{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,30]],"date-time":"2026-08-30T12:22:35Z","timestamp":1788092555368,"version":"build-2803163510"},"reference-count":42,"publisher":"MDPI AG","issue":"10","license":[{"start":{"date-parts":[[2025,10,14]],"date-time":"2025-10-14T00:00:00Z","timestamp":1760400000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"National Institute of Aging of National Institutes of Health","award":["RF1 AG068034"],"award-info":[{"award-number":["RF1 AG068034"]}]},{"name":"National Institute of Aging of National Institutes of Health","award":["RF1 AG068034-01S1"],"award-info":[{"award-number":["RF1 AG068034-01S1"]}]},{"name":"National Institute of Aging of National Institutes of Health","award":["R01 AG068034-02"],"award-info":[{"award-number":["R01 AG068034-02"]}]},{"DOI":"10.13039\/100000050","name":"National Heart, Lung, and Blood Institute","doi-asserted-by":"crossref","award":["2R15HL124338-02A1"],"award-info":[{"award-number":["2R15HL124338-02A1"]}],"id":[{"id":"10.13039\/100000050","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000050","name":"National Heart, Lung, and Blood Institute","doi-asserted-by":"crossref","award":["1R15HL143519-01"],"award-info":[{"award-number":["1R15HL143519-01"]}],"id":[{"id":"10.13039\/100000050","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pharmaceutics"],"abstract":"<jats:p>The blood\u2013brain barrier (BBB) restricts the entry of therapeutic agents into the brain cardiovascular regulatory region, potentially contributing to drug-resistant hypertension. Objective: The objective of this study was to overcome this limitation by modifying PEGylated liposomes with transferrin (Tf) to facilitate Tf receptor binding at the BBB and penetratin (Pen), a cell-penetrating peptide, to enhance neuronal uptake. Methods: This study evaluated the efficacy of Tf-Pen-liposomes in delivering angiotensin-converting enzyme 2 (ACE2) or EGFP (control) genes across the BBB in rats. In addition, the therapeutic effect of intravenous administration of Tf-Pen-Lip carrying plasmid DNA encoding ACE2 (Tf-Pen-Lip-pACE2) was tested in a neurogenic hypertension model induced by intracerebroventricular (ICV) infusion of angiotensin II (Ang II) via osmotic pump implantation and brain cannulation. Results: Conjugation with Tf and Pen significantly enhanced liposome-mediated gene transfection in cultured cells and increased transport across an in vitro BBB model. In vivo, intravenous administration of Tf-Pen-Lip-pACE2 or Tf-Pen-Lip-pGFP successfully elevated ACE2 or EGFP expression, respectively, in the hypothalamic paraventricular nucleus (PVN). Chronic ICV infusion of Ang II produced a sustained increase in blood pressure and heart rate, accompanied by sympathetic overactivation and elevated arginine vasopressin (AVP) secretion, hallmarks of neurogenic hypertension. Notably, intravenous Tf-Pen-Lip-pACE2 treatment dramatically attenuated Ang II\u2013induced neurogenic hypertension, whereas Tf-Pen-Lip-pGFP had no effect on pressor responses, sympathetic activity, or AVP secretion. Conclusions: This dual-functionalized liposomal delivery system effectively transported the ACE2 gene across the BBB into the brain, increased ACE2 expression, and markedly attenuated neurogenic hypertension following systemic administration.<\/jats:p>","DOI":"10.3390\/pharmaceutics17101329","type":"journal-article","created":{"date-parts":[[2025,10,14]],"date-time":"2025-10-14T07:51:28Z","timestamp":1760428288000},"page":"1329","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Engineered Liposomal Delivery of Human ACE2 Across the Blood\u2013Brain Barrier Attenuated Neurogenic Hypertension"],"prefix":"10.3390","volume":"17","author":[{"given":"Yue","family":"Shen","sequence":"first","affiliation":[{"name":"Department of Pharmaceutical Sciences, North Dakota State University, Fargo, ND 58105, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9659-9083","authenticated-orcid":false,"given":"Richard Nii Lante","family":"Lamptey","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Sciences, North Dakota State University, Fargo, ND 58105, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3183-3200","authenticated-orcid":false,"given":"Gowthami Reddy","family":"Mareddy","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Sciences, North Dakota State University, Fargo, ND 58105, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bivek","family":"Chaulagain","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Sciences, North Dakota State University, Fargo, ND 58105, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jagdish","family":"Singh","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Sciences, North Dakota State University, Fargo, ND 58105, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5889-8448","authenticated-orcid":false,"given":"Chengwen","family":"Sun","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Sciences, North Dakota State University, Fargo, ND 58105, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,10,14]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1099","DOI":"10.1038\/s41440-024-01622-w","article-title":"The WHO Global report 2023 on hypertension warning the emerging hypertension burden in globe and its treatment strategy","volume":"47","author":"Kario","year":"2024","journal-title":"Hypertens. Res."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"e2431997","DOI":"10.1001\/jamanetworkopen.2024.31997","article-title":"Examining the hypertension control cascade in adults with uncontrolled hypertension in the US","volume":"7","author":"Richardson","year":"2024","journal-title":"JAMA Netw. Open"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"424","DOI":"10.1161\/HYPERTENSIONAHA.118.12191","article-title":"Prevalence of apparent treatment-resistant hypertension in the United States","volume":"73","author":"Carey","year":"2019","journal-title":"Hypertension"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"e034910","DOI":"10.1161\/JAHA.124.034910","article-title":"Randomized trials of renal denervation for uncontrolled hypertension: An updated meta-analysis","volume":"13","author":"Mufarrih","year":"2024","journal-title":"J. Am. Heart Assoc."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"836","DOI":"10.1161\/HYPERTENSIONAHA.117.09086","article-title":"Sustained reduction of blood pressure with baroreceptor activation therapy: Results of the 6-year open follow-up","volume":"69","author":"Bisognano","year":"2017","journal-title":"Hypertension"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1254","DOI":"10.1016\/j.jacc.2010.03.089","article-title":"Novel baroreflex activation therapy in resistant hypertension: Results of a European multi-center feasibility study","volume":"56","author":"Scheffers","year":"2010","journal-title":"J. Am. Coll. Cardiol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1095","DOI":"10.1007\/s00392-011-0346-8","article-title":"Catheter-based renal sympathetic denervation: Chronic preclinical evidence for renal artery safety","volume":"100","author":"Rippy","year":"2011","journal-title":"Clin. Res. Cardiol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"H1200","DOI":"10.1152\/ajpheart.00216.2018","article-title":"Regulation of sympathetic vasomotor activity by the hypothalamic paraventricular nucleus in normotensive and hypertensive states","volume":"315","author":"Dampney","year":"2018","journal-title":"Am. J. Physiol. Heart Circ. Physiol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1830","DOI":"10.2174\/1871530321666210319120308","article-title":"Angiotensin II-vasopressin interactions in the regulation of cardiovascular functions. Evidence for an impaired hormonal sympathetic reflex in hypertension and congestive heart failure","volume":"21","author":"Iovino","year":"2021","journal-title":"Endocr. Metab. Immune Disord. Drug Targets"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"2535","DOI":"10.1042\/CS20200483","article-title":"Brain angiotensin converting enzyme-2 in central cardiovascular regulation","volume":"134","author":"Mohammed","year":"2020","journal-title":"Clin. Sci."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"505","DOI":"10.1152\/physrev.00023.2016","article-title":"The ACE2\/Angiotensin-(1-7)\/MAS axis of the renin-angiotensin system: Focus on angiotensin-(1-7)","volume":"98","author":"Santos","year":"2018","journal-title":"Physiol. Rev."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"373","DOI":"10.1161\/CIRCRESAHA.109.208645","article-title":"Brain-selective overexpression of human Angiotensin-converting enzyme type 2 attenuates neurogenic hypertension","volume":"106","author":"Feng","year":"2010","journal-title":"Circ. Res."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"926","DOI":"10.1161\/01.HYP.0000259942.38108.20","article-title":"Overexpression of angiotensin-converting enzyme 2 in the rostral ventrolateral medulla causes long-term decrease in blood pressure in the spontaneously hypertensive rats","volume":"49","author":"Yamazato","year":"2007","journal-title":"Hypertension"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"100214","DOI":"10.1016\/j.tjpad.2025.100214","article-title":"Bispecific brain-penetrant antibodies for treatment of Alzheimer\u2019s disease","volume":"12","author":"Sehlin","year":"2025","journal-title":"J. Prev. Alzheimer\u2019s Dis."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"456","DOI":"10.1016\/j.ymthe.2018.12.005","article-title":"Iduronate-2-sulfatase with anti-human transferrin receptor antibody for neuropathic mucopolysaccharidosis II: A phase 1\/2 trial","volume":"27","author":"Okuyama","year":"2019","journal-title":"Mol. Ther."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.neuron.2013.12.023","article-title":"Breaching the blood-brain barrier for drug delivery","volume":"81","author":"Bell","year":"2014","journal-title":"Neuron"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"49","DOI":"10.1016\/j.neuron.2013.10.061","article-title":"Increased brain penetration and potency of a therapeutic antibody using a monovalent molecular shuttle","volume":"81","author":"Niewoehner","year":"2014","journal-title":"Neuron"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1218","DOI":"10.1021\/mp400681n","article-title":"Penetratin, a potentially powerful absorption enhancer for noninvasive intraocular drug delivery","volume":"11","author":"Liu","year":"2014","journal-title":"Mol. Pharm."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"H1116","DOI":"10.1152\/ajpheart.00832.2011","article-title":"Angiotensin-(1-7) attenuates the chronotropic response to angiotensin II via stimulation of PTEN in the spontaneously hypertensive rat neurons","volume":"302","author":"Modgil","year":"2012","journal-title":"Am. J. Physiol. Heart Circ. Physiol."},{"key":"ref_20","first-page":"50079","article-title":"Isolation and culture of mouse cortical astrocytes","volume":"71","author":"Schildge","year":"2013","journal-title":"J. Vis. Exp."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"383","DOI":"10.1007\/978-1-4939-0320-7_31","article-title":"Isolation and culture of primary pericytes from mouse brain","volume":"1135","author":"Boroujerdi","year":"2014","journal-title":"Methods Mol. Biol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"247","DOI":"10.1016\/j.jconrel.2019.06.033","article-title":"Dual functionalized liposomes for efficient co-delivery of anti-cancer chemotherapeutics for the treatment of glioblastoma","volume":"307","author":"Lakkadwala","year":"2019","journal-title":"J. Control. Release"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.jconrel.2013.01.016","article-title":"Cell penetrating peptide tethered bi-ligand liposomes for delivery to brain in vivo: Biodistribution and transfection","volume":"167","author":"Sharma","year":"2013","journal-title":"J. Control. Release"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"2468","DOI":"10.1002\/jps.23152","article-title":"Grafting of cell-penetrating peptide to receptor-targeted liposomes improves their transfection efficiency and transport across blood-brain barrier model","volume":"101","author":"Sharma","year":"2012","journal-title":"J. Pharm. Sci."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1421","DOI":"10.1161\/CIRCRESAHA.108.192302","article-title":"Apelin gene transfer into the rostral ventrolateral medulla induces chronic blood pressure elevation in normotensive rats","volume":"104","author":"Zhang","year":"2009","journal-title":"Circ. Res."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"H1837","DOI":"10.1152\/ajpheart.00354.2009","article-title":"Angiotensin II enhances GABA(B) receptor-mediated responses and expression in nucleus tractus solitarii of rats","volume":"297","author":"Zhang","year":"2009","journal-title":"Am. J. Physiol. Heart Circ. Physiol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"H2712","DOI":"10.1152\/ajpheart.00729.2007","article-title":"Angiotensin II increases GABAB receptor expression in nucleus tractus solitarii of rats","volume":"294","author":"Yao","year":"2008","journal-title":"Am. J. Physiol. Heart Circ. Physiol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"117","DOI":"10.1007\/978-1-4939-6625-7_9","article-title":"Determining the enzymatic activity of angiotensin-converting enzyme 2 (ACE2) in brain tissue and cerebrospinal fluid using a quenched fluorescent substrate","volume":"1527","author":"Sriramula","year":"2017","journal-title":"Methods Mol. Biol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"615","DOI":"10.1038\/nrd2591","article-title":"Strategies in the design of nanoparticles for therapeutic applications","volume":"9","author":"Petros","year":"2010","journal-title":"Nat. Rev. Drug Discov."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"34","DOI":"10.1016\/j.jconrel.2016.05.044","article-title":"Nanoparticle-mediated brain drug delivery: Overcoming blood\u2013brain barrier to treat neurodegenerative diseases","volume":"235","author":"Saraiva","year":"2016","journal-title":"J. Control. Release"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"106267","DOI":"10.1016\/j.ejps.2022.106267","article-title":"How docetaxel entrapment, vesicle size, zeta potential and stability change with liposome composition-A formulation screening study","volume":"177","author":"Wizgird","year":"2022","journal-title":"Eur. J. Pharm. Sci."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"87","DOI":"10.1007\/7651_2020_316","article-title":"Evaluation of blood-brain barrier integrity using vascular permeability markers: Evans blue, sodium fluorescein, albumin-alexa fluor conjugates, and horseradish peroxidase","volume":"2367","author":"Ahishali","year":"2021","journal-title":"Methods Mol. Biol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"417","DOI":"10.1161\/CIRCRESAHA.124.325975","article-title":"RCAN1-mediated calcineurin impairment drives sympathetic outflow in hypertension","volume":"137","author":"Zhou","year":"2025","journal-title":"Circ. Res."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"H1075","DOI":"10.1152\/ajpheart.00822.2016","article-title":"Increased activity of the orexin system in the paraventricular nucleus contributes to salt-sensitive hypertension","volume":"313","author":"Huber","year":"2017","journal-title":"Am. J. Physiol. Heart Circ. Physiol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"R770","DOI":"10.1152\/ajpregu.00435.2017","article-title":"Angiotensin AT1A receptors expressed in vasopressin-producing cells of the supraoptic nucleus contribute to osmotic control of vasopressin","volume":"314","author":"Sandgren","year":"2018","journal-title":"Am. J. Physiol. Regul. Integr. Comp. Physiol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"1688","DOI":"10.1097\/HJH.0000000000003538","article-title":"Sympathetic modulation as a goal of antihypertensive treatment: From drugs to devices","volume":"41","author":"Grassi","year":"2023","journal-title":"J. Hypertens."},{"key":"ref_37","doi-asserted-by":"crossref","unstructured":"Butt, M.H., Zaman, M., Ahmad, A., Khan, R., Mallhi, T.H., Hasan, M.M., Khan, Y.H., Hafeez, S., Massoud, E.E.S., and Rahman, M.H. (2022). Appraisal for the potential of viral and nonviral vectors in gene therapy: A review. Genes, 13.","DOI":"10.3390\/genes13081370"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"20417314221095997","DOI":"10.1177\/20417314221095997","article-title":"Modeling the blood-brain barrier for treatment of central nervous system (CNS) diseases","volume":"13","author":"Rice","year":"2022","journal-title":"J. Tissue Eng."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"RP96161","DOI":"10.7554\/eLife.96161","article-title":"Neurons enhance blood-brain barrier function via upregulating claudin-5 and VE-cadherin expression due to glial cell line-derived neurotrophic factor secretion","volume":"13","author":"Yang","year":"2024","journal-title":"Elife"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"937","DOI":"10.1016\/j.neuron.2020.09.002","article-title":"Neuronal activity regulates blood-brain barrier efflux transport through endothelial circadian genes","volume":"108","author":"Pulido","year":"2020","journal-title":"Neuron"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"1181","DOI":"10.1161\/HYPERTENSIONAHA.119.13133","article-title":"ACE2 and ADAM17 interaction regulates the activity of presympathetic neurons","volume":"74","author":"Mukerjee","year":"2019","journal-title":"Hypertension"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"e22682","DOI":"10.1371\/journal.pone.0022682","article-title":"ACE2-mediated reduction of oxidative stress in the central nervous system is associated with improvement of autonomic function","volume":"6","author":"Xia","year":"2011","journal-title":"PLoS ONE"}],"container-title":["Pharmaceutics"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4923\/17\/10\/1329\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,16]],"date-time":"2025-10-16T04:14:17Z","timestamp":1760588057000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4923\/17\/10\/1329"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,10,14]]},"references-count":42,"journal-issue":{"issue":"10","published-online":{"date-parts":[[2025,10]]}},"alternative-id":["pharmaceutics17101329"],"URL":"https:\/\/doi.org\/10.3390\/pharmaceutics17101329","relation":{},"ISSN":["1999-4923"],"issn-type":[{"value":"1999-4923","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,10,14]]}}}