Silk fibroin microneedles for transdermal drug delivery: where do we stand and how far can we proceed?

Microneedles are a patient-friendly technique for delivering drugs to the site of action in place of traditional oral and injectable administration. Silk fibroin represents an interesting polymeric biomaterial because of its mechanical properties, thermal stability, biocompatibility and possibility of control via genetic engineering. This review focuses on the critical research progress of silk fibroin microneedles since their inception, analyzes in detail the structure and properties of silk fibroin, the types of silk fibroin microneedles, drug delivery applications and clinical trials, and summarizes the future development trend in this field. It also proposes the future research direction of silk fibroin microneedles, including increasing drug loading doses and enriching drug loading types as well as exploring silk fibroin microneedles with stimulation-responsive drug release functions. The safety and effectiveness of silk fibroin microneedles should be further verified in clinical trials at different stages.National Natural Science Foundation of China (Grant No. 51973144), College Nature Science Research Project of Jiangsu Province, China (Grant No. 20KJA540002), PAPD and Six Talent Peaks Project in Jiangsu Province (Grant No. SWYY-038) supported this work

디지털 광원 처리 프린팅을 이용한 실크 피브로인 기반 마이크로니들 및 수광 필름 제조 연구

학위논문(박사) -- 서울대학교대학원 : 농업생명과학대학 바이오시스템.소재학부(바이오소재공학전공), 2023. 2. 현진호.Silk fibroin (SF) is a natural protein material that is currently used in various fields due to its high biocompatibility. Since the photocrosslinking reaction mechanism of SF molecules using riboflavin was recently reported, research on 3D printing of SF using digital light processing (DLP) technology has been actively conducted. As a photoinitiator, riboflavin absorbs light in the 350-450 nm region to form radicals, and at this time, SF forms dityrosine bonds through photo-oxidation to form a hydrogel network. The use of riboflavin in the photocrosslinking reaction of SF is quite challenging and few previously studied cases. However, it is worth researching as it is possible to fabricate a 3D printed structure that is highly biocompatible and maintains its shape only with SF and riboflavin. In addition to exploiting the photocrosslinking reaction of SF, the introduction of an anti-aliasing strategy in DLP printing can improve the resolution of SF patterns by reducing stair effects. Since resolution is a hardware limitation of equipment used in DLP printing, image processing processes such as grayscale processing and image adjustment in pixel units are required to improve resolution. In this study, the hardware limitations of DLP projector resolution were overcome through customized grayscale processing software. The improved resolution of the SF 3D printed structure is evaluated through the smaller and sharper microneedle structure. In this study, a SF microneedle was fabricated and its performance was evaluated through a penetration test on pig skin. In addition, the process of creating a 3D model for DLP printing can be omitted through image adjustment in pixel units. Since the DLP pattern image is a two-dimensional bitmap, the image can be scaled pixel by pixel to print a structure of the desired shape, which is verified by creating a relatively large size structure. In this study, an optical film structure for light harvesting was fabricated and light diffraction characteristics were investigated. It was confirmed that a minimum of 1.62% and a maximum of 6.74% more voltage was produced in the photovoltaic cell using the fabricated SF optical film structure. This research will serve as an opportunity to broaden the application field of protein-based ink by presenting the experimental results for DLP printing of SF.SF (Silk fibroin)는 생체적합성이 높아 현재 다양한 분야에서 사용되는 천연 단백질 재료다. 최근에 리보플라빈을 이용한 SF 분자의 광가교 반응 기작이 보고된 이후로, 디지털 광원 처리 (Digital Light Processing, DLP) 기술을 사용한 SF의 3D 프린팅 연구가 활발히 진행되고 있다. 광개시제로서 리보플라빈은 350-450 nm 영역의 빛을 흡수하여 라디칼을 형성하고, 이 때 SF는 광산화를 통해 디티로신 결합을 형성하여 하이드로겔 네트워크를 만든다. SF의 광가교 반응에 리보플라빈을 사용하는 것은 상당히 도전적이며 기존에 연구된 사례 또한 드물다. 그러나 SF와 리보플라빈만으로 생체적합성이 높으며 형태를 잘 유지하는 3D 프린팅 구조체를 제작할 수 있어 연구의 가치가 있다. SF의 광가교 반응을 활용하기 위함뿐만 아니라, DLP 프린팅에 안티 앨리어싱 전략을 도입하면 계단 현상을 줄여 SF 패턴의 분해능을 높일 수 있다. 분해능은 DLP 프린팅에 사용되는 장비의 하드웨어적 한계인데, 분해능의 개선을 위해서는 그레이스케일 처리, 픽셀 단위의 이미지 조정 등의 이미지 처리 과정을 거쳐야 한다. 본 연구에서는 그레이스케일 처리를 통한 소프트웨어적 개선으로 DLP 프로젝터 분해능의 하드웨어적 한계를 극복했다. 이렇게 SF 3D 프린팅 구조체의 향상된 분해능은 마이크로니들의 더 작고 날카로운 구조를 통해 평가된다. 본 연구에서는 SF 마이크로니들을 제작하고 돼지 피부에 대한 관통 시험을 통해 마이크로니들의 성능을 평가했다. 또한 픽셀 단위 이미지 조정을 통해 DLP 프린팅을 위한 3D 모델 생성 과정을 생략할 수 있다. DLP 패턴 이미지는 2차원 비트맵이므로 이미지를 픽셀 단위로 조정하여 원하는 모양의 구조를 인쇄할 수 있으며, 이는 비교적 큰 크기의 구조물을 생성하여 확인된다. 본 연구에서는 빛 수확을 위한 광학 필름 구조를 제작하고 주요 회절 특성을 조사하였다. 제작한 SF 광학 필름 구조체를 이용해 광전지에서 최소 1.62%, 최대 6.74% 더 많은 전압이 생산되는 것을 확인했다. 본 연구 자료는 SF의 DLP 프린팅 응용을 위한 탐구 결과를 제시함으로써 단백질 기반 잉크의 활용 분야를 더 넓히는 계기가 될 것이다.I. Introduction 1 II. Materials and Methods 8 2.1. Preparation of Silk Fibroin 8 2.1.1. Absorbance of Silk Fibroin Solutions and Films 8 2.1.2. Chemical Sturcture of Silk Fibroin Solutions and Films 8 2.2. Preparation of DLP Printing System 9 2.3. Fabrication of Microneedles 9 2.3.1. Fabrication of Silk Fibroin Microneedles 9 2.3.2. Fluorescence Measurement of Silk Fibroin Hydrogels 10 2.3.3. Morphological Properties of Silk Fibroin Microneedles 10 2.3.4. Mechanical Properties of Silk Fibroin Microneedles 10 2.3.5. Penetration Test of Silk Fibroin Microneedles on Porcine Skin 10 2.4. Fabrication of Optical Film for Light Harvesting 11 2.4.1. Fabrication of Silk Fibroin Optical Film for Light Harvesting 11 2.4.2. Refractive Index of Silk Fibroin Optical Film 12 2.4.3. Various Pattern Formation on Silk Fibroin Optical Film 12 2.4.4. Light Diffraction Properties of Silk Fibroin Optical Film 12 2.4.5. Pattern Images for Fabricaion of Patterned Silk Fibroin Films 12 2.4.6. Gray Value Analysis of Silk Fibroin Optical Film 13 2.4.7. Photovoltaic Panel Voltage Analysis of Silk Fibroin Optical Film 13 III. Results and Discussion 17 3.1. Preparation of Silk Fibroin 17 3.1.1. Properties of SF Solutions 17 3.1.2. Properties of SF Films 19 3.2. DLP Printing System 24 3.3. Silk Fibroin-based Microneedles 29 3.3.1. Fluorescence Measurement of Silk Fibroin Hydrogels 31 3.3.2. Fabrication of Silk Fibroin Microneedles 31 3.3.3. Morphological Properties of Silk Fibroin Microneedles 35 3.3.4. Mechanical Properties of Silk Fibroin Microneedles 43 3.3.5. Penetration Test of Silk Fibroin Microneedles on Porcine Skin 43 3.3.6. Limitations of Measurement Method 44 3.4. Silk Fibroin-based Optical Film for Light Harvesting 48 3.4.1. Fabrication of Silk Fibroin Optical Film for Light Harvesting 48 3.4.2. Refractive Index of Silk Fibroin Optical Film 51 3.4.3. Various Pattern Formation on Silk Fibroin Optical Film 55 3.4.4. Light Diffraction Properties of Silk Fibroin Optical Film 55 3.4.5. Pattern Images for Fabricaion of Patterned Silk Fibroin Films 61 3.4.6. Gray Value Analysis of Silk Fibroin Optical Film 65 3.4.7. Photovoltaic Panel Voltage Analysis of Silk Fibroin Optical Film 71 3.4.8. Limitations of Measurement Method 79 IV. Conclusion 82 V. References 84 VI. Appendix 94박

Silk fibroin nanoparticles : in vitro performance of a putative anticancer nanomedicine

Despite the advantages of nanoparticle-based carriers for anticancer drug delivery, their translation into the clinic has been limited by factors including: (i) poor endocytic uptake and intracellular routing, (ii) macrophage clearance and (iii) a disregard of the tumour microenvironment governing nanoparticle uptake. As a result, there is a continued demand to explore the performance of polymer-based nanoparticles.;The principle hypothesis of this thesis is that silk fibroin nanoparticles can be used as anticancer nanomedicines. To validate this, the mechanisms governing drug release from silk fibroin nanoparticles are explored in Chapter 3. Next, the immunogenicity of silk fibroin nanoparticles towards macrophages is assessed (Chapter 4). Finally, Chapter 5 investigates the endocytosis of silk fibroin nanoparticles in response to the cell cycle and culture substrate mechanics.;This thesis provided the first experimental proof of lysosomotropic anticancer drug delivery from silk fibroin nanoparticles in single human breast cancer cells (Totten et al. 2017. J. Drug Target. 25, pp 865-872) (Chapter 3). Drug loaded silk fibroin nanoparticles were endocytosed by MCF-7 cells and a combination of the acidic lysosomal pH and enzymatic degradation facilitated drug release and subsequent nuclear translocation of the payload within 5 hours of dosing.;Next, nanoparticle-macrophage interactions were studied (Chapter 4). Silk fibroin nanoparticles exerted similar immunogenicity to silica and poly(lactic-co-glycolic acid) nanoparticles (Saborano, Wongpinyochit, Totten, Johnston, Seib and Duarte. 2017. Adv. Healthc. Mater. 6, 1601240). This indicated that silk fibroin nanoparticles can compete with leading healthcare materials in pre-clinical and clinical use.;Further assessment into immunomodulatory potential of silk fibroin nanoparticles revealed that they drive macrophage polarisation towards a pro-inflammatory M1-like state (Totten et al. 2019. ACS Appl. Mater. Interfaces. in press). This effect could be fine-tuned with surface modification (i.e. PEGylation). This observation is important because silk fibroin nanoparticles could act both as carriers for chemotherapeutics and as synergistic attenuators of tumour-associated macrophages in the tumour site.;Finally, advanced analysis of silk fibroin nanoparticle endocytosis was conducted (Chapter 5) by assessing intracellular trafficking in a time-dependent manner. Endocytosis of silk fibroin nanoparticles by breast cancer (MCF-7) cells was influenced by cell cycle progression, but not substrate mechanics. However, substrate mechanics were found to modulate the endocytic behaviour of healthy human (MCF-10A) breast epithelial cells. This relationship warrants further investigation with regard to the cellular response of nanomedicines.;Overall, this thesis accomplished in vitro analysis of silk fibroin nanoparticle drug delivery performance, macrophage interactions and endocytic uptake. These findings indicate that silk fibroin nanoparticles are emerging as an interesting biopolymer for anticancer applications. Work presented in this thesis provides a foundation to now move to pre-clinical in vivo studies.Despite the advantages of nanoparticle-based carriers for anticancer drug delivery, their translation into the clinic has been limited by factors including: (i) poor endocytic uptake and intracellular routing, (ii) macrophage clearance and (iii) a disregard of the tumour microenvironment governing nanoparticle uptake. As a result, there is a continued demand to explore the performance of polymer-based nanoparticles.;The principle hypothesis of this thesis is that silk fibroin nanoparticles can be used as anticancer nanomedicines. To validate this, the mechanisms governing drug release from silk fibroin nanoparticles are explored in Chapter 3. Next, the immunogenicity of silk fibroin nanoparticles towards macrophages is assessed (Chapter 4). Finally, Chapter 5 investigates the endocytosis of silk fibroin nanoparticles in response to the cell cycle and culture substrate mechanics.;This thesis provided the first experimental proof of lysosomotropic anticancer drug delivery from silk fibroin nanoparticles in single human breast cancer cells (Totten et al. 2017. J. Drug Target. 25, pp 865-872) (Chapter 3). Drug loaded silk fibroin nanoparticles were endocytosed by MCF-7 cells and a combination of the acidic lysosomal pH and enzymatic degradation facilitated drug release and subsequent nuclear translocation of the payload within 5 hours of dosing.;Next, nanoparticle-macrophage interactions were studied (Chapter 4). Silk fibroin nanoparticles exerted similar immunogenicity to silica and poly(lactic-co-glycolic acid) nanoparticles (Saborano, Wongpinyochit, Totten, Johnston, Seib and Duarte. 2017. Adv. Healthc. Mater. 6, 1601240). This indicated that silk fibroin nanoparticles can compete with leading healthcare materials in pre-clinical and clinical use.;Further assessment into immunomodulatory potential of silk fibroin nanoparticles revealed that they drive macrophage polarisation towards a pro-inflammatory M1-like state (Totten et al. 2019. ACS Appl. Mater. Interfaces. in press). This effect could be fine-tuned with surface modification (i.e. PEGylation). This observation is important because silk fibroin nanoparticles could act both as carriers for chemotherapeutics and as synergistic attenuators of tumour-associated macrophages in the tumour site.;Finally, advanced analysis of silk fibroin nanoparticle endocytosis was conducted (Chapter 5) by assessing intracellular trafficking in a time-dependent manner. Endocytosis of silk fibroin nanoparticles by breast cancer (MCF-7) cells was influenced by cell cycle progression, but not substrate mechanics. However, substrate mechanics were found to modulate the endocytic behaviour of healthy human (MCF-10A) breast epithelial cells. This relationship warrants further investigation with regard to the cellular response of nanomedicines.;Overall, this thesis accomplished in vitro analysis of silk fibroin nanoparticle drug delivery performance, macrophage interactions and endocytic uptake. These findings indicate that silk fibroin nanoparticles are emerging as an interesting biopolymer for anticancer applications. Work presented in this thesis provides a foundation to now move to pre-clinical in vivo studies

Pro-angiogenic photo-crosslinked silk fibroin hydrogel: a potential candidate for repairing alveolar bone defects

Objective: This study aimed to develop a pro-angiogenic hydrogel with in situ gelation ability for alveolar bone defects repair. Methodology: Silk fibroin was chemically modified by Glycidyl Methacrylate (GMA), which was evaluated by proton nuclear magnetic resonance (1H-NMR). Then, the photo-crosslinking ability of the modified silk fibroin was assessed. Scratch and transwell-based migration assays were conducted to investigate the effect of the photo-crosslinked silk fibroin hydrogel on the migration of human umbilical vein endothelial cells (HUVECs). In vitro angiogenesis was conducted to examine whether the photo-crosslinked silk fibroin hydrogel would affect the tube formation ability of HUVECs. Finally, subcutaneous implantation experiments were conducted to further examine the pro-angiogenic ability of the photo-crosslinked silk fibroin hydrogel, in which the CD31 and α-smooth muscle actin (α-SMA) were stained to assess neovascularization. The tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were also stained to evaluate inflammatory responses after implantation. Results: GMA successfully modified the silk fibroin, which we verified by our 1H-NMR and in vitro photo-crosslinking experiment. Scratch and transwell-based migration assays proved that the photo-crosslinked silk fibroin hydrogel promoted HUVEC migration. The hydrogel also enhanced the tube formation of HUVECs in similar rates to Matrigel®. After subcutaneous implantation in rats for one week, the hydrogel enhanced neovascularization without triggering inflammatory responses. Conclusion: This study found that photo-crosslinked silk fibroin hydrogel showed pro-angiogenic and inflammation inhibitory abilities. Its photo-crosslinking ability makes it suitable for matching irregular alveolar bone defects. Thus, the photo-crosslinkable silk fibroin-derived hydrogel is a potential candidate for constructing scaffolds for alveolar bone regeneration

Fabrication of Silk Fibroin Nanofibres by Needleless Electrospinning

Silk fibroin nanofibres were fabricated using a needleless electrospinning technique. The procedure focused on a new method for the preparation of a spinning solution from silk fibroin. The role of the concentration of silk fibroin solution, applied voltage and spinning distance were investigated as a function of the morphology of the obtained fibres and the spinning performance of the electrospinning process. The biocompatibility of the obtained fibre sheets was evaluated using an in vitro testing method with MG‐63 osteoblasts. The solvent system consisted of formic acid and calcium chloride that can dissolve silk fibroin at room temperature, and a rate of 0.25 g of calcium chloride per 1 g of silk fibroin was required to obtain a completely dissolved silk fibroin solution. The diameters of the silk electrospun fibres obtained from the formic acid–calcium chloride solvent system ranged from 100 to 2400 nm, depending on the spinning parameters. Furthermore, increasing the concentration of the silk fibroin solution and the applied voltage improved spinning ability and spinning performance in needleless electrospinning. In addition, in vitro tests with living cells showed that the obtained electrospun fibre sheets were highly biocompatible with MG‐63 osteoblasts

Macro/microporous silk fibroin scaffolds with potential for articular cartilage and meniscus tissue engineering applications

This study describes the developmental physicochemical properties of silk fibroin scaffolds derived from high concentration aqueous silk fibroin solutions. The silk fibroin scaffolds were prepared with different initial concentrations (8%, 10%, 12% and 16% (wt%)) and obtained by combining the salt-leaching and freeze-drying methodologies. The results indicated that the antiparallel β-pleated sheet (silk-II) conformation was present in the silk fibroin scaffolds. All the scaffolds possessed macro/micro porous structure. Homogeneous porosity distribution was achieved in all the groups of samples. As the silk fibroin concentration increased from 8% to 16%, the mean porosity decreased from 90.8±0.9% to 79.8±0.3%, and the mean interconnectivity decreased from 97.4±0.5% to 92.3±1.3%. The mechanical properties of the scaffolds exhibited a concentration dependence. The dry state compressive modulus increased from 0.81±0.29 MPa to 15.14±1.70 MPa, and the wet state dynamic storage modulus increased around 20-30 folds at each testing frequencies when the silk fibroin concentration increased from 8% to 16%. The water-uptake ratio decreased by means of increasing silk fibroin concentration. The scaffolds present favorable stability as their structure integrity, morphology and mechanical properties were maintained after in vitro degradation for 30 days. Based on these results, the scaffolds developed in this study are herein proposed to be used in meniscus and cartilage tissue engineering scaffolding.Tissue2Tissue project (PTDC/CTM/105703/2008

Adsorption of olive leaf (Olea europaea L.) antioxidants on silk fibroin

The adsorption isotherms of oleuropein and rutin were evaluated at different temperatures, pH values, and solid/liquid ratios. The experimental data of adsorption isotherms were well fitted to a Langmuir model. The maximum adsorption capacities were determined as 108 mg of oleuropein/g of silk fibroin and 21 mg of rutin/g of silk fibroin. After adsorption of oleuropein and rutin, the antioxidant capacity of silk fibroin increased from 1.93 to 3.61 mmol of TEAC/g. Silk fibroin also gained antimicrobial activity against Staphylococcus aureus and Klebsiella pneumoniae after adsorption of olive leaf antioxidants. In a desorption process, 81% of rutin and 85% of oleuropein were removed from the adsorbent surface in 70% aqueous ethanol solution. Consequently, silk fibroin was found to be a promising biomaterial for the production of functional food or dietary supplements and for the purification of oleuropein and rutin from olive leaf extracts

PEGylation-Dependent Metabolic Rewiring of Macrophages with Silk Fibroin Nanoparticles

Silk fibroin nanoparticles are emerging as promising nanomedicines, but their full therapeutic potential is yet to be realized. These nanoparticles can be readily PEGylated to improve colloidal stability and to tune degradation and drug release profiles; however, the relationship between silk fibroin nanoparticle PEGylation and macrophage activation still requires elucidation. Here, we used in vitro assays and nuclear magnetic resonance based metabolomics to examine the inflammatory phenotype and metabolic profiles of macrophages following their exposure to unmodified or PEGylated silk fibroin nanoparticles. The macrophages internalized both types of nanoparticles, but they showed different phenotypic and metabolic responses to each nanoparticle type. Unmodified silk fibroin nanoparticles induced the upregulation of several processes, including production of proinflammatory mediators (e.g., cytokines), release of nitric oxide, and promotion of antioxidant activity. These responses were accompanied by changes in the macrophage metabolomic profiles that were consistent with a proinflammatory state and that indicated an increase in glycolysis and reprogramming of the tricarboxylic acid cycle and the creatine kinase/phosphocreatine pathway. By contrast, PEGylated silk fibroin nanoparticles induced milder changes to both inflammatory and metabolic profiles, suggesting that immunomodulation of macrophages with silk fibroin nanoparticles is PEGylation-dependent. Overall, PEGylation of silk fibroin nanoparticles reduced the inflammatory and metabolic responses initiated by macrophages, and this observation could be used to guide the therapeutic applications of these nanoparticles. © 2019 American Chemical Society

In vitro evaluation of bi-layer silk fibroin scaffolds for gastrointestinal tissue engineering

Silk fibroin scaffolds were investigated for their ability to support attachment, proliferation, and differentiation of human gastrointestinal epithelial and smooth muscle cell lines in order to ascertain their potential for tissue engineering. A bi-layer silk fibroin matrix composed of a porous silk fibroin foam annealed to a homogeneous silk fibroin film was evaluated in parallel with small intestinal submucosa scaffolds. AlamarBlue analysis revealed that silk fibroin scaffolds supported significantly higher levels of small intestinal smooth muscle cell, colon smooth muscle cell, and esophageal smooth muscle cell attachment in comparison to small intestinal submucosa. Following 7 days of culture, relative numbers of each smooth muscle cell population maintained on both scaffold groups were significantly elevated over respective 1-day levels—indicative of cell proliferation. Real-time reverse transcription polymerase chain reaction and immunohistochemical analyses demonstrated that both silk fibroin and small intestinal submucosa scaffolds were permissive for contractile differentiation of small intestinal smooth muscle cell, colon smooth muscle cell, esophageal smooth muscle cell as determined by significant upregulation of α-smooth muscle actin and SM22α messenger RNA and protein expression levels following transforming growth factor-β1 stimulation. AlamarBlue analysis demonstrated that both matrix groups supported similar degrees of attachment and proliferation of gastrointestinal epithelial cell lines including colonic T84 cells and esophageal epithelial cells. Following 14 days of culture on both matrices, spontaneous differentiation of T84 cells toward an enterocyte lineage was confirmed by expression of brush border enzymes, lactase, and maltase, as determined by real-time reverse transcription polymerase chain reaction and immunohistochemical analyses. In contrast to small intestinal submucosa scaffolds, silk fibroin scaffolds supported spontaneous differentiation of esophageal epithelial cells toward a suprabasal cell lineage as indicated by significant upregulation of cytokeratin 4 and cytokeratin 13 messenger RNA transcript levels. In addition, esophageal epithelial cells maintained on silk fibroin scaffolds also produced significantly higher involucrin messenger RNA transcript levels in comparison to small intestinal submucosa counterparts, indicating an increased propensity for superficial, squamous cell specification. Collectively, these data provide evidence for the potential of silk fibroin scaffolds for gastrointestinal tissue engineering applications