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Direct Cytosolic Delivery of Proteins Using Lyophilized and Reconstituted Polymer-Protein Assemblies
Purpose Cytosolic delivery of proteins accesses intracellular targets for chemotherapy and immunomodulation. Current delivery systems utilize inefficient endosomal pathways of uptake and escape that lead to degradation of delivered cargo. Cationic poly(oxanorbornene)imide (PONI) polymers enable high...
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| Published in: | Pharmaceutical research 2022-06, Vol.39 (6), p.1197-1204 |
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| creator | Luther, David C. Nagaraj, Harini Goswami, Ritabrita Çiçek, Yağız Anıl Jeon, Taewon Gopalakrishnan, Sanjana Rotello, Vincent M. |
| description | Purpose
Cytosolic delivery of proteins accesses intracellular targets for chemotherapy and immunomodulation. Current delivery systems utilize inefficient endosomal pathways of uptake and escape that lead to degradation of delivered cargo. Cationic poly(oxanorbornene)imide (PONI) polymers enable highly efficient cytosolic delivery of co-engineered proteins, but aggregation and denaturation in solution limits shelf life. In the present study we evaluate polymer-protein nanocomposite vehicles as candidates for lyophilization and point-of-care resuspension to provide a transferrable technology for cytosolic protein delivery.
Methods
Self-assembled nanocomposites of engineered poly(glutamate)-tagged (E-tagged) proteins and guanidinium-functionalized PONI homopolymers were generated, lyophilized, and stored for 2 weeks. After reconstitution and delivery, cytosolic access of E-tagged GFP cargo (GFP
E15
) was assessed through diffuse cytosolic and nuclear fluorescence
,
and cell killing with chemotherapeutic enzyme Granzyme A (GrA
E10
). Efficiency was quantified between freshly prepared and lyophilized samples.
Results
Reconstituted nanocomposites retained key structural features of freshly prepared assemblies, with minimal loss of material. Cytosolic delivery (> 80% efficiency of freshly prepared nanocomposites) of GFP
E15
was validated in several cell lines, with intracellular access validated and quantified through diffusion into the nucleus. Delivery of GrA
E10
elicited significant tumorigenic cell death. Intracellular access of cytotoxic protein was validated through cell viability.
Conclusion
Reconstituted nanocomposites achieved efficient cytosolic delivery of protein cargo and demonstrated therapeutic applicability with delivery of GrA
E10
. Overall, this strategy represents a versatile and highly translatable method for cytosolic delivery of proteins.
Graphical Abstract |
| doi_str_mv | 10.1007/s11095-022-03226-w |
| format | article |
| fullrecord | <record><control><sourceid>gale_proqu</sourceid><recordid>TN_cdi_proquest_miscellaneous_2640323518</recordid><sourceformat>XML</sourceformat><sourcesystem>PC</sourcesystem><galeid>A747439633</galeid><sourcerecordid>A747439633</sourcerecordid><originalsourceid>FETCH-LOGICAL-c486t-d42c082fcdc0fec1e00eebf46d357b1ca4e61e459afb49e87532386b2b6e71e93</originalsourceid><addsrcrecordid>eNp9kU2LFDEQhoMo7rj6BzxIwIuXXvPVSec4zLoqDLiIC95Cd7oyZunujEl6l95fb9YZPxGpQ0HleV8q9SL0nJIzSoh6nSgluq4IYxXhjMnq9gFa0VrxShPx-SFaEcVE1ShBT9CTlK4JIQ3V4jE64TXTSuhmhfpzH8FmvFlySGHwFp_D4G8gLjg4fBlDBj8lfJX8tMPbJey_-MHfQY_bqccfwYYpZZ_nXCaXYVhGiNVRhNcpwdgNHtJT9Mi1Q4Jnx36Kri7efNq8q7Yf3r7frLeVFY3MVS-YJQ1ztrfEgaVACEDnhOx5rTpqWwGSgqh16zqhoVE1Z7yRHeskKAqan6JXB999DF9nSNmMPlkYhnaCMCfDpCiH4jVtCvryL_Q6zHEq2xVKSa4b_Tu1awcwfnIhx9bem5q1EkpwLTkv1Nk_qFI9jL5cCJwv8z8E7CCwMaQUwZl99GMbF0OJuY_WHKI1JVrzPVpzW0QvjhvP3Qj9T8mPLAvAD0AqT9MO4q8v_cf2G3LDr0A</addsrcrecordid><sourcetype>Aggregation Database</sourcetype><iscdi>true</iscdi><recordtype>article</recordtype><pqid>2676398918</pqid></control><display><type>article</type><title>Direct Cytosolic Delivery of Proteins Using Lyophilized and Reconstituted Polymer-Protein Assemblies</title><source>Springer Nature</source><creator>Luther, David C. ; Nagaraj, Harini ; Goswami, Ritabrita ; Çiçek, Yağız Anıl ; Jeon, Taewon ; Gopalakrishnan, Sanjana ; Rotello, Vincent M.</creator><creatorcontrib>Luther, David C. ; Nagaraj, Harini ; Goswami, Ritabrita ; Çiçek, Yağız Anıl ; Jeon, Taewon ; Gopalakrishnan, Sanjana ; Rotello, Vincent M.</creatorcontrib><description>Purpose
Cytosolic delivery of proteins accesses intracellular targets for chemotherapy and immunomodulation. Current delivery systems utilize inefficient endosomal pathways of uptake and escape that lead to degradation of delivered cargo. Cationic poly(oxanorbornene)imide (PONI) polymers enable highly efficient cytosolic delivery of co-engineered proteins, but aggregation and denaturation in solution limits shelf life. In the present study we evaluate polymer-protein nanocomposite vehicles as candidates for lyophilization and point-of-care resuspension to provide a transferrable technology for cytosolic protein delivery.
Methods
Self-assembled nanocomposites of engineered poly(glutamate)-tagged (E-tagged) proteins and guanidinium-functionalized PONI homopolymers were generated, lyophilized, and stored for 2 weeks. After reconstitution and delivery, cytosolic access of E-tagged GFP cargo (GFP
E15
) was assessed through diffuse cytosolic and nuclear fluorescence
,
and cell killing with chemotherapeutic enzyme Granzyme A (GrA
E10
). Efficiency was quantified between freshly prepared and lyophilized samples.
Results
Reconstituted nanocomposites retained key structural features of freshly prepared assemblies, with minimal loss of material. Cytosolic delivery (> 80% efficiency of freshly prepared nanocomposites) of GFP
E15
was validated in several cell lines, with intracellular access validated and quantified through diffusion into the nucleus. Delivery of GrA
E10
elicited significant tumorigenic cell death. Intracellular access of cytotoxic protein was validated through cell viability.
Conclusion
Reconstituted nanocomposites achieved efficient cytosolic delivery of protein cargo and demonstrated therapeutic applicability with delivery of GrA
E10
. Overall, this strategy represents a versatile and highly translatable method for cytosolic delivery of proteins.
Graphical Abstract</description><identifier>ISSN: 0724-8741</identifier><identifier>EISSN: 1573-904X</identifier><identifier>DOI: 10.1007/s11095-022-03226-w</identifier><identifier>PMID: 35297498</identifier><language>eng</language><publisher>New York: Springer US</publisher><subject>Biochemistry ; Biomedical and Life Sciences ; Biomedical Engineering and Bioengineering ; Biomedicine ; Cancer ; Cell death ; Cell lines ; Cell viability ; Chemotherapy ; Cytosol - metabolism ; Cytotoxicity ; Denaturation ; Endosomes - metabolism ; Enzymes ; Ethylenediaminetetraacetic acid ; Freeze Drying ; Glutamate ; Immunomodulation ; Intracellular ; Medical Law ; Nanocomposites ; Pharmacology/Toxicology ; Pharmacy ; Polymers ; Polymers - chemistry ; Proteins ; Proteins - chemistry ; Research Paper ; Shelf life ; Technology application</subject><ispartof>Pharmaceutical research, 2022-06, Vol.39 (6), p.1197-1204</ispartof><rights>The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2022</rights><rights>2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.</rights><rights>COPYRIGHT 2022 Springer</rights><rights>The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2022.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c486t-d42c082fcdc0fec1e00eebf46d357b1ca4e61e459afb49e87532386b2b6e71e93</citedby><cites>FETCH-LOGICAL-c486t-d42c082fcdc0fec1e00eebf46d357b1ca4e61e459afb49e87532386b2b6e71e93</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,780,784,27924,27925</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/35297498$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Luther, David C.</creatorcontrib><creatorcontrib>Nagaraj, Harini</creatorcontrib><creatorcontrib>Goswami, Ritabrita</creatorcontrib><creatorcontrib>Çiçek, Yağız Anıl</creatorcontrib><creatorcontrib>Jeon, Taewon</creatorcontrib><creatorcontrib>Gopalakrishnan, Sanjana</creatorcontrib><creatorcontrib>Rotello, Vincent M.</creatorcontrib><title>Direct Cytosolic Delivery of Proteins Using Lyophilized and Reconstituted Polymer-Protein Assemblies</title><title>Pharmaceutical research</title><addtitle>Pharm Res</addtitle><addtitle>Pharm Res</addtitle><description>Purpose
Cytosolic delivery of proteins accesses intracellular targets for chemotherapy and immunomodulation. Current delivery systems utilize inefficient endosomal pathways of uptake and escape that lead to degradation of delivered cargo. Cationic poly(oxanorbornene)imide (PONI) polymers enable highly efficient cytosolic delivery of co-engineered proteins, but aggregation and denaturation in solution limits shelf life. In the present study we evaluate polymer-protein nanocomposite vehicles as candidates for lyophilization and point-of-care resuspension to provide a transferrable technology for cytosolic protein delivery.
Methods
Self-assembled nanocomposites of engineered poly(glutamate)-tagged (E-tagged) proteins and guanidinium-functionalized PONI homopolymers were generated, lyophilized, and stored for 2 weeks. After reconstitution and delivery, cytosolic access of E-tagged GFP cargo (GFP
E15
) was assessed through diffuse cytosolic and nuclear fluorescence
,
and cell killing with chemotherapeutic enzyme Granzyme A (GrA
E10
). Efficiency was quantified between freshly prepared and lyophilized samples.
Results
Reconstituted nanocomposites retained key structural features of freshly prepared assemblies, with minimal loss of material. Cytosolic delivery (> 80% efficiency of freshly prepared nanocomposites) of GFP
E15
was validated in several cell lines, with intracellular access validated and quantified through diffusion into the nucleus. Delivery of GrA
E10
elicited significant tumorigenic cell death. Intracellular access of cytotoxic protein was validated through cell viability.
Conclusion
Reconstituted nanocomposites achieved efficient cytosolic delivery of protein cargo and demonstrated therapeutic applicability with delivery of GrA
E10
. Overall, this strategy represents a versatile and highly translatable method for cytosolic delivery of proteins.
Graphical Abstract</description><subject>Biochemistry</subject><subject>Biomedical and Life Sciences</subject><subject>Biomedical Engineering and Bioengineering</subject><subject>Biomedicine</subject><subject>Cancer</subject><subject>Cell death</subject><subject>Cell lines</subject><subject>Cell viability</subject><subject>Chemotherapy</subject><subject>Cytosol - metabolism</subject><subject>Cytotoxicity</subject><subject>Denaturation</subject><subject>Endosomes - metabolism</subject><subject>Enzymes</subject><subject>Ethylenediaminetetraacetic acid</subject><subject>Freeze Drying</subject><subject>Glutamate</subject><subject>Immunomodulation</subject><subject>Intracellular</subject><subject>Medical Law</subject><subject>Nanocomposites</subject><subject>Pharmacology/Toxicology</subject><subject>Pharmacy</subject><subject>Polymers</subject><subject>Polymers - chemistry</subject><subject>Proteins</subject><subject>Proteins - chemistry</subject><subject>Research Paper</subject><subject>Shelf life</subject><subject>Technology application</subject><issn>0724-8741</issn><issn>1573-904X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2022</creationdate><recordtype>article</recordtype><recordid>eNp9kU2LFDEQhoMo7rj6BzxIwIuXXvPVSec4zLoqDLiIC95Cd7oyZunujEl6l95fb9YZPxGpQ0HleV8q9SL0nJIzSoh6nSgluq4IYxXhjMnq9gFa0VrxShPx-SFaEcVE1ShBT9CTlK4JIQ3V4jE64TXTSuhmhfpzH8FmvFlySGHwFp_D4G8gLjg4fBlDBj8lfJX8tMPbJey_-MHfQY_bqccfwYYpZZ_nXCaXYVhGiNVRhNcpwdgNHtJT9Mi1Q4Jnx36Kri7efNq8q7Yf3r7frLeVFY3MVS-YJQ1ztrfEgaVACEDnhOx5rTpqWwGSgqh16zqhoVE1Z7yRHeskKAqan6JXB999DF9nSNmMPlkYhnaCMCfDpCiH4jVtCvryL_Q6zHEq2xVKSa4b_Tu1awcwfnIhx9bem5q1EkpwLTkv1Nk_qFI9jL5cCJwv8z8E7CCwMaQUwZl99GMbF0OJuY_WHKI1JVrzPVpzW0QvjhvP3Qj9T8mPLAvAD0AqT9MO4q8v_cf2G3LDr0A</recordid><startdate>20220601</startdate><enddate>20220601</enddate><creator>Luther, David C.</creator><creator>Nagaraj, Harini</creator><creator>Goswami, Ritabrita</creator><creator>Çiçek, Yağız Anıl</creator><creator>Jeon, Taewon</creator><creator>Gopalakrishnan, Sanjana</creator><creator>Rotello, Vincent M.</creator><general>Springer US</general><general>Springer</general><general>Springer Nature B.V</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>3V.</scope><scope>7RV</scope><scope>7TK</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8AO</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>BENPR</scope><scope>CCPQU</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>K9.</scope><scope>KB0</scope><scope>M0S</scope><scope>M1P</scope><scope>NAPCQ</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>7X8</scope></search><sort><creationdate>20220601</creationdate><title>Direct Cytosolic Delivery of Proteins Using Lyophilized and Reconstituted Polymer-Protein Assemblies</title><author>Luther, David C. ; Nagaraj, Harini ; Goswami, Ritabrita ; Çiçek, Yağız Anıl ; Jeon, Taewon ; Gopalakrishnan, Sanjana ; Rotello, Vincent M.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c486t-d42c082fcdc0fec1e00eebf46d357b1ca4e61e459afb49e87532386b2b6e71e93</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2022</creationdate><topic>Biochemistry</topic><topic>Biomedical and Life Sciences</topic><topic>Biomedical Engineering and Bioengineering</topic><topic>Biomedicine</topic><topic>Cancer</topic><topic>Cell death</topic><topic>Cell lines</topic><topic>Cell viability</topic><topic>Chemotherapy</topic><topic>Cytosol - metabolism</topic><topic>Cytotoxicity</topic><topic>Denaturation</topic><topic>Endosomes - metabolism</topic><topic>Enzymes</topic><topic>Ethylenediaminetetraacetic acid</topic><topic>Freeze Drying</topic><topic>Glutamate</topic><topic>Immunomodulation</topic><topic>Intracellular</topic><topic>Medical Law</topic><topic>Nanocomposites</topic><topic>Pharmacology/Toxicology</topic><topic>Pharmacy</topic><topic>Polymers</topic><topic>Polymers - chemistry</topic><topic>Proteins</topic><topic>Proteins - chemistry</topic><topic>Research Paper</topic><topic>Shelf life</topic><topic>Technology application</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Luther, David C.</creatorcontrib><creatorcontrib>Nagaraj, Harini</creatorcontrib><creatorcontrib>Goswami, Ritabrita</creatorcontrib><creatorcontrib>Çiçek, Yağız Anıl</creatorcontrib><creatorcontrib>Jeon, Taewon</creatorcontrib><creatorcontrib>Gopalakrishnan, Sanjana</creatorcontrib><creatorcontrib>Rotello, Vincent M.</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>ProQuest Central (Corporate)</collection><collection>Nursing & Allied Health Database</collection><collection>Neurosciences Abstracts</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Medical Database (Alumni Edition)</collection><collection>ProQuest Pharma Collection</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>ProQuest Central (Alumni)</collection><collection>ProQuest Central</collection><collection>ProQuest Central</collection><collection>ProQuest One Community College</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>Nursing & Allied Health Database (Alumni Edition)</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>PML(ProQuest Medical Library)</collection><collection>Nursing & Allied Health Premium</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>MEDLINE - Academic</collection><jtitle>Pharmaceutical research</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Luther, David C.</au><au>Nagaraj, Harini</au><au>Goswami, Ritabrita</au><au>Çiçek, Yağız Anıl</au><au>Jeon, Taewon</au><au>Gopalakrishnan, Sanjana</au><au>Rotello, Vincent M.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Direct Cytosolic Delivery of Proteins Using Lyophilized and Reconstituted Polymer-Protein Assemblies</atitle><jtitle>Pharmaceutical research</jtitle><stitle>Pharm Res</stitle><addtitle>Pharm Res</addtitle><date>2022-06-01</date><risdate>2022</risdate><volume>39</volume><issue>6</issue><spage>1197</spage><epage>1204</epage><pages>1197-1204</pages><issn>0724-8741</issn><eissn>1573-904X</eissn><abstract>Purpose
Cytosolic delivery of proteins accesses intracellular targets for chemotherapy and immunomodulation. Current delivery systems utilize inefficient endosomal pathways of uptake and escape that lead to degradation of delivered cargo. Cationic poly(oxanorbornene)imide (PONI) polymers enable highly efficient cytosolic delivery of co-engineered proteins, but aggregation and denaturation in solution limits shelf life. In the present study we evaluate polymer-protein nanocomposite vehicles as candidates for lyophilization and point-of-care resuspension to provide a transferrable technology for cytosolic protein delivery.
Methods
Self-assembled nanocomposites of engineered poly(glutamate)-tagged (E-tagged) proteins and guanidinium-functionalized PONI homopolymers were generated, lyophilized, and stored for 2 weeks. After reconstitution and delivery, cytosolic access of E-tagged GFP cargo (GFP
E15
) was assessed through diffuse cytosolic and nuclear fluorescence
,
and cell killing with chemotherapeutic enzyme Granzyme A (GrA
E10
). Efficiency was quantified between freshly prepared and lyophilized samples.
Results
Reconstituted nanocomposites retained key structural features of freshly prepared assemblies, with minimal loss of material. Cytosolic delivery (> 80% efficiency of freshly prepared nanocomposites) of GFP
E15
was validated in several cell lines, with intracellular access validated and quantified through diffusion into the nucleus. Delivery of GrA
E10
elicited significant tumorigenic cell death. Intracellular access of cytotoxic protein was validated through cell viability.
Conclusion
Reconstituted nanocomposites achieved efficient cytosolic delivery of protein cargo and demonstrated therapeutic applicability with delivery of GrA
E10
. Overall, this strategy represents a versatile and highly translatable method for cytosolic delivery of proteins.
Graphical Abstract</abstract><cop>New York</cop><pub>Springer US</pub><pmid>35297498</pmid><doi>10.1007/s11095-022-03226-w</doi><tpages>8</tpages><oa>free_for_read</oa></addata></record> |
| fulltext | fulltext |
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| ispartof | Pharmaceutical research, 2022-06, Vol.39 (6), p.1197-1204 |
| issn | 0724-8741 1573-904X |
| language | eng |
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| source | Springer Nature |
| subjects | Biochemistry Biomedical and Life Sciences Biomedical Engineering and Bioengineering Biomedicine Cancer Cell death Cell lines Cell viability Chemotherapy Cytosol - metabolism Cytotoxicity Denaturation Endosomes - metabolism Enzymes Ethylenediaminetetraacetic acid Freeze Drying Glutamate Immunomodulation Intracellular Medical Law Nanocomposites Pharmacology/Toxicology Pharmacy Polymers Polymers - chemistry Proteins Proteins - chemistry Research Paper Shelf life Technology application |
| title | Direct Cytosolic Delivery of Proteins Using Lyophilized and Reconstituted Polymer-Protein Assemblies |
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