Temperature-responsive injectable hydrogel derived from elastin-like polypeptide as a cell carrier

Temperature-responsive injectable hydrogel derived from elastin-like polypeptide as a cell carrier | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Temperature-responsive injectable hydrogel derived from elastin-like polypeptide as a cell carrier Mutawakil Al Muqadasi, Keitaro Ii, Kei Nishida, Masayasu Mie, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6676494/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Injectable hydrogels are promising biomaterials for tissue engineering applications because they deliver bioactive compounds or cells with minimal invasiveness. Temperature-responsive hydrogels, which transition from a liquid to a gel in response to environmental temperature, are candidates for injectable hydrogels. Elastin-like polypeptides (ELPs) with temperature-responsive capabilities have been well studied for drug delivery and tissue repair, yet their development as injectable biomaterials remain limited. In our previous study, we designed hydrogels formed from coiled-coil unit bound ELPs (CUBEs), which incorporated ELPs, a poly-aspartic acid (poly D) chain, a coiled-coil peptide (CL), and a functional peptide. However, injectability of CUBEs has not been studied. Here, we evaluated the injectability and cell delivery potential of O-CUBE, (AVGVP) 42 -D 88 -CL, which is a basic CUBE hydrogel system. We injected O-CUBE mixed with human cervical cancer (HeLa) cells into pre-warmed culture medium to initiate in situ gelation. O-CUBE protein was successfully gelled at an approximately 90% gelation rate after injection at 37℃ in a solution with pH 6 to 8. The injected HeLa cells exhibited spheroid morphology, indicating that the hydrogel facilitated cell-cell interactions in three-dimensional culture. Further evaluation with a DNA assay showed that the HeLa cells can proliferate in the O-CUBE hydrogel. Results demonstrate that the CUBE hydrogel system is a promising candidate for an injectable hydrogel cell delivery system with minimal invasiveness. injectable hydrogel temperature-responsive hydrogel three-dimensional culture elastin-like polypeptide Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Hydrogels are three-dimensional (3D) crosslink networks that can absorb significant water due to the hydrophilic components in their polymeric backbone[ 1 ]. The water content within their porous structure enables hydrogels to encapsulate cells and release therapeutic agents such as drugs, proteins, and DNA. Injectable hydrogels have attracted significant attention as a carrier for delivering cells by simple injection[ 2 ]. In situ gelation of injectable hydrogels triggered by various stimuli, including temperature, pH, light, and ion concentration, serve as shape adaptability to access area of the body that are difficult to reach[ 3 , 4 ]. From a clinical perspective, such minimally-invasive injectable hydrogels containing therapeutic cells significantly reduce recovery times and minimize the risk of infection associated with surgical interventions[ 5 ]. To address these challenges, researchers have developed injectable hydrogel to be gelation in response to external stimuli such as pH, electrical and magnetic fields, and temperature, enabling better control of the gelation process[ 5 ]. Among them, temperature-responsive injectable hydrogels are used as a cell carrier due to the gelation triggered by body temperatures following injection[ 6 , 7 ]. Temperature-responsive polymers including poly( N -isopropylacrylamide) (PNIPAM) is a popular component of temperature-responsive injectable hydrogels[ 8 ]. In this regard, temperature-responsive protein-based injectable hydrogels is promising candidates because of biocompatibility, tunable biodegradation, biological mechanical properties, and genetically engineered tunability. The adaptability of hydrogels mimicking the property of native extracellular matrix (ECM) components, such as laminin, hyaluronic acid, glycosaminoglycan, collagen, and elastin, play a potential role in regulating cell growth, differentiation, and tissue interactions[ 9 ]. Elastin-like polypeptides (ELPs) are temperature-responsive biopolymers that are genetically engineered and have been widely used for drug delivery and tissue repair due to their biocompatibility, biodegradability and genetically engineered tunenability.[ 10 ], [ 11 ]. The combination of different ELP-collagen ratios was shown to sustain drug release, which significantly reduced survival and proliferation of glioblastoma cells in vitro [ 12 ]. Chen et al. engineered ELP fused with silk fibroin and successfully improved mature bone and cartilage regeneration with bone mesenchymal stem cells[ 13 ]. Although ELP-based hydrogels have been investigated for various biomedical applications, their development as injectable hydrogels remains relatively limited compared to other biomaterials. In our previous study, we developed a temperature-responsive multifunctional protein hydrogels derived from ELPs comprised of (AVGVP) 42 peptide sequence. ELP was fused with a poly(aspartic acid) (poly D) chain to prevent uncontrolled aggregation via electrostatic interaction; a coiled-coil peptide (CL) that forms an antiparallel tetrameric structure; and a functional peptide (Fig. 1 a). The engineered ELPs, called by coiled-coil unit bound ELP (CUBE), exhibited lower critical solution temperature-type sol-gel transition at physiological temperature and tunable mechanical properties. Moreover, immobilization of heparin-binding angiogenic growth factors into CUBEs fused with an RGD peptide resulted in enhanced angiogenic activity of human umbilical vein endothelial cells (HUVEC)[ 14 ]. Although multifunctional CUBE hydrogel systems represent promising a cell carrier, the injectability of CUBE hydrogels including cells was unknown. In this study, we evaluated the injectability of the CUBE protein and its effectiveness as a cell delivery system. We utilized the basic CUBE hydrogel, referred to as O-CUBE protein, with the peptide sequence (AVGVP) 42 -D 88 -CL. HeLa cells was encapsulated in an O-CUBE protein solution at 4℃ and then injected into pre-warmed culture medium at 37℃ (Fig. 1 b). To assess cell delivery capability, we investigated the cell viability and proliferation of HeLa cells after injection of the O-CUBE hydrogel. 2. Materials and Methods 2.1 Materials Escherichia coli ( E.coli ) KRX was purchased from Promega. BugBuster 10X protein extraction reagent was purchased from Merck Millipore. HeLa cells were purchased from RIKEN Bioresource Center; culture media for DMEM was purchased from FUJIFILM Wako Pure Chemical Corporation. The QuantiFluor® dsDNA System was purchased from Promega. 2.2 Protein expression and purification The plasmid for expression of ELP-poly(D) 88 -CoilLL (O-CUBE) used in this study was constructed in our previous study[ 14 ]. Transformed E. coli KRX were cultured in Luria-Bertani (LB) medium supplemented with 20 µg/mL of kanamycin. Protein expression was induced with rhamnose at a final concentration of 0.2% (w/v) when the culture reached OD 600 of 0.6–0.8 and incubated overnight at 25℃ with shaking at 130 rpm. The cells were collected by centrifugation at 8000 rpm and resuspended in BugBuster protein extract according to the manufacturer’s protocol. The resuspended cells were rotated at room temperature for 15 min, followed by lysis with sonication. The lysate protein was collected by centrifugation and purified by inverse transition cycle (ITC) purification as follows. Soluble proteins were supplemented with NaCl at a final concentration of 0.4 M and incubated at 70℃ for 15 min. Insoluble fractions were collected with centrifugation at 25℃ for 10 min. The insoluble proteins were resuspended in cold phosphate-buffered saline (PBS) and rotated at 4℃ for 30 min. After rotation, the soluble fraction was collected by centrifugation at 4℃ for 5 min. 2.3 Formation of O-CUBE hydrogel The purified O-CUBE protein was lyophilized overnight. The dried protein was diluted by cold PBS to a final concentration of 5% (w/v), followed by rotation at 4℃ for 15 min. O-CUBE protein gelation was formed by incubation at 37℃ for 5 min. 2.4 Injectability of O-CUBE hydrogel Fluorescein was dissolved in N, N-dimethylformamide (DMF) with a final concentration of 50 mg/mL. This solution was diluted 100-fold with ultra-pure water and then 50-fold with PBS. To evaluate the effect of protein concentration, lyophilized O-CUBE protein was dissolved in diluted fluorescein at a concentration of 1–5% (w/v). The fluorescein-encapsulated O-CUBE protein was injected in pre-heated PBS at 37℃. To assess the impact of pH, fluorescein solution was prepared as described above using PBS with pH values ranging from 6.0–8.0 and different speed injection of 25 µL/s, 50 µL/s, and 100 µL/s. Lyophilized O-CUBE protein was dissolved in each fluorescein solution and injected into 500 µL of pre-heated saline buffer at 37℃. The supernatant from each injection was collected, and the fluorescence intensity was measured (λ ex = 494 nm, λ em = 521 nm). The injectable gelation rate of O-CUBE was calculated using the following equation: $$\:Injectablegelationrate=\left(1-\frac{Intensity\:of\:collected\:salinebuffer}{Intensity\:of\:fluorescein\:solution\:without\:CUBE}\right)\times\:100\%$$ 2.5 Cell culture HeLa cells were cultured with DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). The HeLa cells were cultured at 37°C and 5% CO 2 . 2.6 Cell encapsulation within injected O-CUBE hydrogel The purified O-CUBE protein was filtered using a 0.22 µm syringe filter and lyophilized overnight. The lyophilized O-CUBE was diluted by cold cell culture medium to a final concentration of 4% (w/v), followed by rotation at 4℃ for 15 min. The cells were encapsulated in 50 µL of 4% O-CUBE solution, with a final cell number of \(\:2\times\:{10}^{4}\) cells. The O-CUBE solution, 1 mL syringe, and 23G needle were incubated on ice to maintain the CUBE solutions in their liquid state during injection. The culture media for HeLa cells were pre-incubated at 37℃. O-CUBE-loaded cells were then injected into the warm media, and cell morphology and DNA content were evaluated at specific time intervals. 2.7 Cell viability assay The viability of HeLa cells following injection with O-CUBE was evaluated with live/dead fluorescent double staining. Cells were collected from O-CUBE at specific interval times after injection by pipetting the gel. The medium containing the hydrogel was incubated on ice for 15 min and then centrifuged at 12,000 rpm for 5 min. Cells were then collected and incubated with calcein AM (2 µM) and propidium iodide (PI; 4 µM) diluted in serum-free medium for 20 min. The fluorescence of calcein AM and PI were observed under a fluorescence microscope (OLYMPUS IX70). Living cells exhibited green fluorescence (calcein AM), while dead cells exhibited red fluorescence (PI). Percent viability of cell in gell was calculated with the following equation: $$\:Cell\:viability=\left(\frac{Living\:cells}{Living\:cells+Dead\:cells}\right)\times\:100\%$$ 2.8 Proliferation assay by QuantiFluor dsDNA The injected hydrogel encapsulating cells was collected by pipetting and stored in a 1.5 mL microtube. The medium containing the hydrogel was incubated on ice for 15 min and then centrifuged at 12,000 rpm for 5 min. Autoclaved ultra-pure water (100 µL) was added to precipitated cells, and the solution was mixed thoroughly by pipetting and vortexing. The DNA content was extracted by sonication for 10 min, followed by centrifugation at 12,000 rpm for 5 min. The supernatant containing dsDNA was collected and stored at – 20℃. To assess cell proliferation, the DNA content of the injected cells was measured using the QuantiFluor dsDNA assay, following the manufacturer’s protocol. Briefly, 200 µL of working solution was added to a black flat-bottom 96-well plate. The sample containing dsDNA (20 µL) was added to the plate. The fluorescence intensity was measured (λ ex = 504 nm, λ em = 531 nm) with a multilabel microplate reader (Perkin Elmer Enspire 2300) and DNA concentration was quantified using a predefined standard curve. 2.9 Statistical analysis Data are presented as mean ± standard deviation (SD). Multiple quantitative data sets were compared utilizing one-way ANOVA, followed by Bonferroni correction. p < 0.05 was determined as the threshold for statistical significance with n = 3. 3. Results and Discussion 3.1 Temperature-responsive CUBE hydrogels Temperature-responsive hydrogels are promising candidates for injectable applications in tissue engineering, offering significant advantages over non-injectable hydrogels, including minimally invasive administration. In our previous work, we designed temperature-responsive hydrogels derived from ELPs fused with poly-aspartic acid and a coiled-coil peptide, collectively termed coiled-coil unit bound ELPs (CUBEs). In this study, we utilized O-CUBE as the foundational CUBE protein system (Fig. 2 a). The amino sequence of O-CUBE is shown in supplementary Table S1 . The recombinant proteins were successfully expressed in E. coli KRX and purified using an inverse transition cycle (ITC). SDS-PAGE analysis revealed a single band around 50 kDa, which confirmed successful purification by ITC without requiring chromatography (Fig. 2 b). To evaluate gelation ability, the purified O-CUBE protein was diluted in cold PBS (5% (w/v)) and incubated at 37℃. Gelation occurred within 3 min, indicating a rapid in situ gelation (Fig. 2 c). The hydrogel with in situ gelation ability can be administered in liquid form and solidified upon exposure to physiological temperatures[ 15 ]. Furthermore, CUBE hydrogels exhibited a reversible sol-gel transition as they redissolved into their solution phase at 4℃. The reversible sol-gel transition enables easy handling of encapsulated cells prior to injection. The stimuli-responsive behavior of CUBEs is driven by the 42 repeats of the AVGVP peptide, which facilitate temperature-dependent aggregation of ELPs. The poly D chain is incorporated to control the aggregation of ELPs. Poly-aspartic acid enhances hydrophilicity and electrostatic repulsion between ELPs, tuning their phase transition behavior to more favorable solubility and aggregation. The coiled-coil interaction generates tetrameric CUBE proteins, allowing stable hydrogel formation at low concentrations (> 2% (w/v)). The O-CUBE protein has potential to be an injectable hydrogel due to its rapid gelation ability at physiological temperatures. Reversible gelation offers ease of injection and handling, as it can be re-liquefied if needed for modification, reloading and various other applications. In addition, the O-CUBE hydrogel can be simply purified by ITC without chromatography, making it more efficient in reducing the cost and time of production. 3.2 Injectability of O-CUBE protein Gelation efficiency is a critical factor for injection of a hydrogel solution. Rapid gelation can lead to needle blockage, while slow gelation may result in leakage of the encapsulated cells. The O-CUBE protein solution was injected into pre-warmed culture medium to evaluate injectability. Figure 3 a shows a representative image of gelation of the injected O-CUBE protein. O-CUBE at a concentration of 4% (w/v) exhibited an irregular shape, indicating that gelation occurred after injection. This irregular shape is advantageous as it allows the hydrogel to fill irregular cavities in the tissue. O-CUBE at 5% (w/v) showed linear gelation due to premature gelation in the needle. We assessed the optimal O-CUBE concentrations for an injectable hydrogel by dissolving the proteins in a fluorescein-containing solution and subsequently injecting into warm PBS. Release of the fluorescein compound into the PBS was directly correlated with the percentage of failed gelation after injection. The injectable gelation rate of O-CUBE improved as the concentration increased from 1–4% (w/v) (Fig. 3 b). At a concentration of 1% (w/v), O-CUBE exhibited a gelation rate of 23.7 ± 2.7%, while at a concentration of 4% (w/v), 84.7 ± 5.1% of the O-CUBE solution was successfully gelled after injection. At 5% (w/v), O-CUBE exhibited premature gelation in the needle, making injection challenging. The ELP concentration influences the transition temperature (T t ) of the resultant hydrogels. The resultant hydrogels exhibit higher T t at lower concentrations of ELPs, while higher concentrations result in a lower T t . The slight difference between T t affects the gelation rate during injection. We determined the optimal O-CUBE concentration for injectable hydrogels to be 4% (w/v), which was then used for subsequent experiments. Different tissues or diseases, such as those involved in wound healing or characterized by a cancerous environment, exhibit different pH levels from typical physiological conditions[ 16 ], [ 17 ]. To assess the suitability of the O-CUBE hydrogel system for biomedical applications, we evaluated its injection capability across a range of pH levels. Results revealed that CUBE proteins exhibited a robust injectable gelation rate from pH 6 to 8 (Fig. 3 c). At pH 7 and 8, the 25, 50, and 100 µL/s injection rates did not significantly affect the gelation rate. At an injection rate of 25 µL/s, approximately 90% of O-CUBE was successfully gelled after injection into PBS at pH 7 and 8. However, injection rates ranging from 25 to 100 µL/s decreased the gelation ability from 90.7 ± 3.4% to 79.4 ± 3.3% at pH 6. This result indicates that the O-CUBE hydrogel was not highly dependent on pH, making it suitable for diverse applications in environments with varying pH conditions. Taken together, the O-CUBE hydrogel exhibited injectability with robust gelation when injected into pre-warmed medium. O-CUBE at 4% (w/v) was successfully injected into pre-warmed medium with a high gelation rate, and has the potential to fill irregular cavities for tissue engineering applications. In addition, the O-CUBE hydrogel showed a high degree of gelation across a pH range of 6 to 8, which can be useful for broad applications, such as drug delivery for cancer therapy and cell delivery for wound healing. 3.3 Cell morphology of injected HeLa cells within O-CUBE hydrogel Three-dimensional (3D) hydrogel scaffolds offer significant benefits because they mimic the in vivo environment and promote cell behaviors similar to native physiologic conditions[ 18 ]. To serve as an effective cell delivery agent, hydrogels must encapsulate cells in a 3D environment and support their viability after injection. We employed HeLa cells to assess the encapsulation efficiency and distribution of cells within the O-CUBE hydrogel environment. Cells were stained with calcein AM and injected with O-CUBE hydrogel. Their distribution was evaluated using confocal microscopy. O-CUBE successfully gelled upon injection, effectively encapsulating the cells. Confocal laser microscopy further revealed that cells were distributed throughout the bottom, mid, and upper layers of the hydrogel (Fig. 4 a). This result confirmed that the O-CUBE protein solution was successfully injected and uniformly encapsulated in cells. Injectable hydrogels are a promising cell delivery agent based on their ability to retain cells at the targeted site without unintended migration. To optimize cell retention, it is important to identify the maximum number of cells that can be encapsulated within the hydrogel. We evaluated the encapsulation capacity of HeLa cells in the CUBE hydrogel and found the maximum number of HeLa cells encapsulated in O-CUBE to be \(\:\:2.0\times\:{10}^{4}\) cells per 50 µL (Fig. 4 b). Exceeding this cell concentration led to leakage of encapsulated cells from the hydrogel after injection. Figure 4 c shows the cell morphology of HeLa from day 1 to 5 after injection. After 5 days, the sizes of both cell types increased, indicating multicellular aggregation or spheroid formation. Spheroid formation indicates that cell-cell interactions were more dominant than cell-matrix interactions. This result is consistent with the fact that the O-CUBE hydrogel lacks any cell adhesion peptide, which increases cell-cell interactions in 3D culture. Spheroid culture in the absence of a hydrogel matrix has been studied owing to its simplicity, reproducibility, and efficiency. However, matrix-free spheroids also present challenges in mimicking the native tissue environment with specific elasticity and stiffness[ 19 ], [ 20 ]. The injectable O-CUBE hydrogel system exhibited spheroid formation that can be further explored with different protein concentrations and alternative designs. The elasticity and stiffness of the CUBE hydrogels can be controlled by adjusting the CUBE concentration and number of repeating ELP sequences. The ability of O-CUBE hydrogel to provide 3D scaffold for developing physiologically relevant in tumor models, enabling more accurate studies of cancer cell behavior, metastasis, and drug response. 3.4 Cell viability and proliferation of injected HeLa cells within O-CUBE hydrogel We utilized and HeLa cells as model cells, to assess the potential of the O-CUBE hydrogel to deliver cells via simple injection. The incorporation of HeLa cells into a 3D scaffold can provide for physiological characteristics that mimic tumor behavior, metastasis, and drug response. After injection with specific interval times, cells were cultivated from the O-CUBE hydrogels and stained with calcein AM and PI to determine cell viability. Results showed that most of the cells survived (green), while there were only a few dead cells (red), indicating high cell viability after injection with the O-CUBE hydrogel (Fig. 5 a). The cell viability was 96.7 ± 0.5%, 89.4 ± 4.2%, 74.9 ± 1.2% in the O-CUBE hydrogel at 1, 2, and 3 days after injection, respectively (Fig. 5 b). These results suggest that CUBE can be used as injectable gel with encapsulated cells, despite the cells being subjected to low temperatures during hydrogel encapsulation and rapidly transitioning to physiological temperatures during injection. The ability of HeLa cells to survive after encapsulation and injection indicates that O-CUBE is suitable for delivery living cells with minimal loss of viability. To assess cell proliferation, DNA content was measured after cells were collected from the injected O-CUBE hydrogel. HeLa cells showed a significant increase in DNA concentration, indicating high cell proliferation (Fig. 5 c). The DNA concentration increased from 0.8 ± 0.15 ng/µL to 4.4 ± 0.1 ng/µL, on days 1 and 5 following injection, respectively. The high proliferation cells highlight the ability of O-CUBE hydrogel as cell carrier for therapeutic cells in regenerative medicine. Taken together, our O-CUBE hydrogel could maintain cell viability and cell proliferation after injection, which is essential as cell carrier. With further optimization, CUBE hydrogel systems have potential for personalized medicine application, such as patient-specific cancer models for drug screening. Encapsulating cells and locally deliver into specific damaged tissue for wound healing. Moreover, their potential as co-delivery system, which both growth factor and stem cells can be locally delivered in a controlled manner. 5. Conclusion In summary, we evaluated the injectability and biocompatibility of a temperature-responsive O-CUBE hydrogel derived from an elastin-like polypeptide. Results demonstrated that the O-CUBE hydrogel exhibited excellent injectability via in situ gelation at physiological temperature, with a high gelation rate with wide range pH environment. The O-CUBE hydrogel effectively maintained cell survival and proliferation using HeLa cells. Our results demonstrate that O-CUBE has broad potential as multifunctional biomaterial for cancer research, drug testing, and cell-based therapies. Declarations Funding: A part of this work was financially supported by following grants; the Japan Society for the Promotion of Science (JSPS, grant number 23K17209 to Kei Nishida and 23K28427 to Masayasu Mie). Competing interest: The authors declare no competing interest related to this work. Author contribution: All authors contributed to the study conception, design, interpretation of the studies, analysis of the data, and review of the manuscript. Kei Nishida, Masayasu Mie, and Eiry Kobatake designed the research study. Mutawakil Al Muqadasi and Keitaro ii performed the research and analyzed the data. Mutawakil Al Muqadasi wrote the manuscript. Kei Nishida, Masayasu Mie, and Eiry Kobatake reviewed and edited the manuscript. All authors read and approved the final manuscript. Ethical approval: This article contains no studies with human participants or animals performed by any of the authors. Consent to participate: Not applicable Consent to publish: Not applicable Data availability: All data supporting the finding of this study are presented in the article and its Supplementary information. References J. Liu, C. Du, W. Huang, and Y. Lei, “Injectable smart stimuli-responsive hydrogels: pioneering advancements in biomedical applications,” Biomater. Sci. , vol. 12, no. 1, pp. 8–56, 2024, doi: 10.1039/D3BM01352A. Y. Li, H. Y. Yang, and D. S. Lee, “Advances in biodegradable and injectable hydrogels for biomedical applications,” J. 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Gao et al. , “Gelatin-Based Hydrogel for Three-Dimensional Neuron Culture Application,” ACS Omega , vol. 8, no. 48, pp. 45288–45300, Dec. 2023, doi: 10.1021/acsomega.3c03769. S. Žigon-Branc et al. , “Impact of Hydrogel Stiffness on Differentiation of Human Adipose-Derived Stem Cell Microspheroids,” Tissue Eng. Part A , vol. 25, no. 19–20, pp. 1369–1380, Oct. 2019, doi: 10.1089/ten.tea.2018.0237. M. Sheth et al. , “Three-dimensional matrix stiffness modulates mechanosensitive and phenotypic alterations in oral squamous cell carcinoma spheroids,” APL Bioeng. , vol. 8, no. 3, p. 036106, Sep. 2024, doi: 10.1063/5.0210134. Supplementary Files SuplementaryMutawakilAlMuqadasi.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6676494","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":466250050,"identity":"5c6a1358-2cbc-4ecb-9ed7-0265762fbd68","order_by":0,"name":"Mutawakil Al Muqadasi","email":"","orcid":"","institution":"Tokyo Institute of Technology Department of of Life Science and Technology: Tokyo Kogyo Daigaku Seimei Rikogakuin Seimei Rikogakukei","correspondingAuthor":false,"prefix":"","firstName":"Mutawakil","middleName":"Al","lastName":"Muqadasi","suffix":""},{"id":466250051,"identity":"a4d3a4be-62b8-465c-bc48-ba0a2940b28d","order_by":1,"name":"Keitaro Ii","email":"","orcid":"","institution":"Tokyo Institute of Technology: Tokyo Kogyo Daigaku","correspondingAuthor":false,"prefix":"","firstName":"Keitaro","middleName":"","lastName":"Ii","suffix":""},{"id":466250052,"identity":"b2843753-0260-4171-b61a-eb28cd1ece16","order_by":2,"name":"Kei Nishida","email":"","orcid":"","institution":"JAIST: Hokuriku Sentan Kagaku Gijutsu Daigakuin Daigaku","correspondingAuthor":false,"prefix":"","firstName":"Kei","middleName":"","lastName":"Nishida","suffix":""},{"id":466250053,"identity":"07fd4b9f-3d8b-41bf-9577-d38309c07a06","order_by":3,"name":"Masayasu Mie","email":"","orcid":"","institution":"Tokyo Institute of Technology Department of of Life Science and Technology: Tokyo Kogyo Daigaku Seimei Rikogakuin Seimei Rikogakukei","correspondingAuthor":false,"prefix":"","firstName":"Masayasu","middleName":"","lastName":"Mie","suffix":""},{"id":466250054,"identity":"f19f7431-43c5-4d36-964f-661304e353b2","order_by":4,"name":"Eiry Kobatake","email":"data:image/png;base64,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","orcid":"","institution":"Tokyo Institute of Technology Department of of Life Science and Technology: Tokyo Kogyo Daigaku Seimei Rikogakuin Seimei Rikogakukei","correspondingAuthor":true,"prefix":"","firstName":"Eiry","middleName":"","lastName":"Kobatake","suffix":""}],"badges":[],"createdAt":"2025-05-16 02:49:51","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6676494/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6676494/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":84062500,"identity":"9d8970fc-03ab-427a-bfd9-6a5303612493","added_by":"auto","created_at":"2025-06-06 10:28:47","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":720761,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic illustration of injectable O-CUBE hydrogel. \u003cstrong\u003e(a)\u003c/strong\u003e Formation of antiparallel tetramers of O-CUBE protein via interaction of coiled-coils. \u003cstrong\u003e(b)\u003c/strong\u003eTemperature-responsive injectable O-CUBE hydrogel. The figure was created with bioRender\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6676494/v1/d66c20aa3c12373ddc521d4b.png"},{"id":84062490,"identity":"b3451aae-e6cb-408b-8881-8c58b1544ab5","added_by":"auto","created_at":"2025-06-06 10:28:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":689938,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic of O-CUBE protein. \u003cstrong\u003e(a)\u003c/strong\u003e Composition of O-CUBE protein \u003cstrong\u003e(b)\u003c/strong\u003e SDS-PAGE of purified O-CUBE \u003cstrong\u003e(c)\u003c/strong\u003e Temperature-responsive ability of O-CUBE protein.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6676494/v1/678a4a5c0c7ccf287c51b2fa.png"},{"id":84062488,"identity":"9059004c-a953-42fa-a40b-d22e50b94a93","added_by":"auto","created_at":"2025-06-06 10:28:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":808168,"visible":true,"origin":"","legend":"\u003cp\u003eInjectability of O-CUBE protein. \u003cstrong\u003e(a) \u003c/strong\u003eRepresentative images of injectable O-CUBE hydrogel, scale bar 20 mm. \u003cstrong\u003e(b)\u003c/strong\u003e OptimalO-CUBE concentrationsfor injectable hydrogels. \u003cstrong\u003e(c) \u003c/strong\u003eInjectable gelation rate of O-CUBE hydrogel from pH 6 to pH 8 with injection rate of 25 mL/s, 50 mL/s, and 100mL/s. Statistics were obtained by one-way ANOVA with Bonferroni correction test, n = 3, * P \u0026lt; 0.05; ** P \u0026lt; 0.01; ***P \u0026lt; 0.005\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6676494/v1/5d45cbfb14332b6fec322cf7.png"},{"id":84062486,"identity":"658b13e8-e437-4d55-af33-55095cf2df18","added_by":"auto","created_at":"2025-06-06 10:28:44","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1407579,"visible":true,"origin":"","legend":"\u003cp\u003eEncapsulated HeLa cells in O-CUBE hydrogel. \u003cstrong\u003e(a)\u003c/strong\u003e Distribution of encapsulated cells in CUBE hydrogels \u003cstrong\u003e(b)\u003c/strong\u003e The counted cell number after injection in O-CUBE hydrogel and in medium \u0026amp; O-CUBE hydrogel. \u003cstrong\u003e(c) \u003c/strong\u003eRepresentative images of cell morphology of HeLa cells after injection\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6676494/v1/99b604ffe516a1fd1d486321.png"},{"id":84062489,"identity":"00aac766-6d04-4a70-b0f2-3e5e73941cd3","added_by":"auto","created_at":"2025-06-06 10:28:44","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":551978,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a) \u003c/strong\u003eRepresentative images of live/dead staining of recovered cells after injection, scale bar 200 mm \u003cstrong\u003e(b)\u003c/strong\u003e DNA content of HeLa cells injected in the O-CUBE hydrogel.\u003cstrong\u003e (c)\u003c/strong\u003e. DNA content of HeLa cells injected in the O-CUBE hydrogel. Statistics were obtained by one-way ANOVA with Bonferroni correction test, n = 3, * P \u0026lt; 0.05; ** P \u0026lt; 0.01; ***P \u0026lt; 0.005\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6676494/v1/bdcf1fddcbe5782627b6c570.png"},{"id":84063683,"identity":"1b4f49ad-d711-48bb-86cf-c666ba326187","added_by":"auto","created_at":"2025-06-06 10:44:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4595756,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6676494/v1/715a00e7-9430-4ef2-85cb-09e23d08e072.pdf"},{"id":84062835,"identity":"0eb2b4ee-5de9-4123-b094-e7c4b4d161d7","added_by":"auto","created_at":"2025-06-06 10:36:44","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":322194,"visible":true,"origin":"","legend":"","description":"","filename":"SuplementaryMutawakilAlMuqadasi.docx","url":"https://assets-eu.researchsquare.com/files/rs-6676494/v1/6d9e1a297ac09777c5f486e1.docx"}],"financialInterests":"","formattedTitle":"Temperature-responsive injectable hydrogel derived from elastin-like polypeptide as a cell carrier","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eHydrogels are three-dimensional (3D) crosslink networks that can absorb significant water due to the hydrophilic components in their polymeric backbone[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The water content within their porous structure enables hydrogels to encapsulate cells and release therapeutic agents such as drugs, proteins, and DNA. Injectable hydrogels have attracted significant attention as a carrier for delivering cells by simple injection[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. \u003cem\u003eIn situ\u003c/em\u003e gelation of injectable hydrogels triggered by various stimuli, including temperature, pH, light, and ion concentration, serve as shape adaptability to access area of the body that are difficult to reach[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. From a clinical perspective, such minimally-invasive injectable hydrogels containing therapeutic cells significantly reduce recovery times and minimize the risk of infection associated with surgical interventions[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo address these challenges, researchers have developed injectable hydrogel to be gelation in response to external stimuli such as pH, electrical and magnetic fields, and temperature, enabling better control of the gelation process[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Among them, temperature-responsive injectable hydrogels are used as a cell carrier due to the gelation triggered by body temperatures following injection[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Temperature-responsive polymers including poly(\u003cem\u003eN\u003c/em\u003e-isopropylacrylamide) (PNIPAM) is a popular component of temperature-responsive injectable hydrogels[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In this regard, temperature-responsive protein-based injectable hydrogels is promising candidates because of biocompatibility, tunable biodegradation, biological mechanical properties, and genetically engineered tunability. The adaptability of hydrogels mimicking the property of native extracellular matrix (ECM) components, such as laminin, hyaluronic acid, glycosaminoglycan, collagen, and elastin, play a potential role in regulating cell growth, differentiation, and tissue interactions[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Elastin-like polypeptides (ELPs) are temperature-responsive biopolymers that are genetically engineered and have been widely used for drug delivery and tissue repair due to their biocompatibility, biodegradability and genetically engineered tunenability.[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The combination of different ELP-collagen ratios was shown to sustain drug release, which significantly reduced survival and proliferation of glioblastoma cells \u003cem\u003ein vitro\u003c/em\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Chen \u003cem\u003eet al.\u003c/em\u003e engineered ELP fused with silk fibroin and successfully improved mature bone and cartilage regeneration with bone mesenchymal stem cells[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAlthough ELP-based hydrogels have been investigated for various biomedical applications, their development as injectable hydrogels remains relatively limited compared to other biomaterials. In our previous study, we developed a temperature-responsive multifunctional protein hydrogels derived from ELPs comprised of (AVGVP)\u003csub\u003e42\u003c/sub\u003e peptide sequence. ELP was fused with a poly(aspartic acid) (poly D) chain to prevent uncontrolled aggregation via electrostatic interaction; a coiled-coil peptide (CL) that forms an antiparallel tetrameric structure; and a functional peptide (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). The engineered ELPs, called by coiled-coil unit bound ELP (CUBE), exhibited lower critical solution temperature-type sol-gel transition at physiological temperature and tunable mechanical properties. Moreover, immobilization of heparin-binding angiogenic growth factors into CUBEs fused with an RGD peptide resulted in enhanced angiogenic activity of human umbilical vein endothelial cells (HUVEC)[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Although multifunctional CUBE hydrogel systems represent promising a cell carrier, the injectability of CUBE hydrogels including cells was unknown.\u003c/p\u003e \u003cp\u003eIn this study, we evaluated the injectability of the CUBE protein and its effectiveness as a cell delivery system. We utilized the basic CUBE hydrogel, referred to as O-CUBE protein, with the peptide sequence (AVGVP)\u003csub\u003e42\u003c/sub\u003e-D\u003csub\u003e88\u003c/sub\u003e-CL. HeLa cells was encapsulated in an O-CUBE protein solution at 4℃ and then injected into pre-warmed culture medium at 37℃ (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). To assess cell delivery capability, we investigated the cell viability and proliferation of HeLa cells after injection of the O-CUBE hydrogel.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Materials\u003c/h2\u003e \u003cp\u003e \u003cem\u003eEscherichia coli\u003c/em\u003e (\u003cem\u003eE.coli\u003c/em\u003e) KRX was purchased from Promega. BugBuster 10X protein extraction reagent was purchased from Merck Millipore. HeLa cells were purchased from RIKEN Bioresource Center; culture media for DMEM was purchased from FUJIFILM Wako Pure Chemical Corporation. The QuantiFluor\u0026reg; dsDNA System was purchased from Promega.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Protein expression and purification\u003c/h2\u003e \u003cp\u003eThe plasmid for expression of ELP-poly(D)\u003csub\u003e88\u003c/sub\u003e-CoilLL (O-CUBE) used in this study was constructed in our previous study[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Transformed \u003cem\u003eE. coli\u003c/em\u003e KRX were cultured in Luria-Bertani (LB) medium supplemented with 20 \u0026micro;g/mL of kanamycin. Protein expression was induced with rhamnose at a final concentration of 0.2% (w/v) when the culture reached OD 600 of 0.6\u0026ndash;0.8 and incubated overnight at 25℃ with shaking at 130 rpm. The cells were collected by centrifugation at 8000 rpm and resuspended in BugBuster protein extract according to the manufacturer\u0026rsquo;s protocol. The resuspended cells were rotated at room temperature for 15 min, followed by lysis with sonication. The lysate protein was collected by centrifugation and purified by inverse transition cycle (ITC) purification as follows.\u003c/p\u003e \u003cp\u003eSoluble proteins were supplemented with NaCl at a final concentration of 0.4 M and incubated at 70℃ for 15 min. Insoluble fractions were collected with centrifugation at 25℃ for 10 min. The insoluble proteins were resuspended in cold phosphate-buffered saline (PBS) and rotated at 4℃ for 30 min. After rotation, the soluble fraction was collected by centrifugation at 4℃ for 5 min.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Formation of O-CUBE hydrogel\u003c/h2\u003e \u003cp\u003eThe purified O-CUBE protein was lyophilized overnight. The dried protein was diluted by cold PBS to a final concentration of 5% (w/v), followed by rotation at 4℃ for 15 min. O-CUBE protein gelation was formed by incubation at 37℃ for 5 min.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Injectability of O-CUBE hydrogel\u003c/h2\u003e \u003cp\u003eFluorescein was dissolved in N, N-dimethylformamide (DMF) with a final concentration of 50 mg/mL. This solution was diluted 100-fold with ultra-pure water and then 50-fold with PBS. To evaluate the effect of protein concentration, lyophilized O-CUBE protein was dissolved in diluted fluorescein at a concentration of 1\u0026ndash;5% (w/v). The fluorescein-encapsulated O-CUBE protein was injected in pre-heated PBS at 37℃.\u003c/p\u003e \u003cp\u003eTo assess the impact of pH, fluorescein solution was prepared as described above using PBS with pH values ranging from 6.0\u0026ndash;8.0 and different speed injection of 25 \u0026micro;L/s, 50 \u0026micro;L/s, and 100 \u0026micro;L/s. Lyophilized O-CUBE protein was dissolved in each fluorescein solution and injected into 500 \u0026micro;L of pre-heated saline buffer at 37℃. The supernatant from each injection was collected, and the fluorescence intensity was measured (λ\u003csub\u003eex\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;494 nm, λ\u003csub\u003eem\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;521 nm). The injectable gelation rate of O-CUBE was calculated using the following equation:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:Injectablegelationrate=\\left(1-\\frac{Intensity\\:of\\:collected\\:salinebuffer}{Intensity\\:of\\:fluorescein\\:solution\\:without\\:CUBE}\\right)\\times\\:100\\%$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Cell culture\u003c/h2\u003e \u003cp\u003eHeLa cells were cultured with DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). The HeLa cells were cultured at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Cell encapsulation within injected O-CUBE hydrogel\u003c/h2\u003e \u003cp\u003eThe purified O-CUBE protein was filtered using a 0.22 \u0026micro;m syringe filter and lyophilized overnight. The lyophilized O-CUBE was diluted by cold cell culture medium to a final concentration of 4% (w/v), followed by rotation at 4℃ for 15 min. The cells were encapsulated in 50 \u0026micro;L of 4% O-CUBE solution, with a final cell number of \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:2\\times\\:{10}^{4}\\)\u003c/span\u003e\u003c/span\u003e cells. The O-CUBE solution, 1 mL syringe, and 23G needle were incubated on ice to maintain the CUBE solutions in their liquid state during injection. The culture media for HeLa cells were pre-incubated at 37℃. O-CUBE-loaded cells were then injected into the warm media, and cell morphology and DNA content were evaluated at specific time intervals.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Cell viability assay\u003c/h2\u003e \u003cp\u003eThe viability of HeLa cells following injection with O-CUBE was evaluated with live/dead fluorescent double staining. Cells were collected from O-CUBE at specific interval times after injection by pipetting the gel. The medium containing the hydrogel was incubated on ice for 15 min and then centrifuged at 12,000 rpm for 5 min. Cells were then collected and incubated with calcein AM (2 \u0026micro;M) and propidium iodide (PI; 4 \u0026micro;M) diluted in serum-free medium for 20 min. The fluorescence of calcein AM and PI were observed under a fluorescence microscope (OLYMPUS IX70). Living cells exhibited green fluorescence (calcein AM), while dead cells exhibited red fluorescence (PI). Percent viability of cell in gell was calculated with the following equation:\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$$\\:Cell\\:viability=\\left(\\frac{Living\\:cells}{Living\\:cells+Dead\\:cells}\\right)\\times\\:100\\%$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Proliferation assay by QuantiFluor dsDNA\u003c/h2\u003e \u003cp\u003eThe injected hydrogel encapsulating cells was collected by pipetting and stored in a 1.5 mL microtube. The medium containing the hydrogel was incubated on ice for 15 min and then centrifuged at 12,000 rpm for 5 min. Autoclaved ultra-pure water (100 \u0026micro;L) was added to precipitated cells, and the solution was mixed thoroughly by pipetting and vortexing. The DNA content was extracted by sonication for 10 min, followed by centrifugation at 12,000 rpm for 5 min. The supernatant containing dsDNA was collected and stored at \u0026ndash; 20℃.\u003c/p\u003e \u003cp\u003eTo assess cell proliferation, the DNA content of the injected cells was measured using the QuantiFluor dsDNA assay, following the manufacturer\u0026rsquo;s protocol. Briefly, 200 \u0026micro;L of working solution was added to a black flat-bottom 96-well plate. The sample containing dsDNA (20 \u0026micro;L) was added to the plate. The fluorescence intensity was measured (λ\u003csub\u003eex\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;504 nm, λ\u003csub\u003eem\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;531 nm) with a multilabel microplate reader (Perkin Elmer Enspire 2300) and DNA concentration was quantified using a predefined standard curve.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Statistical analysis\u003c/h2\u003e \u003cp\u003eData are presented as mean \u0026plusmn; standard deviation (SD). Multiple quantitative data sets were compared utilizing one-way ANOVA, followed by Bonferroni correction. \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was determined as the threshold for statistical significance with n\u0026thinsp;=\u0026thinsp;3.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Temperature-responsive CUBE hydrogels\u003c/h2\u003e \u003cp\u003eTemperature-responsive hydrogels are promising candidates for injectable applications in tissue engineering, offering significant advantages over non-injectable hydrogels, including minimally invasive administration. In our previous work, we designed temperature-responsive hydrogels derived from ELPs fused with poly-aspartic acid and a coiled-coil peptide, collectively termed coiled-coil unit bound ELPs (CUBEs). In this study, we utilized O-CUBE as the foundational CUBE protein system (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). The amino sequence of O-CUBE is shown in supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. The recombinant proteins were successfully expressed in \u003cem\u003eE. coli\u003c/em\u003e KRX and purified using an inverse transition cycle (ITC). SDS-PAGE analysis revealed a single band around 50 kDa, which confirmed successful purification by ITC without requiring chromatography (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eTo evaluate gelation ability, the purified O-CUBE protein was diluted in cold PBS (5% (w/v)) and incubated at 37℃. Gelation occurred within 3 min, indicating a rapid \u003cem\u003ein situ\u003c/em\u003e gelation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). The hydrogel with \u003cem\u003ein situ\u003c/em\u003e gelation ability can be administered in liquid form and solidified upon exposure to physiological temperatures[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Furthermore, CUBE hydrogels exhibited a reversible sol-gel transition as they redissolved into their solution phase at 4℃. The reversible sol-gel transition enables easy handling of encapsulated cells prior to injection. The stimuli-responsive behavior of CUBEs is driven by the 42 repeats of the AVGVP peptide, which facilitate temperature-dependent aggregation of ELPs. The poly D chain is incorporated to control the aggregation of ELPs. Poly-aspartic acid enhances hydrophilicity and electrostatic repulsion between ELPs, tuning their phase transition behavior to more favorable solubility and aggregation. The coiled-coil interaction generates tetrameric CUBE proteins, allowing stable hydrogel formation at low concentrations (\u0026gt;\u0026thinsp;2% (w/v)).\u003c/p\u003e \u003cp\u003eThe O-CUBE protein has potential to be an injectable hydrogel due to its rapid gelation ability at physiological temperatures. Reversible gelation offers ease of injection and handling, as it can be re-liquefied if needed for modification, reloading and various other applications. In addition, the O-CUBE hydrogel can be simply purified by ITC without chromatography, making it more efficient in reducing the cost and time of production.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Injectability of O-CUBE protein\u003c/h2\u003e \u003cp\u003eGelation efficiency is a critical factor for injection of a hydrogel solution. Rapid gelation can lead to needle blockage, while slow gelation may result in leakage of the encapsulated cells. The O-CUBE protein solution was injected into pre-warmed culture medium to evaluate injectability. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea shows a representative image of gelation of the injected O-CUBE protein. O-CUBE at a concentration of 4% (w/v) exhibited an irregular shape, indicating that gelation occurred after injection. This irregular shape is advantageous as it allows the hydrogel to fill irregular cavities in the tissue. O-CUBE at 5% (w/v) showed linear gelation due to premature gelation in the needle.\u003c/p\u003e \u003cp\u003eWe assessed the optimal O-CUBE concentrations for an injectable hydrogel by dissolving the proteins in a fluorescein-containing solution and subsequently injecting into warm PBS. Release of the fluorescein compound into the PBS was directly correlated with the percentage of failed gelation after injection. The injectable gelation rate of O-CUBE improved as the concentration increased from 1\u0026ndash;4% (w/v) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). At a concentration of 1% (w/v), O-CUBE exhibited a gelation rate of 23.7 \u0026plusmn; 2.7%, while at a concentration of 4% (w/v), 84.7 \u0026plusmn; 5.1% of the O-CUBE solution was successfully gelled after injection. At 5% (w/v), O-CUBE exhibited premature gelation in the needle, making injection challenging. The ELP concentration influences the transition temperature (T\u003csub\u003et\u003c/sub\u003e) of the resultant hydrogels. The resultant hydrogels exhibit higher T\u003csub\u003et\u003c/sub\u003e at lower concentrations of ELPs, while higher concentrations result in a lower T\u003csub\u003et\u003c/sub\u003e. The slight difference between T\u003csub\u003et\u003c/sub\u003e affects the gelation rate during injection. We determined the optimal O-CUBE concentration for injectable hydrogels to be 4% (w/v), which was then used for subsequent experiments.\u003c/p\u003e \u003cp\u003eDifferent tissues or diseases, such as those involved in wound healing or characterized by a cancerous environment, exhibit different pH levels from typical physiological conditions[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. To assess the suitability of the O-CUBE hydrogel system for biomedical applications, we evaluated its injection capability across a range of pH levels. Results revealed that CUBE proteins exhibited a robust injectable gelation rate from pH 6 to 8 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). At pH 7 and 8, the 25, 50, and 100 \u0026micro;L/s injection rates did not significantly affect the gelation rate. At an injection rate of 25 \u0026micro;L/s, approximately 90% of O-CUBE was successfully gelled after injection into PBS at pH 7 and 8. However, injection rates ranging from 25 to 100 \u0026micro;L/s decreased the gelation ability from 90.7 \u0026plusmn; 3.4% to 79.4 \u0026plusmn; 3.3% at pH 6. This result indicates that the O-CUBE hydrogel was not highly dependent on pH, making it suitable for diverse applications in environments with varying pH conditions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTaken together, the O-CUBE hydrogel exhibited injectability with robust gelation when injected into pre-warmed medium. O-CUBE at 4% (w/v) was successfully injected into pre-warmed medium with a high gelation rate, and has the potential to fill irregular cavities for tissue engineering applications. In addition, the O-CUBE hydrogel showed a high degree of gelation across a pH range of 6 to 8, which can be useful for broad applications, such as drug delivery for cancer therapy and cell delivery for wound healing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Cell morphology of injected HeLa cells within O-CUBE hydrogel\u003c/h2\u003e \u003cp\u003eThree-dimensional (3D) hydrogel scaffolds offer significant benefits because they mimic the \u003cem\u003ein vivo\u003c/em\u003e environment and promote cell behaviors similar to native physiologic conditions[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. To serve as an effective cell delivery agent, hydrogels must encapsulate cells in a 3D environment and support their viability after injection. We employed HeLa cells to assess the encapsulation efficiency and distribution of cells within the O-CUBE hydrogel environment. Cells were stained with calcein AM and injected with O-CUBE hydrogel. Their distribution was evaluated using confocal microscopy. O-CUBE successfully gelled upon injection, effectively encapsulating the cells. Confocal laser microscopy further revealed that cells were distributed throughout the bottom, mid, and upper layers of the hydrogel (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). This result confirmed that the O-CUBE protein solution was successfully injected and uniformly encapsulated in cells.\u003c/p\u003e \u003cp\u003eInjectable hydrogels are a promising cell delivery agent based on their ability to retain cells at the targeted site without unintended migration. To optimize cell retention, it is important to identify the maximum number of cells that can be encapsulated within the hydrogel. We evaluated the encapsulation capacity of HeLa cells in the CUBE hydrogel and found the maximum number of HeLa cells encapsulated in O-CUBE to be\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:2.0\\times\\:{10}^{4}\\)\u003c/span\u003e\u003c/span\u003ecells per 50 \u0026micro;L (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Exceeding this cell concentration led to leakage of encapsulated cells from the hydrogel after injection.\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec shows the cell morphology of HeLa from day 1 to 5 after injection. After 5 days, the sizes of both cell types increased, indicating multicellular aggregation or spheroid formation. Spheroid formation indicates that cell-cell interactions were more dominant than cell-matrix interactions. This result is consistent with the fact that the O-CUBE hydrogel lacks any cell adhesion peptide, which increases cell-cell interactions in 3D culture. Spheroid culture in the absence of a hydrogel matrix has been studied owing to its simplicity, reproducibility, and efficiency. However, matrix-free spheroids also present challenges in mimicking the native tissue environment with specific elasticity and stiffness[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The injectable O-CUBE hydrogel system exhibited spheroid formation that can be further explored with different protein concentrations and alternative designs. The elasticity and stiffness of the CUBE hydrogels can be controlled by adjusting the CUBE concentration and number of repeating ELP sequences. The ability of O-CUBE hydrogel to provide 3D scaffold for developing physiologically relevant in tumor models, enabling more accurate studies of cancer cell behavior, metastasis, and drug response.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Cell viability and proliferation of injected HeLa cells within O-CUBE hydrogel\u003c/h2\u003e \u003cp\u003eWe utilized and HeLa cells as model cells, to assess the potential of the O-CUBE hydrogel to deliver cells via simple injection. The incorporation of HeLa cells into a 3D scaffold can provide for physiological characteristics that mimic tumor behavior, metastasis, and drug response.\u003c/p\u003e \u003cp\u003eAfter injection with specific interval times, cells were cultivated from the O-CUBE hydrogels and stained with calcein AM and PI to determine cell viability. Results showed that most of the cells survived (green), while there were only a few dead cells (red), indicating high cell viability after injection with the O-CUBE hydrogel (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). The cell viability was 96.7 \u0026plusmn; 0.5%, 89.4 \u0026plusmn; 4.2%, 74.9 \u0026plusmn; 1.2% in the O-CUBE hydrogel at 1, 2, and 3 days after injection, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). These results suggest that CUBE can be used as injectable gel with encapsulated cells, despite the cells being subjected to low temperatures during hydrogel encapsulation and rapidly transitioning to physiological temperatures during injection. The ability of HeLa cells to survive after encapsulation and injection indicates that O-CUBE is suitable for delivery living cells with minimal loss of viability.\u003c/p\u003e \u003cp\u003eTo assess cell proliferation, DNA content was measured after cells were collected from the injected O-CUBE hydrogel. HeLa cells showed a significant increase in DNA concentration, indicating high cell proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). The DNA concentration increased from 0.8 \u0026plusmn; 0.15 ng/\u0026micro;L to 4.4 \u0026plusmn; 0.1 ng/\u0026micro;L, on days 1 and 5 following injection, respectively. The high proliferation cells highlight the ability of O-CUBE hydrogel as cell carrier for therapeutic cells in regenerative medicine.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTaken together, our O-CUBE hydrogel could maintain cell viability and cell proliferation after injection, which is essential as cell carrier. With further optimization, CUBE hydrogel systems have potential for personalized medicine application, such as patient-specific cancer models for drug screening. Encapsulating cells and locally deliver into specific damaged tissue for wound healing. Moreover, their potential as co-delivery system, which both growth factor and stem cells can be locally delivered in a controlled manner.\u003c/p\u003e \u003c/div\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn summary, we evaluated the injectability and biocompatibility of a temperature-responsive O-CUBE hydrogel derived from an elastin-like polypeptide. Results demonstrated that the O-CUBE hydrogel exhibited excellent injectability via \u003cem\u003ein situ\u003c/em\u003e gelation at physiological temperature, with a high gelation rate with wide range pH environment. The O-CUBE hydrogel effectively maintained cell survival and proliferation using HeLa cells. Our results demonstrate that O-CUBE has broad potential as multifunctional biomaterial for cancer research, drug testing, and cell-based therapies.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA part of this work was financially supported by following grants; the Japan Society for the Promotion of Science (JSPS, grant number 23K17209 to Kei Nishida and 23K28427 to Masayasu Mie).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interest related to this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study conception, design, interpretation of the studies, analysis of the data, and review of the manuscript. Kei Nishida, Masayasu Mie, and Eiry Kobatake designed the research study. Mutawakil Al Muqadasi and Keitaro ii performed the research and analyzed the data. Mutawakil Al Muqadasi wrote the manuscript. Kei Nishida, Masayasu Mie, and Eiry Kobatake reviewed and edited the manuscript. All authors read and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis article contains no studies with human participants or animals performed by any of the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publish:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data supporting the finding of this study are presented in the article and its Supplementary information.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eJ. Liu, C. Du, W. Huang, and Y. 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Gao \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;Gelatin-Based Hydrogel for Three-Dimensional Neuron Culture Application,\u0026rdquo; \u003cem\u003eACS Omega\u003c/em\u003e, vol. 8, no. 48, pp. 45288\u0026ndash;45300, Dec. 2023, doi: 10.1021/acsomega.3c03769.\u003c/li\u003e\n\u003cli\u003eS. Žigon-Branc \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;Impact of Hydrogel Stiffness on Differentiation of Human Adipose-Derived Stem Cell Microspheroids,\u0026rdquo; \u003cem\u003eTissue Eng. Part A\u003c/em\u003e, vol. 25, no. 19\u0026ndash;20, pp. 1369\u0026ndash;1380, Oct. 2019, doi: 10.1089/ten.tea.2018.0237.\u003c/li\u003e\n\u003cli\u003eM. Sheth \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;Three-dimensional matrix stiffness modulates mechanosensitive and phenotypic alterations in oral squamous cell carcinoma spheroids,\u0026rdquo; \u003cem\u003eAPL Bioeng.\u003c/em\u003e, vol. 8, no. 3, p. 036106, Sep. 2024, doi: 10.1063/5.0210134.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"injectable hydrogel, temperature-responsive hydrogel, three-dimensional culture, elastin-like polypeptide","lastPublishedDoi":"10.21203/rs.3.rs-6676494/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6676494/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eInjectable hydrogels are promising biomaterials for tissue engineering applications because they deliver bioactive compounds or cells with minimal invasiveness. Temperature-responsive hydrogels, which transition from a liquid to a gel in response to environmental temperature, are candidates for injectable hydrogels. Elastin-like polypeptides (ELPs) with temperature-responsive capabilities have been well studied for drug delivery and tissue repair, yet their development as injectable biomaterials remain limited. In our previous study, we designed hydrogels formed from coiled-coil unit bound ELPs (CUBEs), which incorporated ELPs, a poly-aspartic acid (poly D) chain, a coiled-coil peptide (CL), and a functional peptide. However, injectability of CUBEs has not been studied. Here, we evaluated the injectability and cell delivery potential of O-CUBE, (AVGVP)\u003csub\u003e42\u003c/sub\u003e-D\u003csub\u003e88\u003c/sub\u003e-CL, which is a basic CUBE hydrogel system. We injected O-CUBE mixed with human cervical cancer (HeLa) cells into pre-warmed culture medium to initiate \u003cem\u003ein situ\u003c/em\u003e gelation. O-CUBE protein was successfully gelled at an approximately 90% gelation rate after injection at 37℃ in a solution with pH 6 to 8. The injected HeLa cells exhibited spheroid morphology, indicating that the hydrogel facilitated cell-cell interactions in three-dimensional culture. Further evaluation with a DNA assay showed that the HeLa cells can proliferate in the O-CUBE hydrogel. Results demonstrate that the CUBE hydrogel system is a promising candidate for an injectable hydrogel cell delivery system with minimal invasiveness.\u003c/p\u003e","manuscriptTitle":"Temperature-responsive injectable hydrogel derived from elastin-like polypeptide as a cell carrier","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-06 10:28:39","doi":"10.21203/rs.3.rs-6676494/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f41cea4f-8320-4e0f-8270-7b4a32372903","owner":[],"postedDate":"June 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-06-06T10:31:20+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-06 10:28:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6676494","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6676494","identity":"rs-6676494","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}