarrier combined with silk fibroin inverse opal scaffold and the temperature-responsive PNIPAM hydrogel for tumor treatment, as shown in Fig. 1. Inverse opal particles with ordered three-dimensional porous microstructures possess the huge specific surface areas and abundant interconnected nanopores, making them ideal scaffolds in the fields of drug delivery and tissue engineering [21–25]. In addition, silk fibroin as a natural protein extracted from silkworm cocoons has attracted extensive attention of researchers because of its low cost, abundant resources and remarkable biomedical value, including excellent
2
H. Zhang, Y. Liu, C. Chen et al. / Applied Materials Today 19 (2020) 100540
Fig. 1. Schematic illustration of the fabrication process and controllable drug release application of the inverse opal particles combined with responsive hydrogel.
biocompatibility and biodegradability [26–32]. Thus, there is reason to believe that the combination of inverse opal scaffold and silk fibroin can construct a novel drug-delivery microcarrier with excellent biological properties. Moreover, the temperature-responsive PNIPAM hydrogel as the second pre-gel could be mixed with the doxorubicin (DOX) and fill into the pores of inverse opal scaffold to achieve drug loading. Due to the temperature responsiveness of PNIPAM, the process of drug release from the microcarriers could be triggered by giving external temperature stimulation, which not only reduces the waste of drug and raises its utilization, but also enhances the safety of tumor therapy [33–37]. These features indicate that the silk fibroin inverse opal microcarriers with temperature-responsive PNIPAM hydrogel are ideal drug-delivery carriers and present a huge potential in the application of biomedical field.
2. Results and discussion In a typical experiment, the silk fibroin pre-gel solutions were filled into the silica colloid crystal bead (SCCB) templates for fabricating the silk fibroin inverse opal scaffolds, as indicated in Fig. 1a-c. The SCCBs with good monodispersity were prepared by the selfassembly of silica nanoparticles in the microfluidic droplets and the size distribution of SCCBs was shown in Fig. S1. The sizes of particles were narrowly distributed in the range of 255 m to 295 m, whose average diameter and standard deviation were calculated to be about 275.51 and 8.28, and the coefficient of variation (CV), which equal to the ratio of the standard deviation to the mean, was calculated to be 2.99%. This result confirmed that the prepared particles were highly monodisperse. To generate the silk fibroin scaffolds, the proper concentrations of the pregel solution should be explored first, because an excessively high concentration of silk fibroin solutions would be difficult to dissolve completely, whereas a low concentration could not supply adequate mechanical strength. It was demonstrated that four optimized groups of silk fibroin with concentrations at 5%, 10%, 20% and 30% were selected for the next experiment, as indicated
in Fig. S2a. In addition, the appropriate concentrations of the PNIPAM solutions were also needed so that this second pregel could successfully infiltrate into the voids of the inverse opal scaffolds. Therefore, the solidification degree of PNIPAM with different concentrations were also researched (Fig. S2b), and four suitable sets of PNIPAM hydrogel concentrations were determined. Due to the ordered arrangement of the spherical silica nanoparticles, the SCCBs were endowed with brilliant structural color and characteristic reflection peaks, as demonstrated in Fig. 2a and Fig. S3. The silica nanoparticles of the SCCBs formed interconnected nanopores that provided spaces for the first pregel solution. To implement this, the prepared SCCBs were first immersed in silk fibroin pregel solution allowing the solution to sufficiently fill into the nanovoids of SCCBs. Through adding the ethanol into the mixture solution and standing overnight, the silk fibroin around and inside the SCCBs was gradually solidified. Then the redundant silk fibroin hydrogel on the surface of template SCCBs was stripped out so that the hybrid SCCBs could be separated from the hydrogel bulk, as shown in Fig. 2b. Finally, the silk fibroin inverse opal scaffolds were fabricated by using hydrofluoric acid to corrode the template SCCBs, while the using hydrofluoric acid had little effect on the silk fibroin scaffold (Fig. 2c). To achieve the controllable release of drug, the resultant silk fibroin scaffolds were soaked in the mixed solution of doxorubicin (DOX) and NIPAM solution. After the voids of the silk fibroin inverse opal scaffold were filled with the prepared mixture solution, the pregel solution was polymerized by exposing to the ultraviolet irradiation. Then, the second pregel solidified rapidly and the anti-cancer drugs were successfully loaded into the scaffolds, as shown in Fig. 2d. Besides, the main peak position could be estimated according to the Bragg’s equation: = 1.633dnaverage where d is the center-to-center distance between two adjacent silica nanoparticles or nanopores and naverage is the average refractive index of the system. Because of the difference in the refractive indexes of SCCBs and hydrogels, the peaks value of the different kinds of particles would shift correspondingly (Fig. S3).
H. Zhang, Y. Liu, C. Chen et al. / Applied Materials Today 19 (2020) 100540
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Fig. 2. (a–d) The characterizations of the template SCCBs (a), silk fibroin hybrid SCCBs (b), silk fibroin inverse opal particles (c), and PNIPAM-filled inverse opal particles (d). The figures showed the reflective images of blue, green, red SCCBs which were generated from the silica nanoparticles with diameters of 215, 260 and 305 nm, respectively, and their microstructures under the SEM, from top to bottom. The scale bars are 300 m in reflective images, and 500 nm in SEM images.
To demonstrate the microstructures of the particles mentioned above, the surface and interior structures of these particles were characterized through using a scanning electron microscopy (SEM), as shown in Fig. 2 and Fig. S4. It could be found that both the silica nanoparticles on the surface and inside of the template SCCBs formed a hexagonal close alignment. In addition, it was obvious that the voids of the SCCBs had been almost filled with the silk fibroin hydrogel, and the silk fibroin inverse opal particles derived from the template SCCBs showed a similarly ordered and porous nanostructure with interconnected nanopores that offered efficient space for drug loading. As expected, it was demonstrated that the nanopores of the drug loaded particles were full of the second pregel (NIPAM solution) both on the surface and interior. PNIPAM hydrogels with volume phase transition temperature (VPTT) about 32 ◦ C could shrink to release the encapsulated drugs when the temperature was above 32 ◦ C. To utilize this feature, the particles and their surrounding environment, the phosphate buffer solution (PBS), were exposed to an environment of 40 ◦ (above VPTT, for 5 min). The performance of the drug-encapsulated microcarriers was evaluated by the fluorescent images and release kinetics, as shown in Fig. 3. From the fluorescent images (Fig. 3a), it was observed that the intrinsic fluorescent intensity of DOX in the particles gradually reduced, indicating a sustained release of drug from the microcarriers. As exhibited in Fig. 3b, it was discovered that about half of the drug in the microcarriers with a low concentration of PNIPAM hydrogel (5%) was released within four temperature cycles, compared to only about 20% drug releasing from the microcarriers with a high concentration of PNIPAM hydrogel (20%). In addition, there was a negative correlation between the release rate of DOX and the concentration of PNIPAM hydrogel at the same temperature cycles. In brief, the drug would release from the microcarriers after giving an external temperature stimulation, while the microcarriers would be locked and the drug-release process would not continue if there is no temperature stimulus. This
Fig. 3. (a) The fluorescent images of DOX after 0, 1, 2, 3, 5 and 7 temperature stimuli cycles. The scale bar is 200 m. (b) Release curves of DOX from the microcarriers under different temperature stimuli cycles. Error bars represent standard deviations.
feature of controllable drug release could not only avoid the waste of drugs but also further promote the safety of clinical treatment.
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Fig. 4. (a–c) The confocal laser scanning images of the 3T3 cells cultured on the microcarriers with silk fibroin concentration of 30% and PNIPAM concentration of 20%. The scale bars are 200 m, 200 m, 300 m.
To explore the biocompatibility of the designed microcarrier, 3T3 cells (fibroblast cell line) were cultured in the media containing the components of the microcarrier, i.e. silk fibroin, PNIPAM and the silk fibroin and PNIPAM mixed materials, respectively. The results of MTT assay, a colorimetric assay for the quantitative investigation of cell viability, were shown in Fig. S5. It was confirmed that each component demonstrated good biocompatibility, which indicates the safety and feasibility of microcarriers for clinical applications to some extent. Attractively, according to the results of the cell culture on the microcarriers for three days obtained by a confocal microscope (Fig. S6 and Fig. 4), it was found that the cells not only grew well around the microcarriers, but also adhered and grew well on the surface of microcarriers. This phenomenon could be attributed to the excellent biocompatibility and the uniform porous microstructures of the silk fibroin inverse opal scaffolds, which provided interconnected nanochannels for cell adhesion and substances exchange. These results indicated that the prepared microcarriers also had a great application potential in the field of tissue engineering in addition to drug loading. As mentioned above, PNIPAM would shrink and extrude the drugs encapsulated in the microcarriers when the temperature exceeded its VPTT at about 32 ◦ C. In order to verify the capacity of the microcarriers for controllable release in cell experiments, an appropriate VPTT for PNIPAM hydrogel between the body temperature at 37 ◦ and hyperthermia temperature at 42 ◦ needs to be regulated, or else the PNIPAM hydrogel will maintain a continuous
release of cancer drug during the cell culture process (the temperature of cell culture environment is 37 ◦ ), resulting in the drug waste and threat to the safety of treatment. Thus, an optimized VPTT of around 39 ◦ was tailored by adding the polyacrylamide (pAAm) into the PNIPAM hydrogel and the concentration ratio of pAAm to PNIPAM was 7.5%–92.5% [38]. Besides, the microcarriers with the concentration of PNIPAM hydrogel at 20 % was selected for cell experiment because of a longer sustained release according to the results of previous investigations. Then, using HepG2 cells as model cells, the cell viability influenced by DOX-loaded microcarriers in different temperature cycles above VPTT (at 40 ◦ for 5 min) and at 37 ◦ (for more than 15 min) were recorded, as shown in Fig. 5. The ordinate of histogram represented the corresponding OD values of the eventual surviving cells. It was found that compared with the control group without any temperature stimulation, most of the HepG2 cells were killed after five temperature cycles due to the anticancer drugs released from the rapid shrinkage of the responsive microcarriers. In addition, the amount of released drug was proportional to the temperature cycles, and consistent with the results obtained via a fluorescence microscopy (Fig. 5ad). These results verified that the microcarriers had reversible and repeatable temperature responsiveness as well as remarkable controllable drug release capacity. On the other hand, to exclude the influence of heating on HepG2 cells, the MTT assay was used for quantitatively analyzing the cell activity influenced by heating. As shown in Fig. 5e, the cell viabilities of the groups in different temperature cycles but no drugs involved were pretty close to that of
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Fig. 5. (a–d) The fluorescent images of the HepG2 cells after controllable release of DOX in different temperature cycles: (a)the control group without temperature stimulation, (b) one cycle, (c) three cycles and (d) five cycles. (e) The results of the MTT assay of the HepG2 cells after the simple heating and controllable release of DOX in different temperature cycles. The scale bar is 50 m.
the control group without any treatment, as also confirmed by the fluorescent images (Fig. S7). 3. Conclusion In summary, we have designed and demonstrated a kind of multifunctional drug-delivery microcarriers based on inverse opal scaffold for controllable drug release. The microcarriers were composed of the silk fibroin inverse opal scaffolds and the temperature-responsive drug-loaded PNIPAM hydrogel. On the one hand, the uniform porous microstructures and interconnected nanopores of the inverse opals could benefit for the infiltration of the second pregel and drug loading. It was confirmed that the excellent biocompatibility and rough surface of the silk fibroin scaffolds contributed to the normal cells adhesion and proliferation, which was advantageous to the tissue regeneration at the tumor site. On the other hand, because of the temperature responsiveness of the PNIPAM hydrogel, the drug release process could be controlled by using different temperature cycles so that not only the drug waste could be reduced but the treatment safety could be improved as well. These features of the multifunctional inverse opal particle-based microcarriers with temperature responsiveness hydrogel make them promising in both drug delivery and tissue engineering. Data availability The raw/processed data required to reproduce these findings cannot be shared at this time as the data also forms part of an ongoing study. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. CRediT authorship contribution statement Hui Zhang: Validation, Formal analysis, Investigation, Data curation, Writing - original draft. Yuxiao Liu: Writing - review & editing. Canwen Chen: Resources, Writing - review & editing. Wenguo Cui: Resources, Writing - review & editing. Chunwu Zhang: Conceptualization, Methodology, Supervision. Fangfu Ye: Conceptualization, Methodology, Supervision. Yuanjin Zhao: Conceptualization, Methodology, Supervision.
Acknowledgements This work was supported by the National Natural Science Foundation of China (grants 61927805 and 51522302), the NSAF Foundation of China (grant U1530260), the Natural Science Foundation of Jiangsu (Grant no. BE2018707), and K.C. Wong Education Foundation.
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Contents lists available at ScienceDirect
Applied Materials Today journal homepage: www.elsevier.com/locate/apmt
Responsive drug-delivery microcarriers based on the silk fibroin inverse opal scaffolds for controllable drug release Hui Zhang a,b,c , Yuxiao Liu a , Canwen Chen c,e , Wenguo Cui a , Chunwu Zhang a,∗ , Fangfu Ye b,d,∗∗ , Yuanjin Zhao a,b,c,∗ a
Department of Orthopaedic Traumatology, The First Affiliated Hospital of Wen Zhou Medical University, Wenzhou, 325035, China Wenzhou Institute, University of Chinese Academy of Sciences, Wenzhou, Zhejiang 325001, China c State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing, 210096, China d Beijing National Laboratory for Condensed Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing, 100190, China e Department of General Surgery, Jinling Hospital, Medical School of Nanjing University, Nanjing, 210002, China b
a r t i c l e
i n f o
Article history: Received 27 October 2019 Received in revised form 11 December 2019 Accepted 17 December 2019 Keywords: Drug delivery Silk fibroin Inverse opal Hydrogel Biomaterial Responsive
a b s t r a c t Tumor has always been a major threat to human health due to its high morbidity and mortality. Among many cancer treatments, drug delivery microcarriers as a newly emerging method has attracted much attention. Here, we present a novel kind of multifunctional drug-delivery microcarriers formed from silk fibroin inverse opal scaffold and temperature-responsive poly (N-isopropylacrylamide) (PNIPAM) hydrogel for controllable and sustained drug release. Because of their uniform porous microstructure and interconnected nanopores, the PNIPAM hydrogel loaded with therapeutic drug can fill into the inverse opal scaffolds. It was demonstrated that these microcarriers had the ability to release drugs controllably and sustainably with temperature changes, which could not only reduce the drug waste but improve the effect of oncotherapy as well. In addition, due to the excellent biocompatibility of the silk fibroin, the inverse opal scaffolds could also support the adhesion and growth of normal cells, which was contributed to the tissue regeneration in the lesions. These features indicate that the designed drug-delivery microcarriers are ideal for oncotherapy and tissue engineering. © 2019 Elsevier Ltd. All rights reserved.
1. Introduction Tumors seriously threaten human life and health due to their high morbidity and mortality [1–3]. With the continuous expansion and aging of the world population, the global burden of tumors is also increasing. Therefore, it is particularly urgent to find better treatments for oncotherapy. In recent years, compared with traditional treatments which are often difficult to maintain a certain drug concentration and have great side effects due to the potential overdose of drugs, microcarriers have shown their great potential as drug carriers since their advantages of non-toxicity, excellent biocompatibility, good targeting and high drug loading efficiency [4–16]. Thus, the methods of tumor therapy based on microcarriers have attracted increasing attentions. Various materials have
∗ Corresponding authors at: Department of Orthopaedic Traumatology, The First Affiliated Hospital of Wen Zhou Medical University, Wenzhou, 325035, China. ∗∗ Corresponding author at: Beijing National Laboratory for Condensed Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing, 100190, China. E-mail addresses: [email protected] (C. Zhang), [email protected] (F. Ye), [email protected] (Y. Zhao). https://doi.org/10.1016/j.apmt.2019.100540 2352-9407/© 2019 Elsevier Ltd. All rights reserved.
been used for developing functional microcarriers, such as chitosan, cellulose, calcium phosphate, etc [17–20]. These microcarriers have improved the drug administration and therapeutic effects of cancer treatment, which significantly contributed to the field of oncotherapy. However, most of them worked solely, each kind of microcarriers could only achieve a single function, and their static way of drug release would lead to drug waste, which greatly limited their therapeutic effects. Hence, the multi-functional microcarriers with controllable release are still anticipated for better oncotherapy. In this paper, we presented a controllable multifunctional drug-delivery microcarrier combined with silk fibroin inverse opal scaffold and the temperature-responsive PNIPAM hydrogel for tumor treatment, as shown in Fig. 1. Inverse opal particles with ordered three-dimensional porous microstructures possess the huge specific surface areas and abundant interconnected nanopores, making them ideal scaffolds in the fields of drug delivery and tissue engineering [21–25]. In addition, silk fibroin as a natural protein extracted from silkworm cocoons has attracted extensive attention of researchers because of its low cost, abundant resources and remarkable biomedical value, including excellent
2
H. Zhang, Y. Liu, C. Chen et al. / Applied Materials Today 19 (2020) 100540
Fig. 1. Schematic illustration of the fabrication process and controllable drug release application of the inverse opal particles combined with responsive hydrogel.
biocompatibility and biodegradability [26–32]. Thus, there is reason to believe that the combination of inverse opal scaffold and silk fibroin can construct a novel drug-delivery microcarrier with excellent biological properties. Moreover, the temperature-responsive PNIPAM hydrogel as the second pre-gel could be mixed with the doxorubicin (DOX) and fill into the pores of inverse opal scaffold to achieve drug loading. Due to the temperature responsiveness of PNIPAM, the process of drug release from the microcarriers could be triggered by giving external temperature stimulation, which not only reduces the waste of drug and raises its utilization, but also enhances the safety of tumor therapy [33–37]. These features indicate that the silk fibroin inverse opal microcarriers with temperature-responsive PNIPAM hydrogel are ideal drug-delivery carriers and present a huge potential in the application of biomedical field.
2. Results and discussion In a typical experiment, the silk fibroin pre-gel solutions were filled into the silica colloid crystal bead (SCCB) templates for fabricating the silk fibroin inverse opal scaffolds, as indicated in Fig. 1a-c. The SCCBs with good monodispersity were prepared by the selfassembly of silica nanoparticles in the microfluidic droplets and the size distribution of SCCBs was shown in Fig. S1. The sizes of particles were narrowly distributed in the range of 255 m to 295 m, whose average diameter and standard deviation were calculated to be about 275.51 and 8.28, and the coefficient of variation (CV), which equal to the ratio of the standard deviation to the mean, was calculated to be 2.99%. This result confirmed that the prepared particles were highly monodisperse. To generate the silk fibroin scaffolds, the proper concentrations of the pregel solution should be explored first, because an excessively high concentration of silk fibroin solutions would be difficult to dissolve completely, whereas a low concentration could not supply adequate mechanical strength. It was demonstrated that four optimized groups of silk fibroin with concentrations at 5%, 10%, 20% and 30% were selected for the next experiment, as indicated
in Fig. S2a. In addition, the appropriate concentrations of the PNIPAM solutions were also needed so that this second pregel could successfully infiltrate into the voids of the inverse opal scaffolds. Therefore, the solidification degree of PNIPAM with different concentrations were also researched (Fig. S2b), and four suitable sets of PNIPAM hydrogel concentrations were determined. Due to the ordered arrangement of the spherical silica nanoparticles, the SCCBs were endowed with brilliant structural color and characteristic reflection peaks, as demonstrated in Fig. 2a and Fig. S3. The silica nanoparticles of the SCCBs formed interconnected nanopores that provided spaces for the first pregel solution. To implement this, the prepared SCCBs were first immersed in silk fibroin pregel solution allowing the solution to sufficiently fill into the nanovoids of SCCBs. Through adding the ethanol into the mixture solution and standing overnight, the silk fibroin around and inside the SCCBs was gradually solidified. Then the redundant silk fibroin hydrogel on the surface of template SCCBs was stripped out so that the hybrid SCCBs could be separated from the hydrogel bulk, as shown in Fig. 2b. Finally, the silk fibroin inverse opal scaffolds were fabricated by using hydrofluoric acid to corrode the template SCCBs, while the using hydrofluoric acid had little effect on the silk fibroin scaffold (Fig. 2c). To achieve the controllable release of drug, the resultant silk fibroin scaffolds were soaked in the mixed solution of doxorubicin (DOX) and NIPAM solution. After the voids of the silk fibroin inverse opal scaffold were filled with the prepared mixture solution, the pregel solution was polymerized by exposing to the ultraviolet irradiation. Then, the second pregel solidified rapidly and the anti-cancer drugs were successfully loaded into the scaffolds, as shown in Fig. 2d. Besides, the main peak position could be estimated according to the Bragg’s equation: = 1.633dnaverage where d is the center-to-center distance between two adjacent silica nanoparticles or nanopores and naverage is the average refractive index of the system. Because of the difference in the refractive indexes of SCCBs and hydrogels, the peaks value of the different kinds of particles would shift correspondingly (Fig. S3).
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Fig. 2. (a–d) The characterizations of the template SCCBs (a), silk fibroin hybrid SCCBs (b), silk fibroin inverse opal particles (c), and PNIPAM-filled inverse opal particles (d). The figures showed the reflective images of blue, green, red SCCBs which were generated from the silica nanoparticles with diameters of 215, 260 and 305 nm, respectively, and their microstructures under the SEM, from top to bottom. The scale bars are 300 m in reflective images, and 500 nm in SEM images.
To demonstrate the microstructures of the particles mentioned above, the surface and interior structures of these particles were characterized through using a scanning electron microscopy (SEM), as shown in Fig. 2 and Fig. S4. It could be found that both the silica nanoparticles on the surface and inside of the template SCCBs formed a hexagonal close alignment. In addition, it was obvious that the voids of the SCCBs had been almost filled with the silk fibroin hydrogel, and the silk fibroin inverse opal particles derived from the template SCCBs showed a similarly ordered and porous nanostructure with interconnected nanopores that offered efficient space for drug loading. As expected, it was demonstrated that the nanopores of the drug loaded particles were full of the second pregel (NIPAM solution) both on the surface and interior. PNIPAM hydrogels with volume phase transition temperature (VPTT) about 32 ◦ C could shrink to release the encapsulated drugs when the temperature was above 32 ◦ C. To utilize this feature, the particles and their surrounding environment, the phosphate buffer solution (PBS), were exposed to an environment of 40 ◦ (above VPTT, for 5 min). The performance of the drug-encapsulated microcarriers was evaluated by the fluorescent images and release kinetics, as shown in Fig. 3. From the fluorescent images (Fig. 3a), it was observed that the intrinsic fluorescent intensity of DOX in the particles gradually reduced, indicating a sustained release of drug from the microcarriers. As exhibited in Fig. 3b, it was discovered that about half of the drug in the microcarriers with a low concentration of PNIPAM hydrogel (5%) was released within four temperature cycles, compared to only about 20% drug releasing from the microcarriers with a high concentration of PNIPAM hydrogel (20%). In addition, there was a negative correlation between the release rate of DOX and the concentration of PNIPAM hydrogel at the same temperature cycles. In brief, the drug would release from the microcarriers after giving an external temperature stimulation, while the microcarriers would be locked and the drug-release process would not continue if there is no temperature stimulus. This
Fig. 3. (a) The fluorescent images of DOX after 0, 1, 2, 3, 5 and 7 temperature stimuli cycles. The scale bar is 200 m. (b) Release curves of DOX from the microcarriers under different temperature stimuli cycles. Error bars represent standard deviations.
feature of controllable drug release could not only avoid the waste of drugs but also further promote the safety of clinical treatment.
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Fig. 4. (a–c) The confocal laser scanning images of the 3T3 cells cultured on the microcarriers with silk fibroin concentration of 30% and PNIPAM concentration of 20%. The scale bars are 200 m, 200 m, 300 m.
To explore the biocompatibility of the designed microcarrier, 3T3 cells (fibroblast cell line) were cultured in the media containing the components of the microcarrier, i.e. silk fibroin, PNIPAM and the silk fibroin and PNIPAM mixed materials, respectively. The results of MTT assay, a colorimetric assay for the quantitative investigation of cell viability, were shown in Fig. S5. It was confirmed that each component demonstrated good biocompatibility, which indicates the safety and feasibility of microcarriers for clinical applications to some extent. Attractively, according to the results of the cell culture on the microcarriers for three days obtained by a confocal microscope (Fig. S6 and Fig. 4), it was found that the cells not only grew well around the microcarriers, but also adhered and grew well on the surface of microcarriers. This phenomenon could be attributed to the excellent biocompatibility and the uniform porous microstructures of the silk fibroin inverse opal scaffolds, which provided interconnected nanochannels for cell adhesion and substances exchange. These results indicated that the prepared microcarriers also had a great application potential in the field of tissue engineering in addition to drug loading. As mentioned above, PNIPAM would shrink and extrude the drugs encapsulated in the microcarriers when the temperature exceeded its VPTT at about 32 ◦ C. In order to verify the capacity of the microcarriers for controllable release in cell experiments, an appropriate VPTT for PNIPAM hydrogel between the body temperature at 37 ◦ and hyperthermia temperature at 42 ◦ needs to be regulated, or else the PNIPAM hydrogel will maintain a continuous
release of cancer drug during the cell culture process (the temperature of cell culture environment is 37 ◦ ), resulting in the drug waste and threat to the safety of treatment. Thus, an optimized VPTT of around 39 ◦ was tailored by adding the polyacrylamide (pAAm) into the PNIPAM hydrogel and the concentration ratio of pAAm to PNIPAM was 7.5%–92.5% [38]. Besides, the microcarriers with the concentration of PNIPAM hydrogel at 20 % was selected for cell experiment because of a longer sustained release according to the results of previous investigations. Then, using HepG2 cells as model cells, the cell viability influenced by DOX-loaded microcarriers in different temperature cycles above VPTT (at 40 ◦ for 5 min) and at 37 ◦ (for more than 15 min) were recorded, as shown in Fig. 5. The ordinate of histogram represented the corresponding OD values of the eventual surviving cells. It was found that compared with the control group without any temperature stimulation, most of the HepG2 cells were killed after five temperature cycles due to the anticancer drugs released from the rapid shrinkage of the responsive microcarriers. In addition, the amount of released drug was proportional to the temperature cycles, and consistent with the results obtained via a fluorescence microscopy (Fig. 5ad). These results verified that the microcarriers had reversible and repeatable temperature responsiveness as well as remarkable controllable drug release capacity. On the other hand, to exclude the influence of heating on HepG2 cells, the MTT assay was used for quantitatively analyzing the cell activity influenced by heating. As shown in Fig. 5e, the cell viabilities of the groups in different temperature cycles but no drugs involved were pretty close to that of
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Fig. 5. (a–d) The fluorescent images of the HepG2 cells after controllable release of DOX in different temperature cycles: (a)the control group without temperature stimulation, (b) one cycle, (c) three cycles and (d) five cycles. (e) The results of the MTT assay of the HepG2 cells after the simple heating and controllable release of DOX in different temperature cycles. The scale bar is 50 m.
the control group without any treatment, as also confirmed by the fluorescent images (Fig. S7). 3. Conclusion In summary, we have designed and demonstrated a kind of multifunctional drug-delivery microcarriers based on inverse opal scaffold for controllable drug release. The microcarriers were composed of the silk fibroin inverse opal scaffolds and the temperature-responsive drug-loaded PNIPAM hydrogel. On the one hand, the uniform porous microstructures and interconnected nanopores of the inverse opals could benefit for the infiltration of the second pregel and drug loading. It was confirmed that the excellent biocompatibility and rough surface of the silk fibroin scaffolds contributed to the normal cells adhesion and proliferation, which was advantageous to the tissue regeneration at the tumor site. On the other hand, because of the temperature responsiveness of the PNIPAM hydrogel, the drug release process could be controlled by using different temperature cycles so that not only the drug waste could be reduced but the treatment safety could be improved as well. These features of the multifunctional inverse opal particle-based microcarriers with temperature responsiveness hydrogel make them promising in both drug delivery and tissue engineering. Data availability The raw/processed data required to reproduce these findings cannot be shared at this time as the data also forms part of an ongoing study. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. CRediT authorship contribution statement Hui Zhang: Validation, Formal analysis, Investigation, Data curation, Writing - original draft. Yuxiao Liu: Writing - review & editing. Canwen Chen: Resources, Writing - review & editing. Wenguo Cui: Resources, Writing - review & editing. Chunwu Zhang: Conceptualization, Methodology, Supervision. Fangfu Ye: Conceptualization, Methodology, Supervision. Yuanjin Zhao: Conceptualization, Methodology, Supervision.
Acknowledgements This work was supported by the National Natural Science Foundation of China (grants 61927805 and 51522302), the NSAF Foundation of China (grant U1530260), the Natural Science Foundation of Jiangsu (Grant no. BE2018707), and K.C. Wong Education Foundation.
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