Enhanced in vivo blood brain barrier transcytosis of macromolecular cargo using an engineered pH-sensitive mouse transferrin receptor binding nanobody
Abstract Background The blood brain barrier limits entry of macromolecular diagnostic and therapeutic cargos. Blood brain barrier transcytosis via receptor mediated transport systems, such as the transferrin receptor, can be used to carry macromolecular cargos with variable efficiency. Transcytosis...
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| Format: | Article |
| Language: | English |
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BMC
2023-08-01
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| Series: | Fluids and Barriers of the CNS |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s12987-023-00462-z |
| _version_ | 1797451746623094784 |
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| author | Thomas J. Esparza Shiran Su Caroline M. Francescutti Elvira Rodionova Joong Hee Kim David L. Brody |
| author_facet | Thomas J. Esparza Shiran Su Caroline M. Francescutti Elvira Rodionova Joong Hee Kim David L. Brody |
| author_sort | Thomas J. Esparza |
| collection | DOAJ |
| description | Abstract Background The blood brain barrier limits entry of macromolecular diagnostic and therapeutic cargos. Blood brain barrier transcytosis via receptor mediated transport systems, such as the transferrin receptor, can be used to carry macromolecular cargos with variable efficiency. Transcytosis involves trafficking through acidified intracellular vesicles, but it is not known whether pH-dependent unbinding of transport shuttles can be used to improve blood brain barrier transport efficiency. Methods A mouse transferrin receptor binding nanobody, NIH-mTfR-M1, was engineered to confer greater unbinding at pH 5.5 vs 7.4 by introducing multiple histidine mutations. The histidine mutant nanobodies were coupled to neurotensin for in vivo functional blood brain barrier transcytosis testing via central neurotensin-mediated hypothermia in wild-type mice. Multi-nanobody constructs including the mutant M1R56H, P96H, Y102H and two copies of the P2X7 receptor-binding 13A7 nanobody were produced to test proof-of-concept macromolecular cargo transport in vivo using quantitatively verified capillary depleted brain lysates and in situ histology. Results The most effective histidine mutant, M1R56H, P96H, Y102H-neurotensin, caused > 8 °C hypothermia after 25 nmol/kg intravenous injection. Levels of the heterotrimeric construct M1R56H, P96H, Y102H-13A7-13A7 in capillary depleted brain lysates peaked at 1 h and were 60% retained at 8 h. A control construct with no brain targets was only 15% retained at 8 h. Addition of the albumin-binding Nb80 nanobody to make M1R56H, P96H, Y102H-13A7-13A7-Nb80 extended blood half-life from 21 min to 2.6 h. At 30–60 min, biotinylated M1R56H, P96H, Y102H-13A7-13A7-Nb80 was visualized in capillaries using in situ histochemistry, whereas at 2–16 h it was detected in diffuse hippocampal and cortical cellular structures. Levels of M1R56H, P96H, Y102H-13A7-13A7-Nb80 reached more than 3.5 percent injected dose/gram of brain tissue after 30 nmol/kg intravenous injection. However, higher injected concentrations did not result in higher brain levels, compatible with saturation and an apparent substrate inhibitory effect. Conclusion The pH-sensitive mouse transferrin receptor binding nanobody M1R56H, P96H, Y102H may be a useful tool for rapid and efficient modular transport of diagnostic and therapeutic macromolecular cargos across the blood brain barrier in mouse models. Additional development will be required to determine whether this nanobody-based shuttle system will be useful for imaging and fast-acting therapeutic applications. |
| first_indexed | 2024-03-09T14:58:54Z |
| format | Article |
| id | doaj.art-3df86e54db8a43ec9c44e0591d61d114 |
| institution | Directory Open Access Journal |
| issn | 2045-8118 |
| language | English |
| last_indexed | 2024-03-09T14:58:54Z |
| publishDate | 2023-08-01 |
| publisher | BMC |
| record_format | Article |
| series | Fluids and Barriers of the CNS |
| spelling | doaj.art-3df86e54db8a43ec9c44e0591d61d1142023-11-26T14:00:49ZengBMCFluids and Barriers of the CNS2045-81182023-08-0120111710.1186/s12987-023-00462-zEnhanced in vivo blood brain barrier transcytosis of macromolecular cargo using an engineered pH-sensitive mouse transferrin receptor binding nanobodyThomas J. Esparza0Shiran Su1Caroline M. Francescutti2Elvira Rodionova3Joong Hee Kim4David L. Brody5National Institute of Neurological Disorders and StrokeNational Institute of Neurological Disorders and StrokeNational Institute of Neurological Disorders and StrokeNational Institute of Neurological Disorders and StrokeNational Institute of Neurological Disorders and StrokeNational Institute of Neurological Disorders and StrokeAbstract Background The blood brain barrier limits entry of macromolecular diagnostic and therapeutic cargos. Blood brain barrier transcytosis via receptor mediated transport systems, such as the transferrin receptor, can be used to carry macromolecular cargos with variable efficiency. Transcytosis involves trafficking through acidified intracellular vesicles, but it is not known whether pH-dependent unbinding of transport shuttles can be used to improve blood brain barrier transport efficiency. Methods A mouse transferrin receptor binding nanobody, NIH-mTfR-M1, was engineered to confer greater unbinding at pH 5.5 vs 7.4 by introducing multiple histidine mutations. The histidine mutant nanobodies were coupled to neurotensin for in vivo functional blood brain barrier transcytosis testing via central neurotensin-mediated hypothermia in wild-type mice. Multi-nanobody constructs including the mutant M1R56H, P96H, Y102H and two copies of the P2X7 receptor-binding 13A7 nanobody were produced to test proof-of-concept macromolecular cargo transport in vivo using quantitatively verified capillary depleted brain lysates and in situ histology. Results The most effective histidine mutant, M1R56H, P96H, Y102H-neurotensin, caused > 8 °C hypothermia after 25 nmol/kg intravenous injection. Levels of the heterotrimeric construct M1R56H, P96H, Y102H-13A7-13A7 in capillary depleted brain lysates peaked at 1 h and were 60% retained at 8 h. A control construct with no brain targets was only 15% retained at 8 h. Addition of the albumin-binding Nb80 nanobody to make M1R56H, P96H, Y102H-13A7-13A7-Nb80 extended blood half-life from 21 min to 2.6 h. At 30–60 min, biotinylated M1R56H, P96H, Y102H-13A7-13A7-Nb80 was visualized in capillaries using in situ histochemistry, whereas at 2–16 h it was detected in diffuse hippocampal and cortical cellular structures. Levels of M1R56H, P96H, Y102H-13A7-13A7-Nb80 reached more than 3.5 percent injected dose/gram of brain tissue after 30 nmol/kg intravenous injection. However, higher injected concentrations did not result in higher brain levels, compatible with saturation and an apparent substrate inhibitory effect. Conclusion The pH-sensitive mouse transferrin receptor binding nanobody M1R56H, P96H, Y102H may be a useful tool for rapid and efficient modular transport of diagnostic and therapeutic macromolecular cargos across the blood brain barrier in mouse models. Additional development will be required to determine whether this nanobody-based shuttle system will be useful for imaging and fast-acting therapeutic applications.https://doi.org/10.1186/s12987-023-00462-zBlood brain barrierTransferrin receptorNanobodyNeurotensinHistidineP2X7 receptor |
| spellingShingle | Thomas J. Esparza Shiran Su Caroline M. Francescutti Elvira Rodionova Joong Hee Kim David L. Brody Enhanced in vivo blood brain barrier transcytosis of macromolecular cargo using an engineered pH-sensitive mouse transferrin receptor binding nanobody Fluids and Barriers of the CNS Blood brain barrier Transferrin receptor Nanobody Neurotensin Histidine P2X7 receptor |
| title | Enhanced in vivo blood brain barrier transcytosis of macromolecular cargo using an engineered pH-sensitive mouse transferrin receptor binding nanobody |
| title_full | Enhanced in vivo blood brain barrier transcytosis of macromolecular cargo using an engineered pH-sensitive mouse transferrin receptor binding nanobody |
| title_fullStr | Enhanced in vivo blood brain barrier transcytosis of macromolecular cargo using an engineered pH-sensitive mouse transferrin receptor binding nanobody |
| title_full_unstemmed | Enhanced in vivo blood brain barrier transcytosis of macromolecular cargo using an engineered pH-sensitive mouse transferrin receptor binding nanobody |
| title_short | Enhanced in vivo blood brain barrier transcytosis of macromolecular cargo using an engineered pH-sensitive mouse transferrin receptor binding nanobody |
| title_sort | enhanced in vivo blood brain barrier transcytosis of macromolecular cargo using an engineered ph sensitive mouse transferrin receptor binding nanobody |
| topic | Blood brain barrier Transferrin receptor Nanobody Neurotensin Histidine P2X7 receptor |
| url | https://doi.org/10.1186/s12987-023-00462-z |
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