{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,24]],"date-time":"2026-07-24T19:24:49Z","timestamp":1784921089346,"version":"3.55.0"},"reference-count":49,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2021,8,28]],"date-time":"2021-08-28T00:00:00Z","timestamp":1630108800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["1R03EB020944-01"],"award-info":[{"award-number":["1R03EB020944-01"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000057","name":"National Institute of General Medical Sciences","doi-asserted-by":"publisher","award":["P30 GM103332"],"award-info":[{"award-number":["P30 GM103332"]}],"id":[{"id":"10.13039\/100000057","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pharmaceutics"],"abstract":"<jats:p>Frequent and inappropriate usage of antibiotics has changed the natural evolution of bacteria by reducing susceptibility and increasing resistance towards antibacterial agents. New resistance mechanisms evolved in the response to host defenses and pharmaceutical interventions are threatening our ability to treat common infections, resulting in increased mortality. In the face of this rising epidemic, antibiotic drug discovery, which has long been overlooked by big pharma, is reaching a critical low. Thus, the development of an infection-responsive drug delivery system, which may mitigate multidrug resistance and preserve the lifetime of our current antibiotic arsenal, has garnered the attention of both popular science and funding agencies. The present work describes the development of a thrombin-sensitive linker embedded into a recombinant spider silk copolymer to create a nanosphere drug delivery vehicle. Recent studies have suggested that there is an increase in thrombin-like activity during Staphylococcus aureus infection; thus, drug release from this new \u201csmart\u201d nanosphere can be triggered in the presence of infection. A thrombin sensitive peptide (TSP) was synthesized, and the thrombin cleavage sensitivity was determined by HPLC. The results showed no cleavage of the peptide when exposed to human serum whereas the peptide was cleaved when incubated with S. aureus exudate. Subsequently, the peptide was coupled with a silk copolymer via EDC-NHS chemistry and formulated into nanospheres encapsulating antibiotic vancomycin. These nanospheres were evaluated for in vitro infection-responsive drug release and antimicrobial activity. Finally, the drug responsive nanospheres were assessed for efficacy in an in vivo septic arthritis model. Our study provides evidence that the protein conjugate was enzyme responsive and can be used to formulate targeted drug release to combat infections against multidrug-resistant bacterial strains.<\/jats:p>","DOI":"10.3390\/pharmaceutics13091358","type":"journal-article","created":{"date-parts":[[2021,8,31]],"date-time":"2021-08-31T04:07:28Z","timestamp":1630382848000},"page":"1358","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":25,"title":["Infection Responsive Smart Delivery of Antibiotics Using Recombinant Spider Silk Nanospheres"],"prefix":"10.3390","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7876-069X","authenticated-orcid":false,"given":"Pranothi","family":"Mulinti","sequence":"first","affiliation":[{"name":"Department of Pharmaceutical Sciences, North Dakota State University, Fargo, ND 58102, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6472-9608","authenticated-orcid":false,"given":"Jacob","family":"Shreffler","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Sciences, North Dakota State University, Fargo, ND 58102, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7706-0106","authenticated-orcid":false,"given":"Raquib","family":"Hasan","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Sciences, North Dakota State University, Fargo, ND 58102, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michael","family":"Dea","sequence":"additional","affiliation":[{"name":"College of Osteopathic Medicine, Rocky Vista University, Ivins, UT 84734, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9785-5969","authenticated-orcid":false,"given":"Amanda E.","family":"Brooks","sequence":"additional","affiliation":[{"name":"Department of Molecular Biology, Rocky Vista University, Ivins, UT 84734, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2021,8,28]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"S34","DOI":"10.1002\/jhm.978","article-title":"Infections, bacterial resistance, and antimicrobial stewardship: The emerging role of hospitalists","volume":"7","author":"Rosenberg","year":"2012","journal-title":"J. Hosp. Med."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"293","DOI":"10.1016\/j.ijmm.2013.02.004","article-title":"Antibiotics and antibiotic resistance: A bitter fight against evolution","volume":"303","author":"Couce","year":"2013","journal-title":"Int. J. Med. Microbiol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"14","DOI":"10.1016\/j.addr.2014.10.027","article-title":"Therapeutic strategies to combat antibiotic resistance","volume":"78","author":"Brooks","year":"2014","journal-title":"Adv. Drug Deliv. Rev."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"5","DOI":"10.1111\/j.1469-0691.2006.01492.x","article-title":"The emergence of antibiotic resistance by mutation","volume":"13","author":"Woodford","year":"2007","journal-title":"Clin. Microbiol. Infect."},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Budimir, A. (2018). Fighting Antimicrobial Resistance, IAPC Publishing.","DOI":"10.5599\/obp.15.0"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"2450","DOI":"10.1016\/j.biomaterials.2005.11.031","article-title":"Drug\/device combinations for local drug therapies and infection prophylaxis","volume":"27","author":"Wu","year":"2006","journal-title":"Biomaterials"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"13","DOI":"10.1590\/S1984-82502013000100003","article-title":"Osteomyelitis: An overview of antimicrobial therapy","volume":"49","author":"Gomes","year":"2013","journal-title":"Braz. J. Pharm. Sci."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"6566","DOI":"10.1021\/ja101554a","article-title":"A Thin Film Detection\/Response System for Pathogenic Bacteria","volume":"132","author":"Zhou","year":"2010","journal-title":"J. Am. Chem. Soc."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"6175","DOI":"10.1002\/adma.201202847","article-title":"Bacteria-Responsive Multifunctional Nanogel for Targeted Antibiotic Delivery","volume":"24","author":"Xiong","year":"2012","journal-title":"Adv. Mater."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"967","DOI":"10.1016\/j.addr.2012.01.002","article-title":"Enzyme-responsive nanoparticles for drug release and diagnostics","volume":"64","author":"Aili","year":"2012","journal-title":"Adv. Drug Deliv. Rev."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"(2016). Enzyme-responsive materials for wound infection diagnosis. Biotechnol. Bioeng., 113, 2534.","DOI":"10.1002\/bit.25803"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"26","DOI":"10.1016\/S0008-6363(02)00857-X","article-title":"Infection and inflammation and the coagulation system","volume":"60","author":"Levi","year":"2003","journal-title":"Cardiovasc. Res."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"510","DOI":"10.1021\/js980418j","article-title":"A novel microbial infection-responsive drug release system","volume":"88","author":"Tanihara","year":"1999","journal-title":"J. Pharm. Sci."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"112","DOI":"10.1002\/(SICI)1097-4636(199810)42:1<112::AID-JBM14>3.0.CO;2-N","article-title":"A new drug delivery system with controlled release of antibiotic only in the presence of infection","volume":"42","author":"Suzuki","year":"1998","journal-title":"J. Biomed. Mater. Res."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1179","DOI":"10.1074\/jbc.M507956200","article-title":"Fibrinogen Substrate Recognition by Staphylocoagulase\u00b7(Pro)thrombin Complexes","volume":"281","author":"Panizzi","year":"2006","journal-title":"J. Biol. Chem."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1107","DOI":"10.1160\/TH11-12-0891","article-title":"Fibrin formation by staphylothrombin facilitates Staphylococcus aureus-induced platelet aggregation","volume":"107","author":"Vanassche","year":"2012","journal-title":"Thromb. Haemost."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"421","DOI":"10.1016\/S0196-9781(97)00420-8","article-title":"Thrombin-sensitive peptide linkers for biological signal-responsive drug release systems","volume":"19","author":"Tanihara","year":"1998","journal-title":"Peptides"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"401","DOI":"10.1016\/S0142-9612(02)00353-8","article-title":"Silk-based biomaterials","volume":"24","author":"Altman","year":"2003","journal-title":"Biomaterials"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"17","DOI":"10.1016\/j.actbio.2015.11.034","article-title":"Silk protein-based hydrogels: Promising advanced materials for biomedical applications","volume":"31","author":"Kapoor","year":"2016","journal-title":"Acta Biomater."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"86","DOI":"10.1016\/j.progpolymsci.2015.02.001","article-title":"Structures, mechanical properties and applications of silk fibroin materials","volume":"46","author":"Koh","year":"2015","journal-title":"Prog. Polym. Sci."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"6064","DOI":"10.1016\/j.biomaterials.2006.07.008","article-title":"Stem cell-based tissue engineering with silk biomaterials","volume":"27","author":"Wang","year":"2006","journal-title":"Biomaterials"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1810","DOI":"10.1002\/adma.200602709","article-title":"Engineered Microcapsules Fabricated from Reconstituted Spider Silk","volume":"19","author":"Hermanson","year":"2007","journal-title":"Adv. Mater."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"4592","DOI":"10.1002\/anie.200800683","article-title":"An Engineered Spider Silk Protein Forms Microspheres","volume":"47","author":"Slotta","year":"2008","journal-title":"Angew. Chem. Int. Ed."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"229","DOI":"10.1021\/bm800930x","article-title":"Self-Assembly of Genetically Engineered Spider Silk Block Copolymers","volume":"10","author":"Rabotyagova","year":"2009","journal-title":"Biomacromolecules"},{"key":"ref_25","first-page":"e4191","article-title":"Synthetic Spider Silk Production on a Laboratory Scale","volume":"65","author":"Hsia","year":"2012","journal-title":"J. Vis. Exp."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"341","DOI":"10.1038\/nprot.2008.250","article-title":"A protocol for the production of recombinant spider silk-like proteins for artificial fiber spinning","volume":"4","author":"Cooper","year":"2009","journal-title":"Nat. Protoc."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"1495","DOI":"10.1093\/oxfordjournals.jbchem.a131840","article-title":"New Fluorogenic Substrates for \u03b1-Thrombin, Factor Xa, Kallikreins, and Urokinase1","volume":"82","author":"Morita","year":"1977","journal-title":"J. Biochem."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"10119","DOI":"10.1021\/la2022177","article-title":"Preparation of Peptide-Functionalized Gold Nanoparticles Using One Pot EDC\/Sulfo-NHS Coupling","volume":"27","author":"Bartczak","year":"2011","journal-title":"Langmuir"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"55","DOI":"10.1007\/978-1-60761-670-2_3","article-title":"Amine coupling through EDC\/NHS: A practical approach","volume":"Volume 627","author":"Mol","year":"2010","journal-title":"Surface Plasmon Resonance; Methods in Molecular Biology"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"195","DOI":"10.1016\/0022-1759(90)90048-Z","article-title":"An improved conjugation method for controlled covalent coupling of synthetic peptides to proteins using glutaraldehyde in a dialysis method","volume":"130","author":"Zegers","year":"1990","journal-title":"J. Immunol. Methods"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"248","DOI":"10.1016\/j.jcis.2005.07.006","article-title":"Pyrene absorption can be a convenient method for probing critical micellar concentration (cmc) and indexing micellar polarity","volume":"294","author":"Ray","year":"2006","journal-title":"J. Colloid Interface Sci."},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Hasan, R., Wohlers, A., Shreffler, J., Mulinti, P., Ostlie, H., Schaper, C., Brooks, B., and Brooks, A. (2020). An Antibiotic-Releasing Bone Void Filling (ABVF) Putty for the Treatment of Osteomyelitis. Materials, 13.","DOI":"10.3390\/ma13225080"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"82","DOI":"10.2174\/1877912311202020082","article-title":"Novel Nanomicellar Formulation Approaches for Anterior and Posterior Segment Ocular Drug Delivery","volume":"2","author":"Cholkar","year":"2012","journal-title":"Recent Pat. Nanomed."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"96","DOI":"10.1016\/j.jconrel.2017.01.012","article-title":"Polymeric micelles for ocular drug delivery: From structural frameworks to recent preclinical studies","volume":"248","author":"Mandal","year":"2017","journal-title":"J. Control. Release"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"169","DOI":"10.1016\/j.jconrel.2005.09.034","article-title":"Block copolymer micelles: Preparation, characterization and application in drug delivery","volume":"109","author":"Gaucher","year":"2005","journal-title":"J. Control. Release"},{"key":"ref_36","unstructured":"(2018, July 29). The Method of Purifying Bioengineered Spider Silk Determines the Silk Sphere Properties | Scientific Reports. Available online: https:\/\/www.nature.com\/articles\/srep28106."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"136","DOI":"10.1093\/jac\/dkt308","article-title":"Determining accurate vancomycin MIC values for methicillin-resistant Staphylococcus aureus by the microdilution method","volume":"69","author":"Lepe","year":"2013","journal-title":"J. Antimicrob. Chemother."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"4279","DOI":"10.1021\/nn3008383","article-title":"Surface Charge-Switching Polymeric Nanoparticles for Bacterial Cell Wall-Targeted Delivery of Antibiotics","volume":"6","author":"Lu","year":"2012","journal-title":"ACS Nano"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"629","DOI":"10.1080\/09205063.2015.1053170","article-title":"Alendronate-decorated biodegradable polymeric micelles for potential bone-targeted delivery of vancomycin","volume":"26","author":"Cong","year":"2015","journal-title":"J. Biomater. Sci. Polym. Ed."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"196","DOI":"10.22203\/eCM.v027a15","article-title":"A systematic review of animal models for Staphylococcus aureus osteomyelitis","volume":"27","author":"Reizner","year":"2014","journal-title":"Eur. Cells Mater."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"42","DOI":"10.1590\/S0102-86502004000700009","article-title":"Model of septic arthritis by intravenous inoculation of Staphylococcus aureus in Wistar rats","volume":"19","author":"Batista","year":"2004","journal-title":"Acta Cir. Bras."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"19","DOI":"10.1186\/1678-9199-20-19","article-title":"Septic arthritis: Immunopathogenesis, experimental models and therapy","volume":"20","author":"Colavite","year":"2014","journal-title":"J. Venom. Anim. Toxins Incl. Trop. Dis."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"2479","DOI":"10.1128\/JCM.37.8.2479-2482.1999","article-title":"PCR and Blood Culture for Detection of Escherichia coli Bacteremia in Rats","volume":"37","author":"Heininger","year":"1999","journal-title":"J. Clin. Microbiol."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"1228","DOI":"10.1021\/la303733w","article-title":"Hydrophobic Core\/Hydrophilic Shell Structured Mesoporous Silica Nanospheres: Enhanced Adsorption of Organic Compounds from Water","volume":"29","author":"Li","year":"2013","journal-title":"Langmuir"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"154","DOI":"10.4161\/pri.2.4.7490","article-title":"The elaborate structure of spider silk","volume":"2","author":"Scheibel","year":"2008","journal-title":"Prion"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"1445","DOI":"10.1056\/NEJM199611073351907","article-title":"Antimicrobial-Drug Resistance","volume":"335","author":"Gold","year":"1996","journal-title":"N. Engl. J. Med."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"1","DOI":"10.4172\/2155-952X.1000e111","article-title":"Proteases as Selective Activators Of Triggered Drug Release: A Potential Answer To The Problem Of Biomaterial-Associated Infections?","volume":"2","author":"Gilmore","year":"2012","journal-title":"J. Biotechnol. Biomater."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"413","DOI":"10.1002\/cssc.200800030","article-title":"Processing Conditions for the Formation of Spider Silk Microspheres","volume":"1","author":"Lammel","year":"2008","journal-title":"ChemSusChem"},{"key":"ref_49","unstructured":"(2019, August 08). Septic Arthritis\u2014Diagnosis and Treatment\u2014Mayo Clinic. Available online: https:\/\/www.mayoclinic.org\/diseases-conditions\/bone-and-joint-infections\/diagnosis-treatment\/drc-20350760."}],"container-title":["Pharmaceutics"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4923\/13\/9\/1358\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T06:54:38Z","timestamp":1760165678000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4923\/13\/9\/1358"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,8,28]]},"references-count":49,"journal-issue":{"issue":"9","published-online":{"date-parts":[[2021,9]]}},"alternative-id":["pharmaceutics13091358"],"URL":"https:\/\/doi.org\/10.3390\/pharmaceutics13091358","relation":{},"ISSN":["1999-4923"],"issn-type":[{"value":"1999-4923","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,8,28]]}}}