Liposome Photosensitizer with Enzyme from Black Soybean Tempeh: Formula Optimization and In Vitro Thrombolytic Activity Evaluation
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Keywords:
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Black Soybean Tempeh, Liposome, Photosensitizer, Photothermal Therapy, Thrombolytic
Abstract
Stroke and myocardial infarction contributed significantly as the leading causes of global mortality rate, both commonly caused by thrombosis. Black soybean tempeh (BSBT), a traditional Indonesian food fermented with Rhizopus oligosporus fungus is rich in proteolytic enzymes, with potential to be utilized for thrombosis related ailments. Herein, we report the first findings of BSBT enzymatic activity and its subsequent formulation into liposomal system as a thrombolytic. Additionally, we incorporated photosensitizer dyes into the liposomes, phycocyanin and fluoresecein, creating a photothermally active therapeutic delivery system. Liposomes containing BSBTwere formulated using soy lecithin and tween 80, whichwere then subjected to evaluations including size, PDI, zeta potential, morphological, and stability studies. Furthermore, we observed their photothermal efficiency and thrombolytic activity using whole blood clot in vitro model. BSBT crude and purified extract produced satisfactory enzymatic activity, stable at neutral pH (∼7) and maintained stable activity at temperatures of ∼60◦C. Liposome formulation was spherical with a particle size of 607.8 nm; PDI of 0.339; and zeta potential of -24.2 mV. BSBT crude extract and purified enzyme at a concentration of 100% gave 51.28 and 56.05% thrombolytic activity. Based on the test results obtained, the optimum formula of photosensitizer liposomes produced had high encapsulation efficiency, with photothermal efficiency of 57.66 and 44.23% for Lip-Flu and Lip-Phy respectively. The formulations with laser exposure generated good thrombolytic activity (∼55-56%) comparable with nattokinase. Based on these findings, liposomal delivery of BSBT enzymes can maintain proteolytic activity, providing the first insights for thrombolytic purposes of BSBT enzymes.
References
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Levine, R. M., T. R. Pearce, M. Adil, and E. Kokkoli (2013). Preparation and Characterization of Liposome-Encapsulated Plasmid DNA for Gene Delivery. Langmuir, 29(29); 9208–9215
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Lu, Y. J., T. S. Anilkumar, C. C. Chuang, and J. P. Chen (2021). Liposomal IR-780 as a Highly Stable Nanotheranostic Agent for Improved Photothermal/Photodynamic Therapy of Brain Tumors by Convection-Enhanced Delivery. Cancers, 13(15); 3690
Mardiyanto, M., R. Mohadi, N. A. Fithri, G. Kurniawan, et al. (2024). Optimization of Nanoemulsion Formula Containing Erythromycin with VCO and Varying Concentrations of Tween-80 and PEG-400. Science and Technology Indonesia, 9(3); 697–709
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Mcleod, A. M. J., S. A. E. Barwell, T. Holyoak, and R. E. Thorne (2024). A Structural Perspective on the Temperature-Dependent Activity of Enzymes. bioRxiv, 8; 1–31
Nadea, N. S. W. P., A. Indrayati, and F. Leviana (2023). Potensi Ekstrak Kasar Enzim dari Tempe Kedelai Hitam (Glycine soja (L.) Merr.) sebagai Obat Fibrinolitik Alami dengan Metode Clot Lysis In Vitro. Jurnal Sains Dan Kesehatan, 5(2); 115–125
Ortel, T. L., I. Neumann, W. Ageno, R. Beyth, N. P. Clark, A. Cuker, et al. (2020). American Society of Hematology 2020 Guidelines for Management of Venous Thromboembolism: Treatment of Deep Vein Thrombosis and Pulmonary Embolism. Blood Advances, 4(19); 4693–4738
Pandey, A., S. Negi, and C. R. Soccol (2016). Current Developments in Biotechnology and Bioengineering: Production, Isolation and Purification of Industrial Products. Elsevier, Amsterdam, Netherlands
Polat, E. and K. Kang (2021). Natural Photosensitizers in Antimicrobial Photodynamic Therapy. Biomedicines, 9(6); 584
Psimadas, D., P. Georgoulias, V. Valotassiou, and G. Loudos (2012). Molecular Nanomedicine Towards Cancer. Journal of Pharmaceutical Sciences, 101(7); 2271–2280
Sahoo, S. K. and S. Sahoo (2020). Exploitation of Fibrinolytic Enzymes in Combating Blood Clotting Disorders – Recent Advances and Strategies: A Comprehensive Review. In J. Patra, A. Shukla, and G. Das, editors, Advances in Pharmaceutical Biotechnology. Springer, Singapore, pages 383–395
Sasmita, I. R. A., A. Sutrisno, E. Zubaidah, and A. K. Wardani (2018). Purification and Characterization of a Fibrinolytic Enzyme from Tempeh Bongkrek as an Alternative of Thrombolytic Agents. In IOP Conference Series: Earth and Environmental Science, volume 131
Ta, H. T., N. P. Truong, A. K. Whittaker, T. P. Davis, and K. Peter (2018). The Effects of Particle Size, Shape, Density and Flow Characteristics on Particle Margination to Vascular Walls in Cardiovascular Diseases. Expert Opinion on Drug Delivery, 15(1); 33–45
Tortajada, A., G. L. Righetti, A. McGinley, M. Mu, M. García-Melchor, and E. Hevia (2024). Alkene Isomerisation Catalysed by a Superbasic Sodium Amide. Angewandte Chemie International Edition, 136(38); e202407262
Tran, H. D. N., S. S. Moonshi, Z. P. Xu, and H. T. Ta (2022). Influence of Nanoparticles on the Haemostatic Balance: Between Thrombosis and Haemorrhage. Biomaterials Science, 10(1); 10–50
Tsengam, I. K. M., M. Omarova, E. G. Kelley, A. McCormick, G. D. Bothun, S. R. Raghavan, et al. (2022). Transformation of Lipid Vesicles into Micelles by Adding Nonionic Surfactants: Elucidating the Structural Pathway and the Intermediate Structures. Journal of Physical Chemistry B, 126(11); 2208–2216
Wang, K., Y. Gao, J. Zhao, Y. Wu, J. Sun, G. Niu, F. Zuo, and X. Zheng (2022). Effects of in Vitro Digestion on Protein Degradation, Phenolic Compound Release, and Bioactivity of Black Bean Tempeh. Frontiers in Nutrition, 9; 1017765
Weber, G., B. Buhren, H. Schrumpf, J. Wohlrab, and P. Gerber (2019). Clinical Applications of Hyaluronidase. In N. Labrou, editor, Therapeutic Enzymes: Function and Clinical Implications. Advances in Experimental Medicine and Biology. Springer, Singapore, pages 255–277
Yamada, K., A. Kawanabe, S. Yoshizawa, K. Inoue, K. Kogure, and H. Kandori (2012). Anomalous pH Effect of Blue Proteorhodopsin. Journal of Physical Chemistry Letters, 3(7); 800–804
Yu, Y., Y. Guan, H. Wen, Y. Zhang, J. Liu, and T. Zhang (2021). Mild Heating Assisted Alkaline pH Shifting Modify the EggWhite Protein: The Mechanism and the Enhancement of Emulsifying Properties. LWT–Food Science and Technology, 151; 112094
Zhao, X., J. Liu, J. Fan, H. Chao, and X. Peng (2021). Recent Progress in Photosensitizers for Overcoming the Challenges of Photodynamic Therapy: From Molecular Design to Application. Chemical Society Reviews, 50(6); 4185–4219
Zhou, H., H. Pan, F. Raza, H. Zafar, Y. Ge, N. Wang, R. Zheng, D. Zhang, and Y. Yang (2024). Thermosensitive Drug-Loaded Liposomes for Photothermal and Chemotherapeutic Treatment of Colon Cancer. Materials Advances, 5(6); 2456–2469
Andra, V. V. S. N. L., S. V. N. Pammi, L. V. K. P. Bhatraju, and L. K. Ruddaraju (2022). Correction to: A Comprehensive Review on Novel Liposomal Methodologies, Commercial Formulations, Clinical Trials and Patents. BioNanoScience, 12(2); 274–291
Arvand, M., A. N. Masouleh, and S. M. J. Moghimi (2020). Development and Validation of an Ion Chromatography Method for Quantification of Ammonium Ions in STEALTH® Liposomes. Analytical and Bioanalytical Chemistry Research, 7(1); 17–31
Bartusik-Aebisher, D., P. Woźnicki, K. Dynarowicz, and D. Aebisher (2023). Photosensitizers for Photodynamic Therapy of Brain Cancers-a Review. Brain Sciences, 13(9); 1299
Cardali, S. M., G. Ricciardo, G. Garufi, G. Raffa, F. Messineo, G. Scalia, A. Conti, and A. Germanò (2022). Fluorescein-Guided Surgery for Intradural Spinal Tumors: A Single-Center Experience. Brain and Spine, 2; 100908
Chimbekujwo, K. I., M. I. Ja’afaru, and O. M. Adeyemo (2020). Purification, Characterization and Optimization Conditions of Protease Produced by Aspergillus brasiliensis Strain BCW2. Scientific African, 8; e00398
Dipiro, J. (2020). Pharmacotherapy: A Pathophysiology Approach. McGraw-Hill Education, New York, USA, 11 edition
Ellent, S. S. C., L. Dewi, and M. C. Tapilouw (2022). Karakteristik Mutu Tempe Kedelai (Glycine max L.) yang Dikemas dengan Klobot. AGRITEKNO, 11(1); 32–40 (in Indonesian)
Enciso, P., F. M. Cabrerizo, J. S. Gancheff, P. A. Denis, and M. F. Cerdá (2013). Phycocyanin as Potential Natural Dye for Its Use in Photovoltaic Cells. Journal of Applied Solution Chemistry and Modeling, 2(4); 225–233
Eugster, R. and P. Luciani (2024). Liposomes: Bridging the Gap from Lab to Pharmaceuticals. Current Opinion in Colloid & Interface Science, 75(10); 101875
Fithri, N. A., Mardiyanto, A. Fadilah, A. Pratiwi, S. S. Alisyahbana, M. F. Alhafiz, and N. P. Maharani (2025). Uncaria gambir Based Green Synthesis of Inorganic Nanoparticles for Photothermal Induced Thrombolytic and Antibacterial Applications. Science and Technology Indonesia, 10(1); 303–312
Fithri, N. A., A. Rahmah, and N. M. Annisa (2024). Formulation and Evaluation of Gambier (Uncaria gambir)-Chitosan Microparticle Intranasal Delivery for Alzheimer’s Diseases. Science and Technology Indonesia, 9(2); 284–298
Fithri, N. A., Y. Wu, G. Cowin, F. Akther, H. D. N. Tran, B. Tse, and H. T. Ta (2023). Gold-Iron Oxide Nanoparticle: A Unique Multimodal Theranostic Approach for Thrombosis. Applied Materials Today, 31; 101750
Gu, Y. (2024). The Effect of Buffer pH on Enzyme Activity. Theoretical and Natural Science, 33(1); 144–154
Hmood, S. A. and G. M. Aziz (2016). Purification and Characterization of Nattokinase Produced by Local Isolate of Bacillus sp. B24. Iraqi Journal of Biotechnology, 15(2); 93–108
Howard, F. B. and I. W. Levin (2010). Lipid Vesicle Aggregation Induced by Cooling. International Journal of Molecular Sciences, 11(2); 754–761
Jaffer, I. H. and J. I. Weitz (2018). Antithrombotic Drugs. In R. Hoffman, E. J. Benz, L. E. Silberstein, H. E. Heslop, J. I. Weitz, J. Anastasi, et al., editors, Hematology. Elsevier, 7 edition, pages 2168–2188
Kabir, M. F. and L. K. Ju (2023). On Optimization of Enzymatic Processes: Temperature Effects on Activity and Long-Term Deactivation Kinetics. Process Biochemistry, 130; 734–746
Kubrak, T. P., P. Kołodziej, J. Sawicki, A. Mazur, K. Koziorowska, and D. Aebisher (2022). Some Natural Photosensitizers and Their Medicinal Properties for Use in Photodynamic Therapy. Molecules, 27(4); 1192
Le, N. T. T., V. Cao, T. N. Q. Nguyen, T. T. H. Le, T. T. Tran, and T. T. H. Thi (2019). Soy Lecithin-Derived Liposomal Delivery Systems: Surface Modification and Current Applications. International Journal of Molecular Sciences, 20(19); 4706
Levine, R. M., T. R. Pearce, M. Adil, and E. Kokkoli (2013). Preparation and Characterization of Liposome-Encapsulated Plasmid DNA for Gene Delivery. Langmuir, 29(29); 9208–9215
Li, S., Q. Deng, Y. Zhang, X. Li, G. Wen, X. Cui, et al. (2020). Rational Design of Conjugated Small Molecules for Superior Photothermal Theranostics in the NIR-II Biowindow. Advanced Materials, 32(33); 1–8
Liu, Q., R. Geng, J. Zhao, Q. Chen, and B. Kong (2015). Structural and Gel Textural Properties of Soy Protein Isolate When Subjected to Extreme Acid pH-Shifting and Mild Heating Processes. Journal of Agricultural and Food Chemistry, 63(19); 4853–4861
Lu, Y. J., T. S. Anilkumar, C. C. Chuang, and J. P. Chen (2021). Liposomal IR-780 as a Highly Stable Nanotheranostic Agent for Improved Photothermal/Photodynamic Therapy of Brain Tumors by Convection-Enhanced Delivery. Cancers, 13(15); 3690
Mardiyanto, M., R. Mohadi, N. A. Fithri, G. Kurniawan, et al. (2024). Optimization of Nanoemulsion Formula Containing Erythromycin with VCO and Varying Concentrations of Tween-80 and PEG-400. Science and Technology Indonesia, 9(3); 697–709
Martin, S. S., A. W. Aday, Z. I. Almarzooq, C. A. M. Anderson, P. Arora, C. L. Avery, et al. (2024). Heart Disease and Stroke Statistics: A Report of US and Global Data From the American Heart Association. Circulation, 149(8); e347–e913
Mcleod, A. M. J., S. A. E. Barwell, T. Holyoak, and R. E. Thorne (2024). A Structural Perspective on the Temperature-Dependent Activity of Enzymes. bioRxiv, 8; 1–31
Nadea, N. S. W. P., A. Indrayati, and F. Leviana (2023). Potensi Ekstrak Kasar Enzim dari Tempe Kedelai Hitam (Glycine soja (L.) Merr.) sebagai Obat Fibrinolitik Alami dengan Metode Clot Lysis In Vitro. Jurnal Sains Dan Kesehatan, 5(2); 115–125
Ortel, T. L., I. Neumann, W. Ageno, R. Beyth, N. P. Clark, A. Cuker, et al. (2020). American Society of Hematology 2020 Guidelines for Management of Venous Thromboembolism: Treatment of Deep Vein Thrombosis and Pulmonary Embolism. Blood Advances, 4(19); 4693–4738
Pandey, A., S. Negi, and C. R. Soccol (2016). Current Developments in Biotechnology and Bioengineering: Production, Isolation and Purification of Industrial Products. Elsevier, Amsterdam, Netherlands
Polat, E. and K. Kang (2021). Natural Photosensitizers in Antimicrobial Photodynamic Therapy. Biomedicines, 9(6); 584
Psimadas, D., P. Georgoulias, V. Valotassiou, and G. Loudos (2012). Molecular Nanomedicine Towards Cancer. Journal of Pharmaceutical Sciences, 101(7); 2271–2280
Sahoo, S. K. and S. Sahoo (2020). Exploitation of Fibrinolytic Enzymes in Combating Blood Clotting Disorders – Recent Advances and Strategies: A Comprehensive Review. In J. Patra, A. Shukla, and G. Das, editors, Advances in Pharmaceutical Biotechnology. Springer, Singapore, pages 383–395
Sasmita, I. R. A., A. Sutrisno, E. Zubaidah, and A. K. Wardani (2018). Purification and Characterization of a Fibrinolytic Enzyme from Tempeh Bongkrek as an Alternative of Thrombolytic Agents. In IOP Conference Series: Earth and Environmental Science, volume 131
Ta, H. T., N. P. Truong, A. K. Whittaker, T. P. Davis, and K. Peter (2018). The Effects of Particle Size, Shape, Density and Flow Characteristics on Particle Margination to Vascular Walls in Cardiovascular Diseases. Expert Opinion on Drug Delivery, 15(1); 33–45
Tortajada, A., G. L. Righetti, A. McGinley, M. Mu, M. García-Melchor, and E. Hevia (2024). Alkene Isomerisation Catalysed by a Superbasic Sodium Amide. Angewandte Chemie International Edition, 136(38); e202407262
Tran, H. D. N., S. S. Moonshi, Z. P. Xu, and H. T. Ta (2022). Influence of Nanoparticles on the Haemostatic Balance: Between Thrombosis and Haemorrhage. Biomaterials Science, 10(1); 10–50
Tsengam, I. K. M., M. Omarova, E. G. Kelley, A. McCormick, G. D. Bothun, S. R. Raghavan, et al. (2022). Transformation of Lipid Vesicles into Micelles by Adding Nonionic Surfactants: Elucidating the Structural Pathway and the Intermediate Structures. Journal of Physical Chemistry B, 126(11); 2208–2216
Wang, K., Y. Gao, J. Zhao, Y. Wu, J. Sun, G. Niu, F. Zuo, and X. Zheng (2022). Effects of in Vitro Digestion on Protein Degradation, Phenolic Compound Release, and Bioactivity of Black Bean Tempeh. Frontiers in Nutrition, 9; 1017765
Weber, G., B. Buhren, H. Schrumpf, J. Wohlrab, and P. Gerber (2019). Clinical Applications of Hyaluronidase. In N. Labrou, editor, Therapeutic Enzymes: Function and Clinical Implications. Advances in Experimental Medicine and Biology. Springer, Singapore, pages 255–277
Yamada, K., A. Kawanabe, S. Yoshizawa, K. Inoue, K. Kogure, and H. Kandori (2012). Anomalous pH Effect of Blue Proteorhodopsin. Journal of Physical Chemistry Letters, 3(7); 800–804
Yu, Y., Y. Guan, H. Wen, Y. Zhang, J. Liu, and T. Zhang (2021). Mild Heating Assisted Alkaline pH Shifting Modify the EggWhite Protein: The Mechanism and the Enhancement of Emulsifying Properties. LWT–Food Science and Technology, 151; 112094
Zhao, X., J. Liu, J. Fan, H. Chao, and X. Peng (2021). Recent Progress in Photosensitizers for Overcoming the Challenges of Photodynamic Therapy: From Molecular Design to Application. Chemical Society Reviews, 50(6); 4185–4219
Zhou, H., H. Pan, F. Raza, H. Zafar, Y. Ge, N. Wang, R. Zheng, D. Zhang, and Y. Yang (2024). Thermosensitive Drug-Loaded Liposomes for Photothermal and Chemotherapeutic Treatment of Colon Cancer. Materials Advances, 5(6); 2456–2469
Authors
Azzahra, F. D., Mulyani, L. N., Sabrina, T., & Fithri, N. A. (2025). Liposome Photosensitizer with Enzyme from Black Soybean Tempeh: Formula Optimization and In Vitro Thrombolytic Activity Evaluation. Science and Technology Indonesia, 10(3), 903–915. https://doi.org/10.26554/sti.2025.10.3.903-915
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