{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,4]],"date-time":"2026-06-04T05:06:01Z","timestamp":1780549561803,"version":"3.54.1"},"reference-count":46,"publisher":"MDPI AG","issue":"6","license":[{"start":{"date-parts":[[2026,5,31]],"date-time":"2026-05-31T00:00:00Z","timestamp":1780185600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Vesselon, Inc.","award":["SP14951"],"award-info":[{"award-number":["SP14951"]}]},{"name":"National Institute of General Medical Sciences of the National Institutes of Health","award":["4 RO1 AG 079336-02"],"award-info":[{"award-number":["4 RO1 AG 079336-02"]}]},{"name":"National Institute of General Medical Sciences of the National Institutes of Health","award":["5U54GM115428"],"award-info":[{"award-number":["5U54GM115428"]}]},{"name":"National Institute of General Medical Sciences of the National Institutes of Health","award":["P20GM121334"],"award-info":[{"award-number":["P20GM121334"]}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"crossref","id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pharmaceutics"],"abstract":"<jats:p>Background\/Objectives: Immune checkpoint inhibitors targeting the PD-1\/PD-L1 axis have transformed cancer treatment, yet therapeutic responses remain limited in many solid tumors due to poor and uneven drug distribution within the tumor microenvironment (TME). Here, we evaluated whether co-formulation of an anti-PD-1 antibody (RMP1-14, murine surrogate for pembrolizumab) with Imagent\u00ae microbubble\/liposome (MBLP) complexes and ultrasound activation could enhance tumor-specific delivery while reducing systemic exposure. Methods: Immunocompetent MC-38 colorectal tumor-bearing mice (B6(Cg)-Tyrc-2J\/J, 7-week-old females) received isotype control, isotype\/MBLP\/US, RMP1-14 alone, RMP1-14\/MBLP, or RMP1-14\/MBLP\/US. Survival was analyzed by Kaplan\u2013Meier curves, tumor necrosis by H&amp;E staining, antibody biodistribution by immunohistochemistry, and tumor perfusion by laser speckle imaging. Results: No significant differences in tumor size or body weight were observed between groups. Survival analysis showed significant improvements in the RMP1-14 (p = 0.013) and RMP1-14\/MBLP\/US (p = 0.047) groups versus isotype controls, with the RMP1-14\/MBLP\/US group achieving the longest mean survival (57.8 days vs. 26.5 days for RMP1-14 alone) and complete tumor regression in 2\/8 mice. The RMP1-14\/MBLP\/US group demonstrated significantly greater tumor necrosis than all other groups. Immunohistochemical analysis confirmed a 6.1-fold increase in intratumoral antibody accumulation with MBLP\/US versus RMP1-14 alone (p = 0.0003), alongside significantly reduced off-target exposure in spleen, liver, kidney, and heart. Laser speckle imaging revealed a transient ~30% increase in tumor perfusion during MBLP\/US treatment, consistent with cavitation-mediated hemodynamic effects. Conclusions: These findings demonstrate that MBLP\/US co-formulation enhances intratumoral delivery of checkpoint inhibitors, improves survival, and reduces systemic organ exposure, representing a promising platform to improve the efficacy and safety profile of antibody-based immunotherapy.<\/jats:p>","DOI":"10.3390\/pharmaceutics18060690","type":"journal-article","created":{"date-parts":[[2026,6,3]],"date-time":"2026-06-03T09:33:14Z","timestamp":1780479194000},"page":"690","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Co-Formulation of Pembrolizumab Murine Surrogate RMP1-14 with Imagent\u00ae Ultrasound Contrast Agent Enhances Intratumoral Antibody Delivery Through a Transient Increase in Tumor Blood Perfusion"],"prefix":"10.3390","volume":"18","author":[{"given":"Imani A.","family":"Kirven","sequence":"first","affiliation":[{"name":"Department of Pharmacology & Toxicology, University of Mississippi Medical Center, Jackson, MS 39216, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7703-0070","authenticated-orcid":false,"given":"Patrice","family":"Penfornis","sequence":"additional","affiliation":[{"name":"Department of Pharmacology & Toxicology, University of Mississippi Medical Center, Jackson, MS 39216, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Muhammad R.","family":"Siddiqui","sequence":"additional","affiliation":[{"name":"University of Mississippi Medical Center, Jackson, MS 39216, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3198-8835","authenticated-orcid":false,"given":"Kenneth R.","family":"Butler","sequence":"additional","affiliation":[{"name":"Department of Pharmacology & Toxicology, University of Mississippi Medical Center, Jackson, MS 39216, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5581-0217","authenticated-orcid":false,"given":"Richard J.","family":"Roman","sequence":"additional","affiliation":[{"name":"Department of Pharmacology & Toxicology, University of Mississippi Medical Center, Jackson, MS 39216, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Clayton T.","family":"Larsen","sequence":"additional","affiliation":[{"name":"Vesselon, Inc., Wilton, CT 06897, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6537-3551","authenticated-orcid":false,"given":"Candace M.","family":"Howard","sequence":"additional","affiliation":[{"name":"Department of Radiology, University of Mississippi Medical Center, Jackson, MS 39216, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7790-1622","authenticated-orcid":false,"given":"Pier Paolo","family":"Claudio","sequence":"additional","affiliation":[{"name":"Department of Pharmacology & Toxicology, University of Mississippi Medical Center, Jackson, MS 39216, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,5,31]]},"reference":[{"key":"ref_1","first-page":"209","article-title":"Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries","volume":"71","author":"Sung","year":"2021","journal-title":"CA Cancer J. Clin."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"669","DOI":"10.1001\/jama.2021.0106","article-title":"Diagnosis and Treatment of Metastatic Colorectal Cancer: A Review","volume":"325","author":"Biller","year":"2021","journal-title":"JAMA"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"222","DOI":"10.1016\/j.tips.2023.01.003","article-title":"Metastatic colorectal cancer: Mechanisms and emerging therapeutics","volume":"44","author":"Shin","year":"2023","journal-title":"Trends Pharmacol. Sci."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"16","DOI":"10.1038\/s41698-023-00353-4","article-title":"Survival improvement for patients with metastatic colorectal cancer over twenty years","volume":"7","author":"Zeineddine","year":"2023","journal-title":"npj Precis. Oncol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"277","DOI":"10.1016\/j.annonc.2024.11.012","article-title":"Pembrolizumab versus chemotherapy in microsatellite instability-high or mismatch repair-deficient metastatic colorectal cancer: 5-year follow-up from the randomized phase III KEYNOTE-177 study","volume":"36","author":"Shiu","year":"2025","journal-title":"Ann. Oncol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"11","DOI":"10.1056\/NEJMoa2407417","article-title":"Final, 10-Year Outcomes with Nivolumab plus Ipilimumab in Advanced Melanoma","volume":"392","author":"Wolchok","year":"2025","journal-title":"N. Engl. J. Med."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"3085","DOI":"10.1111\/1759-7714.14193","article-title":"Pembrolizumab provides long-term survival benefits in advanced non-small cell lung cancer: The 5-year outcomes of the KEYNOTE-024 trial","volume":"12","author":"Li","year":"2021","journal-title":"Thorac. Cancer"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"2777","DOI":"10.1080\/21645515.2016.1199310","article-title":"Pembrolizumab (Keytruda)","volume":"12","author":"Kwok","year":"2016","journal-title":"Hum. Vaccin. Immunother."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"49","DOI":"10.1002\/psp4.12139","article-title":"Model-Based Characterization of the Pharmacokinetics of Pembrolizumab: A Humanized Anti-PD-1 Monoclonal Antibody in Advanced Solid Tumors","volume":"6","author":"Ahamadi","year":"2017","journal-title":"CPT Pharmacomet. Syst. Pharmacol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"809","DOI":"10.1038\/s41416-019-0599-y","article-title":"Immune checkpoint inhibitors for the treatment of MSI-H\/MMR-D colorectal cancer and a perspective on resistance mechanisms","volume":"121","author":"Sahin","year":"2019","journal-title":"Br. J. Cancer"},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Yan, S., Wang, W., Feng, Z., Xue, J., Liang, W., Wu, X., Tan, Z., Zhang, X., Zhang, S., and Li, X. (2024). Immune checkpoint inhibitors in colorectal cancer: Limitation and challenges. Front. Immunol., 15.","DOI":"10.3389\/fimmu.2024.1403533"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"501","DOI":"10.1007\/s40199-021-00404-w","article-title":"Durable complete response to pembrolizumab in microsatellite stable colorectal cancer","volume":"29","author":"Gomar","year":"2021","journal-title":"DARU J. Pharm. Sci."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"154","DOI":"10.4103\/apjon.apjon_3_19","article-title":"Management of Immune-Related Adverse Events Associated with Immune Checkpoint Inhibitor Therapy: A Minireview of Current Clinical Guidelines","volume":"6","author":"Trinh","year":"2019","journal-title":"Asia-Pac. J. Oncol. Nurs."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"e009902","DOI":"10.1136\/jitc-2024-009902","article-title":"Reporting of late-onset immune-related adverse events with immune checkpoint inhibitors in VigiBase","volume":"12","author":"Noseda","year":"2024","journal-title":"J. Immunother. Cancer"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"e14708","DOI":"10.1200\/JCO.2024.42.16_suppl.e14708","article-title":"Immune checkpoint inhibitor-associated gastrointestinal adverse events in patients with colorectal cancer","volume":"42","author":"Shatila","year":"2024","journal-title":"J. Clin. Oncol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"2939","DOI":"10.1111\/cas.14983","article-title":"Barriers to antibody therapy in solid tumors, and their solutions","volume":"112","author":"Matsumura","year":"2021","journal-title":"Cancer Sci."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"2276","DOI":"10.1007\/s13346-021-00923-8","article-title":"Overcoming biological barriers to improve solid tumor immunotherapy","volume":"11","author":"Ukidve","year":"2021","journal-title":"Drug Deliv. Transl. Res."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"2430","DOI":"10.1007\/s13346-021-01036-y","article-title":"Overcoming physical stromal barriers to cancer immunotherapy","volume":"11","author":"Chung","year":"2021","journal-title":"Drug Deliv. Transl. Res."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"860","DOI":"10.1002\/jps.22801","article-title":"Dose dependence of intratumoral perivascular distribution of monoclonal antibodies","volume":"101","author":"Rhoden","year":"2012","journal-title":"J. Pharm. Sci."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"e000938","DOI":"10.1136\/jitc-2020-000938","article-title":"89Zr-pembrolizumab biodistribution is influenced by PD-1-mediated uptake in lymphoid organs","volume":"8","author":"Giesen","year":"2020","journal-title":"J. Immunother. Cancer."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"162","DOI":"10.2967\/jnumed.116.177857","article-title":"Preclinical Pharmacokinetics and Biodistribution Studies of 89Zr-Labeled Pembrolizumab","volume":"58","author":"England","year":"2017","journal-title":"J. Nucl. Med."},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Brown, E.L., DeWeerd, R.A., Zidel, A., and Pereira, P.M.R. (2022). Preclinical antibody-PET imaging of PD-L1. Front. Nucl. Med., 2.","DOI":"10.3389\/fnume.2022.953202"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"5325","DOI":"10.1158\/1078-0432.CCR-21-0453","article-title":"First-in-Human Study of the Biodistribution and Pharmacokinetics of 89Zr-CX-072, a Novel Immunopet Tracer Based on an Anti-PD-L1 Probody","volume":"27","author":"Giesen","year":"2021","journal-title":"Clin. Cancer Res."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"346","DOI":"10.1038\/s41698-025-01123-0","article-title":"A real-world cohort study of immune-related adverse events in patients receiving immune checkpoint inhibitors","volume":"9","author":"Wang","year":"2025","journal-title":"npj Precis. Oncol."},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Sandker, G.G.W., Adema, G., Molkenboer-Kuenen, J., Wierstra, P., Bussink, J., Heskamp, S., and Aarntzen, E.H.J.G. (2022). PD-L1 Antibody Pharmacokinetics and Tumor Targeting in Mouse Models for Infectious Diseases. Front. Immunol., 13.","DOI":"10.3389\/fimmu.2022.837370"},{"key":"ref_26","first-page":"1","article-title":"Ultrasound and microbubble-mediated drug delivery and immunotherapy","volume":"2001","author":"Omata","year":"2022","journal-title":"J. Med. Ultrason."},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Wu, Y., Li, J., Shu, L., Tian, Z., Wu, S., and Wu, Z. (2024). Ultrasound combined with microbubble mediated immunotherapy for tumor microenvironment. Front. Pharmacol., 15.","DOI":"10.3389\/fphar.2024.1304502"},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Yuan, J., Ye, D., Chen, S., and Chen, H. (2021). Therapeutic Ultrasound-Enhanced Immune Checkpoint Inhibitor Therapy. Front. Phys., 9.","DOI":"10.3389\/fphy.2021.636985"},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Navarro-Becerra, J.A., and Borden, M.A. (2023). Targeted Microbubbles for Drug, Gene, and Cell Delivery in Therapy and Immunotherapy. Pharmaceutics, 15.","DOI":"10.3390\/pharmaceutics15061625"},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"He, J., Liu, Z., Zhu, X., Xia, H., Gao, H., and Lu, J. (2022). Ultrasonic Microbubble Cavitation Enhanced Tissue Permeability and Drug Diffusion in Solid Tumor Therapy. Pharmaceutics, 14.","DOI":"10.3390\/pharmaceutics14081642"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"4079","DOI":"10.7150\/thno.70372","article-title":"Theranostics in the vasculature: Bioeffects of ultrasound and microbubbles to induce vascular shutdown","volume":"13","author":"Padilla","year":"2023","journal-title":"Theranostics"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"2100154","DOI":"10.1002\/adtp.202100154","article-title":"Sonoporation-Enhanced Delivery of STING Agonist Induced Robust Immune Modulation and Tumor Regression","volume":"4","author":"Xu","year":"2021","journal-title":"Adv. Ther."},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Wu, N., Cao, Y., Liu, Y., Zhou, Y., He, H., Tang, R., Wan, L., Wang, C., Xiong, X., and Zhong, L. (2023). Low-intensity focused ultrasound targeted microbubble destruction reduces tumor blood supply and sensitizes anti-PD-L1 immunotherapy. Front. Bioeng. Biotechnol., 11.","DOI":"10.3389\/fbioe.2023.1173381"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"5220","DOI":"10.7150\/thno.105857","article-title":"ATP released by ultrasound targeted microbubble cavitation induces vascular inflammation and improves immune checkpoint blockade efficacy","volume":"15","author":"Jahangiri","year":"2025","journal-title":"Theranostics"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"413","DOI":"10.1002\/jcp.20736","article-title":"Ultrasound guided site specific gene delivery system using adenoviral vectors and commercial ultrasound contrast agents","volume":"209","author":"Howard","year":"2006","journal-title":"J. Cell. Physiol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"163","DOI":"10.2174\/1566523211313030001","article-title":"Microbubble-assisted p53, RB, and p130 gene transfer in combination with radiation therapy in prostate cancer","volume":"13","author":"Nande","year":"2013","journal-title":"Curr. Gene Ther."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"295","DOI":"10.1038\/mt.2009.252","article-title":"Eradication of therapy-resistant human prostate tumors using an ultrasound-guided site-specific cancer terminator virus delivery approach","volume":"18","author":"Greco","year":"2010","journal-title":"Mol. Ther."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"19","DOI":"10.1186\/s12967-019-1771-0","article-title":"Microbubble-mediated delivery of human adenoviruses does not elicit innate and adaptive immunity response in an immunocompetent mouse model of prostate cancer","volume":"17","author":"Thomas","year":"2019","journal-title":"J. Transl. Med."},{"key":"ref_39","doi-asserted-by":"crossref","unstructured":"Sitta, J., De Carlo, F., Kirven, I., Tackett, J.H., Penfornis, P., Dobbins, G.C., Barbier, M., Del Valle, L., Larsen, C.T., and Schutt, E.G. (2024). Microbubble-Protected Oncolytic Virotherapy Targeted by Sonoporation Induces Tumor Necrosis and T-Lymphocyte Infiltration in Humanized Mice Bearing Triple-Negative Breast Cancer. Int. J. Mol. Sci., 25.","DOI":"10.3390\/ijms252413697"},{"key":"ref_40","doi-asserted-by":"crossref","unstructured":"Adam, K., Iuga, A., Tocheva, A.S., and Mor, A. (2021). A novel mouse model for checkpoint inhibitor-induced adverse events. PLoS ONE, 16.","DOI":"10.1371\/journal.pone.0246168"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"57889","DOI":"10.18632\/oncotarget.18361","article-title":"Pseudoprogression in microsatellite instability-high colorectal cancer during treatment with combination T cell mediated immunotherapy: A case report and literature review","volume":"8","author":"Chae","year":"2017","journal-title":"Oncotarget"},{"key":"ref_42","doi-asserted-by":"crossref","unstructured":"Han, Y., Sun, J., Wei, H., Hao, J., Liu, W., and Wang, X. (2022). Ultrasound-Targeted Microbubble Destruction: Modulation in the Tumor Microenvironment and Application in Tumor Immunotherapy. Front. Immunol., 13.","DOI":"10.3389\/fimmu.2022.937344"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"715","DOI":"10.1038\/s41416-022-02076-y","article-title":"Ultrasound-targeted microbubble destruction remodels tumour microenvironment to improve immunotherapeutic effect","volume":"128","author":"Liu","year":"2023","journal-title":"Br. J. Cancer"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"1069","DOI":"10.1021\/acs.bioconjchem.1c00203","article-title":"Acoustically Detonated Microbubbles Coupled with Low Frequency Insonation: Multiparameter Evaluation of Low Energy Mechanical Ablation","volume":"33","author":"Bismuth","year":"2022","journal-title":"Bioconjug. Chem."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"113847","DOI":"10.1016\/j.addr.2021.113847","article-title":"Ultrasound and microbubbles to beat barriers in tumors: Improving delivery of nanomedicine","volume":"177","author":"Snipstad","year":"2021","journal-title":"Adv. Drug Deliv. Rev."},{"key":"ref_46","doi-asserted-by":"crossref","unstructured":"Herr, F.L., Antons, M.J., Blume, L.V., Hirner-Eppeneder, H., Kloiber-Langhorst, S., Cimic, A., Stueckl, J., Tardy, I., Burkard, T., and Ricke, J. (2025). Contrast-enhanced ultrasound with VEGFR2-targeted microbubbles for monitoring combined anti-PD-L1\/anti-CTLA-4 immunotherapy effects in a murine melanoma model with immunohistochemical validation. 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