O2-Nanobubble of Iron-Porphyrin conjugated Polyaspartamide for Molecular Ultrasound Contrast Effect

O2-Nanobubble of Iron-Porphyrin conjugated Polyaspartamide for Molecular Ultrasound Contrast Effect | 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 O2-Nanobubble of Iron-Porphyrin conjugated Polyaspartamide for Molecular Ultrasound Contrast Effect Yoon Na Cho, Jun Woo Lim, Seung Joo Oh, Sa Ra Han, Sungwoo Cho, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4634291/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 19 Feb, 2025 Read the published version in Biotechnology Letters → Version 1 posted 5 You are reading this latest preprint version Abstract Objective This study aimed to prepare oxygen-nanobubbles incorporating ferrous porphyrin to emulate the oxygen-capturing ability of hemoglobin porphyrin in red blood cells. Results We synthesized poly(2-hydroxyethyl aspartamide) (PHEA) grafted with ferrous porphyrins (Iron-P-PHEA) and created nanobubbles using 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). These nanobubbles trapped oxygen and retained it over a 2-hour period. The O2-nanobubbles demonstrated an enhanced photoacoustic effect as an ultrasound contrast agent, as confirmed by Doppler ultrasound testing. Conclusions The innovative strategy for O2-nanobubble preparation enhances the efficiency of targeted delivery in molecular optical and ultrasound imaging. Iron-Porphyrin Nanobubbles Polyaspartamide Targeted Delivery Ultrasound Contrast Agent Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction The acoustic properties of ultrasound contrast agent microbubbles have undergone extensive investigation to enhance targeted drug delivery and molecular imaging capabilities (Tran et al. 2003; Quaia 2007; Ferrara et al. 2007; Yang et al. 2008; Yang et al. 2019). Typically sized between 1 and 10 µm, ultrasound contrast agents can efficiently navigate through capillaries (Sirsi and Borden 2009). The composition of the microbubble shell, such as lipids and albumin, is often manipulated to enhance stability (Mehta et al. 2017). Additionally, perfluorinated compounds like perfluoropropane (C3F8), sulfur hexafluoride (SF6), and perfluorocarbons (PFC) are incorporated to significantly augment the acoustic effect (Riess 2006; Schneider 1999; Suzuki et al. 2007). Despite these advancements, environmental concerns arise from the use of gases in ultrasound contrast agents that contribute to global warming, and the contrast effect is limited in the arterial vascular system (Ravishankara et al. 1993; Rafailidis et al. 2020). In response to these challenges, there is a pressing need for the development of a stable ultrasound contrast agent capable of continuously enhancing ultrasonic contrast without relying on perfluorinated gases. Therefore, we propose the design of oxygen(O 2 )-nanobubble ultrasonic contrast agents, inspired by the oxygen-capturing ability of hemoglobin in red blood cells (Jeong et al. 2012; Cho et al. 2016). We synthesized poly(2-hydroxyethyl aspartamide) (PHEA) grafted with ferrous porphyrins, forming self-assemblies in an aqueous solution (Jeong et al. 2005). Importantly, the prolonged retention of captured oxygen over a 2-hour period within the nanobubbles signifies a notable outcome. The O 2 -nanobubbles were prepared through self-assembly with ferrous-porphyrin-conjugated PHEA (Iron-P-PHEA) and lipid molecules (Fig. 1 a). Maintaining a stable spherical shape with a diameter below the micrometer scale was achieved by adjusting the degree of ferrous porphyrin substitution to 5 mol%. Furthermore, the O 2 -nanobubbles demonstrated a robust contrast effect in tissue phantoms (Fig. 2 b). In conclusion, this innovative strategy for O 2 -nanobubble preparation holds promise in enhancing the efficiency of targeted delivery for molecular optical and ultrasound imaging across various biomedical modalities. Materials and Methods Synthesis of ferrous porphyrin-conjugated Poly(2-hydroxy-ethyl aspartamide) Poly(succinimide) (PSI) was synthesized through acid-catalyzed polycondensation of L-aspartic acid (Sigma) (Jeong et al. 2005; Jeong et al. 2012). Ethanolamine (Sigma) was subsequently added to open the PSI ring and produce poly(2-hydroxy-ethyl aspartamide) (PHEA) (Yang et al. 2003). Ferrous porphyrin was then conjugated to PHEA using a DCC-mediated reaction, forming ester linkages. The substitution of Iron-Protoporphyrin IX to PHEA was carried out at room temperature for 24 hours, resulting in the synthesis of ferrous porphyrin-conjugated PHEA (Iron-P-PHEA) with varying degrees of substitution (4.8–24 mol%). The morphology of Iron-P-PHEA self-assemblies was confirmed in an aqueous solution (Table 1). Preparation and Characterization of O 2 -Microbubbles Microbubbles for ultrasound imaging were prepared by mixing 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC, Sigma) solution (10 mg/mL chloroform) with Iron-P-PHEA in PBS at a mass ratio of 5:1 using the thin film hydration method. The mixture was then evaporated to dryness at 80 ℃ using rotary evaporation, allowing DPPC to be uniformly coated. Liposomal bubbles (2.5 mg/mL DPPC) were formed by adding 4 mL PBS to the round flask, spreading the DPPC widely under an O 2 atmosphere for 1 hour. The mixture was sonicated for ten minutes using a sonicator (Powersonic 410) after one minute of shaking. The O 2 -microbubbles were characterized by size distribution and TEM images. Size measurements were obtained through DLS at 10 and 120 minutes to confirm the maintenance of O 2 . The morphology of O 2 -microbubbles at these time points was observed using transmission electron microscopy (FE-TEM, JEOL) at 120 kV. Ultrasound Imaging Using O 2 -Microbubbles A 5 wt% agar solution was dissolved at 120°C and cooled to prepare an agar gel phantom according to Fig. 4 (a). A Teflon tube with an 800 µm diameter was introduced to simulate the vascular flow system within the agar gel phantom. The O 2 -bubble solution was perfused through the Teflon tube at a flow rate of 0.04 cm/s using a syringe pump. Ultrasound imaging to evaluate the contrast effect of O 2 -bubbles was conducted using the LOGIQ9 scanner (GE Medical Systems, Milwaukee, WI, USA) with a 12 MHz linear transducer in Cheil General Hospital. The transducer was placed at the center of the 5.0% agar gel phantom with a horizontally hollow center hole. Results and Discussion First, Iron-P-PHEA was synthesized through a top-down approach, varying the degree of substitution (DS) from 4.8 to 24.0 mol%. This was carried out to ascertain the optimal DS for creating a stable self-assembly, confirming its chemical structure and morphology (Yang et al. 2003). The increment in DS of iron-porphyrins induced a structural transition, resulting in specific size distribution through inter/intra-molecular π-π stacking of iron-porphyrin. This phenomenon suggests that grafting iron-porphyrins with a macrocyclic ring to the hydrophilic backbone propels the structural transition to rod-like assemblies. This transition aligns with the micelle-to-bilayer transition observed in graft copolymer systems, as proposed by Discher and Eisenberg (Discher and Eisenberg 2002; Jeong et al. 2005). Among the Iron-P-PHEA self-assemblies, the DS 4.8 mol% self-assembly exhibited the most stable form, appearing as a sphere with a diameter of approximately 54 nm (Table 1). DPPC and Iron-P-PHEA were self-assembled in an aqueous solution under an O 2 atmosphere. Microbubbles made solely from DPPC solution were labeled as D-MB, while those made from DPPC and Iron-P-PHEA solution were labeled as IP-MB. The size of D-MB significantly increased after O 2 purging, reaching 7.5 µm, compared to the self-assemblies (Fig. 2 c). Subsequently, within the initial 60 minutes, the microbubble's size rapidly decreased from 7.5 µm to 1 µm, demonstrating a swift release of captured oxygen (Fig. 2 a). This observed phenomenon is attributed to the rapid depletion of captured oxygen within the first 60 minutes. As shown in Fig. 2 d, 120 minutes later, the TEM image revealed that D-MB exhibited a liposomal morphology without oxygen, resembling the pure liposomes. This indicates that oxygen had already been depleted, and the structure transformed from a microbubble to a liposome. In contrast, the size of IP-MBs remained nearly constant even after O 2 purging (Fig. 3 a). This stability in size, persisting up to 120 minutes, is attributed to the effective retention of oxygen by IP-MBs (Fig. 2 b). Particularly noteworthy is the burst-shaped morphology observed in TEM images of IP-MBs after 120 minutes, occurring during the drying process (Fig. 2 d). This phenomenon underscores the ability of ferrous porphyrin to retain oxygen within the IP-MB, evident as the bubble bursts when subjected to the drying process for TEM imaging. The stability of the IP-MBs demonstrates the critical role of ferrous porphyrin in enhancing oxygen retention within microbubbles, a feature that could significantly improve the efficacy of ultrasound contrast agents. This enhanced stability and oxygen retention suggest potential applications in prolonged imaging sessions and more precise targeting in therapeutic delivery, marking a notable advancement in the design of ultrasound contrast agents. The contrast agent's effectiveness was validated through ultrasound Doppler imaging in an in vitro test (Jeong et al. 2012; Shen et al. 2007). The combination of lipid and Iron-P-PHEA induced a strong contrast effect in an agar gel tissue phantom emulating capillaries with adjustable vessel size and flow rate (Fig. 4 a). At 18 ml/h, Doppler ultrasound imaging of D-NB did not exhibit any acoustic flow (Fig. 4 b- 2 ), while P-NB displayed a significant acoustic enhancement (Fig. 4 b- 3 ). Therefore, the O 2 -nanobubbles containing ferrous porphyrin, capable of combining with oxygen, hold promise in enhancing the efficiency of targeted delivery for optical and ultrasound imaging. The results demonstrate that the degree of substitution of iron-porphyrins significantly influences the structural properties and stability of the resulting microbubbles. The DS 4.8 mol% exhibited optimal stability, forming spherical structures that retained oxygen effectively over time. This ability to maintain structural integrity and retain oxygen suggests that Iron-P-PHEA microbubbles are promising candidates for ultrasound contrast agents. Moreover, the enhanced acoustic contrast observed in the Doppler ultrasound imaging confirms their potential application in biomedical imaging, particularly in targeted delivery systems. The study highlights the importance of controlling the DS in the design of functionalized nanomaterials for medical applications. Conclusion In conclusion, our study successfully crafted oxygen-nanobubbles with ferrous porphyrin, mimicking the oxygen-capturing ability of red blood cell hemoglobin. We precisely adjusted ferrous porphyrin substitution to control the hydrophobic segment of amphiphilic copolymers, enabling stable nanobubble formation with efficient oxygen uptake. Utilizing DPPC liposomes, we created liposomal bubbles, resembling biomembrane structures. The nanobubbles, showcasing stable size and spherical form, exhibited a remarkable acoustic effect, as evidenced by Doppler ultrasound testing. This innovative O 2 -nanobubble strategy holds substantial promise for enhancing targeted delivery efficiency in molecular optical and ultrasound imaging across various biomedical applications. Declarations Acknowledgments This work was supported by the National Research Foundation of Korea (RS-2024-00358960), the Ministry of Education (NRF–2020R1A6A1A03044977) and the Ministry of SMEs and Startups (RS-2023-00256750). References Brentrup L, Onken U, Measurement of bubble size distribution in fermentors, Biotechnology Letters, 1 (1979) 427-432. Creighton T. E., Proteins; structure and molecular principles, W. H. Freeman, New York, 1983. Jeong J. H., Kang H. S., Yang S. R., Park K., Kim J. D., Biodegradable Poly(asparagine) grafted with Poly(caprolactone) and the Effect of Substitution on Self-aggregation, Colloids Surf. A, 264, (2005) 187-194. Jeong J. H., Cha C., Kaczmarowski A., Haan J., Oh S., Kong H. J., Polyaspartamide Vesicles Induced by Metallic Nanoparticles, Soft Matter, 8 (2012) 2237-2242. Cho Y. N., Kim H. J., Cho S. W., Shin S. G., Jeong J. H., Biomimetic self-assembly of porphyrin-conjugated polyaspartamide in aqueous solution, Polymer(Korea), 40 (2016) 163-166. Mehta K. S., Lee J. J., Taha A. A., Avgerinos E., Chaer R. A., Vascular applications of contrast-enhanced ultrasound imaging, Journal of Vascular Surgery, 66(1), (2017) 266-274. Quaia E., Microbubble ultrasound contrast agents: an update, European Radiology, 17(8) (2007) 1995-2008. Ravishankara A. R., Solomon S., Turnipseed A. A., Warren R. F., Atmospheric lifetimes of long-lived halogenated species, Science, 259(5092), (1993) 194-199. Rafailidis V., Huang D. Y., Yusuf G. T., Sidhu P. S., General principles and overview of vascular contrast-enhanced ultrasonography, Ultrasonography, 39(1), (2020) 22-42. Tran B. C., Seo J., Hall T. L., Fowlkes J. B., Cain C. A., Microbubble-enhanced cavitation for noninvasive ultrasound surgery, IEEE Transactions on Ultrasonics, Ferroelectrics, and Frequency Control, 50(10) (2003) 1296-1304. Sirsi S. R., Borden M. A., Microbubble compositions, properties and biomedical applications, Bubble Science, Engineering & Technology 1(1-2) (2009) 3-17. Ferrara K., Rachel P., Mark B., Ultrasound microbubble contrast agents: fundamentals and application to gene and drug delivery, Annual Review of Biomedical Engineering 9 (2007) 415-447. Schneider M., SonoVue, a new ultrasound contrast agent, European Radiology, 9(3) (1999) S347-S348. Yang X., Wang C., Ding M., Zhang C., Wang W., Cao Z., Indocyanine green (ICG)–Menthol loaded cerasomal nanoparticles for ultrasound imaging and photothermal therapy against tumor, Materials Letters, 255, (2019) 126524. Yang F., Gu A., Chen Z., Gu N., Ji M., Multiple emulsion microbubbles for ultrasound imaging, Materials Letters, 62(1) (2008) 121-124. Riess J. G., Perfluorocarbon-based oxygen delivery, Artificial Cells, Blood Substitutes, and Biotechnology, 34(6) (2006) 567-580. Table Table 1 is available in the Supplementary Files section. Supplementary Files Table1.png Cite Share Download PDF Status: Published Journal Publication published 19 Feb, 2025 Read the published version in Biotechnology Letters → Version 1 posted Editorial decision: Major revisions 02 Jan, 2025 Reviewers agreed at journal 02 Sep, 2024 Reviewers invited by journal 02 Sep, 2024 Editor assigned by journal 18 Aug, 2024 First submitted to journal 17 Aug, 2024 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-4634291","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":348364300,"identity":"b7265cfd-7c45-4c5b-ad39-cae7ea6fb6d3","order_by":0,"name":"Yoon Na Cho","email":"","orcid":"","institution":"Soongsil University","correspondingAuthor":false,"prefix":"","firstName":"Yoon","middleName":"Na","lastName":"Cho","suffix":""},{"id":348364301,"identity":"e3fb3f16-ba91-4be6-be57-577b5cb0b91d","order_by":1,"name":"Jun Woo Lim","email":"","orcid":"","institution":"Soongsil University","correspondingAuthor":false,"prefix":"","firstName":"Jun","middleName":"Woo","lastName":"Lim","suffix":""},{"id":348364302,"identity":"53c70ff5-4d2f-4682-84ae-c49e147bf9e5","order_by":2,"name":"Seung Joo Oh","email":"","orcid":"","institution":"Soongsil University","correspondingAuthor":false,"prefix":"","firstName":"Seung","middleName":"Joo","lastName":"Oh","suffix":""},{"id":348364303,"identity":"e46567b3-8f17-4984-b4d9-2b6f3c4b1f4e","order_by":3,"name":"Sa Ra Han","email":"","orcid":"","institution":"Soongsil University","correspondingAuthor":false,"prefix":"","firstName":"Sa","middleName":"Ra","lastName":"Han","suffix":""},{"id":348364304,"identity":"dc5b9b5e-6775-4929-8f16-6eca66fb5cbf","order_by":4,"name":"Sungwoo Cho","email":"","orcid":"","institution":"Soongsil University","correspondingAuthor":false,"prefix":"","firstName":"Sungwoo","middleName":"","lastName":"Cho","suffix":""},{"id":348364305,"identity":"2717b31f-445b-465c-9dcd-fd91e2e47fcf","order_by":5,"name":"Jimin Jeong","email":"","orcid":"","institution":"Soongsil University","correspondingAuthor":false,"prefix":"","firstName":"Jimin","middleName":"","lastName":"Jeong","suffix":""},{"id":348364306,"identity":"9ad9b80e-c9f0-41f7-a44e-bbc9201e3c12","order_by":6,"name":"Byoung Hee Han","email":"","orcid":"","institution":"Gangneung Asan Hospital","correspondingAuthor":false,"prefix":"","firstName":"Byoung","middleName":"Hee","lastName":"Han","suffix":""},{"id":348364307,"identity":"0782d187-7f80-4a4b-915e-394f760ea04a","order_by":7,"name":"Jae Hyun Jeong","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxUlEQVRIiWNgGAWjYDAC5gMMBpINNjBuAhFa2BJAWtJI1MLA2HCYBC3mbMwHCix3nLeXn5HA+OEHQ1o+QS2WbWwJBpJnbiduuJHALNnDkGPZQEiLwf0eAwPJttsJBhIJDNIMDBUGBG0xOMb/AajlHMhhzL+J1MIDDLG2A4wNNxLYgLbkEKOFDeSw5MQNZx62WfYYpBGjhfmZsWSbnb18e/LhGz8qkglrAQI2YwkwzdgANIEYDcAk8/ADcQpHwSgYBaNgpAIAbXs3RK7lXzoAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-4263-408X","institution":"Soongsil University","correspondingAuthor":true,"prefix":"","firstName":"Jae","middleName":"Hyun","lastName":"Jeong","suffix":""}],"badges":[],"createdAt":"2024-06-25 07:19:50","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4634291/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4634291/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10529-025-03571-x","type":"published","date":"2025-02-19T15:57:11+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":66945240,"identity":"77e4d61f-77fc-4f2c-8cad-f3f035669b0e","added_by":"auto","created_at":"2024-10-18 09:38:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1094395,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Preparation of micro and nanobubbles: D-MB with only DPPC, and IP-MB with DPPC and ferrous porphyrin-conjugated PHEA for ultrasound contrast agents. (b) Schematic diagram of color flow Doppler mapping using O\u003csub\u003e2\u003c/sub\u003e-nanobubbles (IP-MB) in ultrasound imaging.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4634291/v1/a6ab60f3bde0801b8ead7129.png"},{"id":66945239,"identity":"d8b40c83-98a9-4a84-af7b-1f38b4ac3196","added_by":"auto","created_at":"2024-10-18 09:38:45","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1265603,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Size distribution of DPPC self-assemblies and DPPC microbubble (D-MB) after O\u003csub\u003e2\u003c/sub\u003e purging at 10 and 120 min. (b) Size variation of O2-microbubble with D-MB over time. (c) Optical bright-field image of D-MB immediately after O\u003csub\u003e2\u003c/sub\u003e purging in DPPC self-assemblies. (d) TEM images of self-assemblies of D-MB in aqueous solution, taken 120 min post-purging.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4634291/v1/7980ba94f2bec7c15f5de41a.png"},{"id":66945222,"identity":"16b924e6-3992-4d2e-bcdb-401ac965351f","added_by":"auto","created_at":"2024-10-18 09:38:43","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1168645,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Size distribution of DPPC self-assemblies and Iron-porphyrin microbubble (IP-MB) after O\u003csub\u003e2\u003c/sub\u003e purging at 10 and 120 min. (b) Size variation of O\u003csub\u003e2\u003c/sub\u003e-nanobubble with IP-MB over time. (c) Optical bright-field image of IP-MB immediately after O\u003csub\u003e2\u003c/sub\u003e purging in DPPC self-assemblies. (d) TEM images of self-assemblies of IP-MB in aqueous solution, taken 120 min post-purging.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4634291/v1/3d01d8d0aeaed18fc34979ca.png"},{"id":66945262,"identity":"71fa5402-94fe-49b6-937c-737e0265d4fa","added_by":"auto","created_at":"2024-10-18 09:38:47","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1254765,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Schematic representation of tissue-mimicking phantom for ultrasound imaging using agar and a Teflon tube with adjustable vessel size and flow rate. (b) Doppler ultrasound images of the blood vessel mimic with an inner diameter of 800 μm: (b-1) before microbubble injection, (b-2) after PBS injection (D-NB), and (b-3) after P-NB injection using the LOGIQ 9 ultrasound imaging system (GE Medical Systems, Milwaukee, WI, USA) with a 12 MHz linear transducer. (c) Velocity measurement of the solution with O\u003csub\u003e2\u003c/sub\u003e nanobubbles in the tissue-mimicking phantom at the point (x=0 ~ -3 cm).\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-4634291/v1/2cd530a02ddea39924ac88a5.png"},{"id":77052515,"identity":"5586987a-6b32-42a9-a3f1-4025c142c3f8","added_by":"auto","created_at":"2025-02-24 16:13:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6868033,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4634291/v1/5b5d343a-2fee-40ea-a33e-73584b31626b.pdf"},{"id":66945235,"identity":"969c9ed1-a5d0-426d-a4fa-d01eb2469af8","added_by":"auto","created_at":"2024-10-18 09:38:44","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":233740,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.png","url":"https://assets-eu.researchsquare.com/files/rs-4634291/v1/2fb3b92c378b875bfd9dd4e4.png"}],"financialInterests":"","formattedTitle":"O2-Nanobubble of Iron-Porphyrin conjugated Polyaspartamide for Molecular Ultrasound Contrast Effect","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe acoustic properties of ultrasound contrast agent microbubbles have undergone extensive investigation to enhance targeted drug delivery and molecular imaging capabilities (Tran et al. 2003; Quaia 2007; Ferrara et al. 2007; Yang et al. 2008; Yang et al. 2019). Typically sized between 1 and 10 \u0026micro;m, ultrasound contrast agents can efficiently navigate through capillaries (Sirsi and Borden 2009). The composition of the microbubble shell, such as lipids and albumin, is often manipulated to enhance stability (Mehta et al. 2017). Additionally, perfluorinated compounds like perfluoropropane (C3F8), sulfur hexafluoride (SF6), and perfluorocarbons (PFC) are incorporated to significantly augment the acoustic effect (Riess 2006; Schneider 1999; Suzuki et al. 2007). Despite these advancements, environmental concerns arise from the use of gases in ultrasound contrast agents that contribute to global warming, and the contrast effect is limited in the arterial vascular system (Ravishankara et al. 1993; Rafailidis et al. 2020).\u003c/p\u003e \u003cp\u003eIn response to these challenges, there is a pressing need for the development of a stable ultrasound contrast agent capable of continuously enhancing ultrasonic contrast without relying on perfluorinated gases. Therefore, we propose the design of oxygen(O\u003csub\u003e2\u003c/sub\u003e)-nanobubble ultrasonic contrast agents, inspired by the oxygen-capturing ability of hemoglobin in red blood cells (Jeong et al. 2012; Cho et al. 2016). We synthesized poly(2-hydroxyethyl aspartamide) (PHEA) grafted with ferrous porphyrins, forming self-assemblies in an aqueous solution (Jeong et al. 2005). Importantly, the prolonged retention of captured oxygen over a 2-hour period within the nanobubbles signifies a notable outcome. The O\u003csub\u003e2\u003c/sub\u003e-nanobubbles were prepared through self-assembly with ferrous-porphyrin-conjugated PHEA (Iron-P-PHEA) and lipid molecules (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Maintaining a stable spherical shape with a diameter below the micrometer scale was achieved by adjusting the degree of ferrous porphyrin substitution to 5 mol%. Furthermore, the O\u003csub\u003e2\u003c/sub\u003e-nanobubbles demonstrated a robust contrast effect in tissue phantoms (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). In conclusion, this innovative strategy for O\u003csub\u003e2\u003c/sub\u003e-nanobubble preparation holds promise in enhancing the efficiency of targeted delivery for molecular optical and ultrasound imaging across various biomedical modalities.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSynthesis of ferrous porphyrin-conjugated Poly(2-hydroxy-ethyl aspartamide)\u003c/h2\u003e \u003cp\u003ePoly(succinimide) (PSI) was synthesized through acid-catalyzed polycondensation of L-aspartic acid (Sigma) (Jeong et al. 2005; Jeong et al. 2012). Ethanolamine (Sigma) was subsequently added to open the PSI ring and produce poly(2-hydroxy-ethyl aspartamide) (PHEA) (Yang et al. 2003). Ferrous porphyrin was then conjugated to PHEA using a DCC-mediated reaction, forming ester linkages. The substitution of Iron-Protoporphyrin IX to PHEA was carried out at room temperature for 24 hours, resulting in the synthesis of ferrous porphyrin-conjugated PHEA (Iron-P-PHEA) with varying degrees of substitution (4.8\u0026ndash;24 mol%). The morphology of Iron-P-PHEA self-assemblies was confirmed in an aqueous solution (Table\u0026nbsp;1).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePreparation and Characterization of O\u003csub\u003e2\u003c/sub\u003e-Microbubbles\u003c/h2\u003e \u003cp\u003eMicrobubbles for ultrasound imaging were prepared by mixing 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC, Sigma) solution (10 mg/mL chloroform) with Iron-P-PHEA in PBS at a mass ratio of 5:1 using the thin film hydration method. The mixture was then evaporated to dryness at 80 ℃ using rotary evaporation, allowing DPPC to be uniformly coated. Liposomal bubbles (2.5 mg/mL DPPC) were formed by adding 4 mL PBS to the round flask, spreading the DPPC widely under an O\u003csub\u003e2\u003c/sub\u003e atmosphere for 1 hour. The mixture was sonicated for ten minutes using a sonicator (Powersonic 410) after one minute of shaking. The O\u003csub\u003e2\u003c/sub\u003e-microbubbles were characterized by size distribution and TEM images. Size measurements were obtained through DLS at 10 and 120 minutes to confirm the maintenance of O\u003csub\u003e2\u003c/sub\u003e. The morphology of O\u003csub\u003e2\u003c/sub\u003e-microbubbles at these time points was observed using transmission electron microscopy (FE-TEM, JEOL) at 120 kV.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eUltrasound Imaging Using O\u003csub\u003e2\u003c/sub\u003e-Microbubbles\u003c/h2\u003e \u003cp\u003eA 5 wt% agar solution was dissolved at 120\u0026deg;C and cooled to prepare an agar gel phantom according to Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e(a). A Teflon tube with an 800 \u0026micro;m diameter was introduced to simulate the vascular flow system within the agar gel phantom. The O\u003csub\u003e2\u003c/sub\u003e-bubble solution was perfused through the Teflon tube at a flow rate of 0.04 cm/s using a syringe pump. Ultrasound imaging to evaluate the contrast effect of O\u003csub\u003e2\u003c/sub\u003e-bubbles was conducted using the LOGIQ9 scanner (GE Medical Systems, Milwaukee, WI, USA) with a 12 MHz linear transducer in Cheil General Hospital. The transducer was placed at the center of the 5.0% agar gel phantom with a horizontally hollow center hole.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003eFirst, Iron-P-PHEA was synthesized through a top-down approach, varying the degree of substitution (DS) from 4.8 to 24.0 mol%. This was carried out to ascertain the optimal DS for creating a stable self-assembly, confirming its chemical structure and morphology (Yang et al. 2003). The increment in DS of iron-porphyrins induced a structural transition, resulting in specific size distribution through inter/intra-molecular π-π stacking of iron-porphyrin. This phenomenon suggests that grafting iron-porphyrins with a macrocyclic ring to the hydrophilic backbone propels the structural transition to rod-like assemblies. This transition aligns with the micelle-to-bilayer transition observed in graft copolymer systems, as proposed by Discher and Eisenberg (Discher and Eisenberg 2002; Jeong et al. 2005). Among the Iron-P-PHEA self-assemblies, the DS 4.8 mol% self-assembly exhibited the most stable form, appearing as a sphere with a diameter of approximately 54 nm (Table\u0026nbsp;1).\u003c/p\u003e \u003cp\u003eDPPC and Iron-P-PHEA were self-assembled in an aqueous solution under an O\u003csub\u003e2\u003c/sub\u003e atmosphere. Microbubbles made solely from DPPC solution were labeled as D-MB, while those made from DPPC and Iron-P-PHEA solution were labeled as IP-MB. The size of D-MB significantly increased after O\u003csub\u003e2\u003c/sub\u003e purging, reaching 7.5 \u0026micro;m, compared to the self-assemblies (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Subsequently, within the initial 60 minutes, the microbubble's size rapidly decreased from 7.5 \u0026micro;m to 1 \u0026micro;m, demonstrating a swift release of captured oxygen (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). This observed phenomenon is attributed to the rapid depletion of captured oxygen within the first 60 minutes. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed, 120 minutes later, the TEM image revealed that D-MB exhibited a liposomal morphology without oxygen, resembling the pure liposomes. This indicates that oxygen had already been depleted, and the structure transformed from a microbubble to a liposome. In contrast, the size of IP-MBs remained nearly constant even after O\u003csub\u003e2\u003c/sub\u003e purging (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). This stability in size, persisting up to 120 minutes, is attributed to the effective retention of oxygen by IP-MBs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Particularly noteworthy is the burst-shaped morphology observed in TEM images of IP-MBs after 120 minutes, occurring during the drying process (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). This phenomenon underscores the ability of ferrous porphyrin to retain oxygen within the IP-MB, evident as the bubble bursts when subjected to the drying process for TEM imaging. The stability of the IP-MBs demonstrates the critical role of ferrous porphyrin in enhancing oxygen retention within microbubbles, a feature that could significantly improve the efficacy of ultrasound contrast agents. This enhanced stability and oxygen retention suggest potential applications in prolonged imaging sessions and more precise targeting in therapeutic delivery, marking a notable advancement in the design of ultrasound contrast agents.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe contrast agent's effectiveness was validated through ultrasound Doppler imaging in an in vitro test (Jeong et al. 2012; Shen et al. 2007). The combination of lipid and Iron-P-PHEA induced a strong contrast effect in an agar gel tissue phantom emulating capillaries with adjustable vessel size and flow rate (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). At 18 ml/h, Doppler ultrasound imaging of D-NB did not exhibit any acoustic flow (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eb-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), while P-NB displayed a significant acoustic enhancement (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eb-\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Therefore, the O\u003csub\u003e2\u003c/sub\u003e-nanobubbles containing ferrous porphyrin, capable of combining with oxygen, hold promise in enhancing the efficiency of targeted delivery for optical and ultrasound imaging. The results demonstrate that the degree of substitution of iron-porphyrins significantly influences the structural properties and stability of the resulting microbubbles. The DS 4.8 mol% exhibited optimal stability, forming spherical structures that retained oxygen effectively over time. This ability to maintain structural integrity and retain oxygen suggests that Iron-P-PHEA microbubbles are promising candidates for ultrasound contrast agents. Moreover, the enhanced acoustic contrast observed in the Doppler ultrasound imaging confirms their potential application in biomedical imaging, particularly in targeted delivery systems. The study highlights the importance of controlling the DS in the design of functionalized nanomaterials for medical applications.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, our study successfully crafted oxygen-nanobubbles with ferrous porphyrin, mimicking the oxygen-capturing ability of red blood cell hemoglobin. We precisely adjusted ferrous porphyrin substitution to control the hydrophobic segment of amphiphilic copolymers, enabling stable nanobubble formation with efficient oxygen uptake. Utilizing DPPC liposomes, we created liposomal bubbles, resembling biomembrane structures. The nanobubbles, showcasing stable size and spherical form, exhibited a remarkable acoustic effect, as evidenced by Doppler ultrasound testing. This innovative O\u003csub\u003e2\u003c/sub\u003e-nanobubble strategy holds substantial promise for enhancing targeted delivery efficiency in molecular optical and ultrasound imaging across various biomedical applications.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Research Foundation of Korea (RS-2024-00358960), the Ministry of Education (NRF\u0026ndash;2020R1A6A1A03044977) and the Ministry of SMEs and Startups (RS-2023-00256750).\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eBrentrup L, Onken U, Measurement of bubble size distribution in fermentors, Biotechnology Letters, 1 (1979) 427-432.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eCreighton T. E., Proteins; structure and molecular principles, W. H. Freeman, New York, 1983.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eJeong J. H., Kang H. S., Yang S. R., Park K., Kim J. D., Biodegradable Poly(asparagine) grafted with Poly(caprolactone) and the Effect of Substitution on Self-aggregation, Colloids Surf. A, 264, (2005) 187-194.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eJeong J. H., Cha C., Kaczmarowski A., Haan J., Oh S., Kong H. J., Polyaspartamide Vesicles Induced by Metallic Nanoparticles, Soft Matter, 8 (2012) 2237-2242.\u003c/li\u003e\n \u003cli\u003eCho Y. N., Kim H. J., Cho S. W., Shin S. G., Jeong J. H., Biomimetic self-assembly of porphyrin-conjugated polyaspartamide in aqueous solution, Polymer(Korea), 40 (2016) 163-166.\u003c/li\u003e\n \u003cli\u003eMehta K. S., Lee J. J., Taha A. A., Avgerinos E., Chaer R. A., Vascular applications of contrast-enhanced ultrasound imaging, Journal of Vascular Surgery, 66(1), (2017) 266-274.\u003c/li\u003e\n \u003cli\u003eQuaia E., Microbubble ultrasound contrast agents: an update, European Radiology, 17(8) (2007) 1995-2008.\u003c/li\u003e\n \u003cli\u003eRavishankara A. R., Solomon S., Turnipseed A. A., Warren R. F., Atmospheric lifetimes of long-lived halogenated species, Science, 259(5092), (1993) 194-199.\u003c/li\u003e\n \u003cli\u003eRafailidis V., Huang D. Y., Yusuf G. T., Sidhu P. S., General principles and overview of vascular contrast-enhanced ultrasonography, Ultrasonography, 39(1), (2020) 22-42.\u003c/li\u003e\n \u003cli\u003eTran B. C., Seo J., Hall T. L., Fowlkes J. B., Cain C. A., Microbubble-enhanced cavitation for noninvasive ultrasound surgery, IEEE Transactions on Ultrasonics, Ferroelectrics, and Frequency Control, 50(10) (2003) 1296-1304.\u003c/li\u003e\n \u003cli\u003eSirsi S. R., Borden M. A., Microbubble compositions, properties and biomedical applications, Bubble Science, Engineering \u0026amp; Technology 1(1-2) (2009) 3-17.\u003c/li\u003e\n \u003cli\u003eFerrara K., Rachel P., Mark B., Ultrasound microbubble contrast agents: fundamentals and application to gene and drug delivery, Annual Review of Biomedical Engineering 9 (2007) 415-447.\u003c/li\u003e\n \u003cli\u003eSchneider M., SonoVue, a new ultrasound contrast agent, European Radiology, 9(3) (1999) S347-S348.\u003c/li\u003e\n \u003cli\u003eYang X., Wang C., Ding M., Zhang C., Wang W., Cao Z., Indocyanine green (ICG)\u0026ndash;Menthol loaded cerasomal nanoparticles for ultrasound imaging and photothermal therapy against tumor, Materials Letters, 255, (2019) 126524.\u003c/li\u003e\n \u003cli\u003eYang F., Gu A., Chen Z., Gu N., Ji M., Multiple emulsion microbubbles for ultrasound imaging, Materials Letters, 62(1) (2008) 121-124.\u003c/li\u003e\n \u003cli\u003eRiess J. G., Perfluorocarbon-based oxygen delivery, Artificial Cells, Blood Substitutes, and Biotechnology, 34(6) (2006) 567-580.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"biotechnology-letters","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bile","sideBox":"Learn more about [Biotechnology Letters](https://www.springer.com/journal/10529)","snPcode":"10529","submissionUrl":"https://submission.nature.com/new-submission/10529/3","title":"Biotechnology Letters","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Iron-Porphyrin, Nanobubbles, Polyaspartamide, Targeted Delivery, Ultrasound Contrast Agent","lastPublishedDoi":"10.21203/rs.3.rs-4634291/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4634291/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjective\u003c/h2\u003e \u003cp\u003eThis study aimed to prepare oxygen-nanobubbles incorporating ferrous porphyrin to emulate the oxygen-capturing ability of hemoglobin porphyrin in red blood cells.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eWe synthesized poly(2-hydroxyethyl aspartamide) (PHEA) grafted with ferrous porphyrins (Iron-P-PHEA) and created nanobubbles using 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). These nanobubbles trapped oxygen and retained it over a 2-hour period. The O2-nanobubbles demonstrated an enhanced photoacoustic effect as an ultrasound contrast agent, as confirmed by Doppler ultrasound testing.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThe innovative strategy for O2-nanobubble preparation enhances the efficiency of targeted delivery in molecular optical and ultrasound imaging.\u003c/p\u003e","manuscriptTitle":"O2-Nanobubble of Iron-Porphyrin conjugated Polyaspartamide for Molecular Ultrasound Contrast Effect","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-18 09:38:12","doi":"10.21203/rs.3.rs-4634291/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revisions","date":"2025-01-02T12:36:04+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-09-02T12:13:14+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-09-02T11:23:54+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-08-18T06:55:56+00:00","index":"","fulltext":""},{"type":"submitted","content":"Biotechnology Letters","date":"2024-08-17T08:35:44+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"biotechnology-letters","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bile","sideBox":"Learn more about [Biotechnology Letters](https://www.springer.com/journal/10529)","snPcode":"10529","submissionUrl":"https://submission.nature.com/new-submission/10529/3","title":"Biotechnology Letters","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"132a25d4-7be2-4c33-ab82-1ecf165aa820","owner":[],"postedDate":"October 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-02-24T16:00:10+00:00","versionOfRecord":{"articleIdentity":"rs-4634291","link":"https://doi.org/10.1007/s10529-025-03571-x","journal":{"identity":"biotechnology-letters","isVorOnly":false,"title":"Biotechnology Letters"},"publishedOn":"2025-02-19 15:57:11","publishedOnDateReadable":"February 19th, 2025"},"versionCreatedAt":"2024-10-18 09:38:12","video":"","vorDoi":"10.1007/s10529-025-03571-x","vorDoiUrl":"https://doi.org/10.1007/s10529-025-03571-x","workflowStages":[]},"version":"v1","identity":"rs-4634291","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4634291","identity":"rs-4634291","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}