Multivalent HER2-binding polymer conjugates facilitate rapid endocytosis and enhance intracellular drug delivery

D. Christopher Radford; Jiyuan Yang; Mai C. Doan; Lian Li; Andrew S. Dixon; Shawn C. Owen; Jindřich Kopeček

doi:10.1016/j.jconrel.2019.12.049

Multivalent HER2-binding polymer conjugates facilitate rapid endocytosis and enhance intracellular drug delivery

D. Christopher Radford, Jiyuan Yang, Mai C. Doan, Lian Li, Andrew S. Dixon, Shawn C. Owen, Jindřich Kopeček

Research output: Contribution to journal › Article › peer-review

32 Scopus citations

Abstract

Incorporating targeting moieties that recognize cancer-specific cellular markers can enhance specificity of anticancer nanomedicines. The HER2 receptor is overexpressed on numerous cancers, making it an attractive target. However, unlike many receptors that trigger endocytosis upon ligand binding, HER2 is an internalization-resistant receptor. As most chemotherapeutics act on intracellular targets, this presents a significant challenge for exploiting HER2 overexpression for improved tumor killing. However, hyper-crosslinking of HER2 has been shown to override the receptor's native behavior and trigger internalization. This research co-opts this crosslinking-mediated internalization for efficient intracellular delivery of an anticancer nanomedicine – specifically a HPMA copolymer-based drug delivery system. This polymeric carrier was conjugated with a small (7 kDa) HER2-binding affibody peptide to produce a panel of polymer-affibody conjugates with valences from 2 to 10 peptides per polymer chain. The effect of valence on surface binding and uptake was evaluated separately. All conjugates demonstrated similar (nanomolar) binding affinity towards HER2-positive ovarian carcinoma cells, but higher-valence conjugates induced more rapid endocytosis, with over 90% of the surface-bound conjugate internalized within 4 h. Furthermore, this enhancement was sensitive to crowding – high surface loading reduced conjugates' ability to crosslink receptors. Collectively, this evidence strongly supports a crosslinking-mediated endocytosis mechanism. Lead candidates from this panel achieved high intracellular delivery even at picomolar treatment concentrations; untargeted HPMA copolymers required 1000-fold higher treatment concentrations to achieve similar levels of intracellular accumulation. This increased intracellular delivery also translated to a more potent nanomedicine against HER2-positive cells; incorporation of the chemotherapeutic paclitaxel into this targeted carrier enhanced cytotoxicity over untargeted polymer-drug conjugate.

Original language	English
Pages (from-to)	285-299
Number of pages	15
Journal	Journal of Controlled Release
Volume	319
DOIs	https://doi.org/10.1016/j.jconrel.2019.12.049
State	Published - Mar 10 2020
Externally published	Yes

Funding

The work was supported in part by NIH grants R42 CA156933 (to JK), F31 CA203476 (to DCR), and J.K.'s development funds. ESI mass spectrometry analysis was performed at the Mass Spectrometry and Proteomics Core Facility at the University of Utah. Mass spectrometry equipment was obtained through a Shared Instrumentation Grant 1S10 OD018210 01A1. JY and JK are co-inventors of US patent 9,289,510 related to the technology used in the manuscript that the University of Utah licensed to TheraTarget. Otherwise, the authors declare no competing financial interests. The work was supported in part by NIH grants R42 CA156933 (to JK), F31 CA203476 (to DCR), and J.K.'s development funds. ESI mass spectrometry analysis was performed at the Mass Spectrometry and Proteomics Core Facility at the University of Utah. Mass spectrometry equipment was obtained through a Shared Instrumentation Grant 1S10 OD018210 01A1 .

Funders	Funder number
Mass Spectrometry and Proteomics Core Facility
National Institutes of Health	F31 CA203476
National Cancer Institute	R42CA156933
University of Utah	1S10 OD018210 01A1

Keywords

Affibody
Endocytosis
HER2
HPMA copolymers
Paclitaxel
Receptor crosslinking

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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10.1016/j.jconrel.2019.12.049

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title = "Multivalent HER2-binding polymer conjugates facilitate rapid endocytosis and enhance intracellular drug delivery",

abstract = "Incorporating targeting moieties that recognize cancer-specific cellular markers can enhance specificity of anticancer nanomedicines. The HER2 receptor is overexpressed on numerous cancers, making it an attractive target. However, unlike many receptors that trigger endocytosis upon ligand binding, HER2 is an internalization-resistant receptor. As most chemotherapeutics act on intracellular targets, this presents a significant challenge for exploiting HER2 overexpression for improved tumor killing. However, hyper-crosslinking of HER2 has been shown to override the receptor's native behavior and trigger internalization. This research co-opts this crosslinking-mediated internalization for efficient intracellular delivery of an anticancer nanomedicine – specifically a HPMA copolymer-based drug delivery system. This polymeric carrier was conjugated with a small (7 kDa) HER2-binding affibody peptide to produce a panel of polymer-affibody conjugates with valences from 2 to 10 peptides per polymer chain. The effect of valence on surface binding and uptake was evaluated separately. All conjugates demonstrated similar (nanomolar) binding affinity towards HER2-positive ovarian carcinoma cells, but higher-valence conjugates induced more rapid endocytosis, with over 90% of the surface-bound conjugate internalized within 4 h. Furthermore, this enhancement was sensitive to crowding – high surface loading reduced conjugates' ability to crosslink receptors. Collectively, this evidence strongly supports a crosslinking-mediated endocytosis mechanism. Lead candidates from this panel achieved high intracellular delivery even at picomolar treatment concentrations; untargeted HPMA copolymers required 1000-fold higher treatment concentrations to achieve similar levels of intracellular accumulation. This increased intracellular delivery also translated to a more potent nanomedicine against HER2-positive cells; incorporation of the chemotherapeutic paclitaxel into this targeted carrier enhanced cytotoxicity over untargeted polymer-drug conjugate.",

keywords = "Affibody, Endocytosis, HER2, HPMA copolymers, Paclitaxel, Receptor crosslinking",

author = "Radford, {D. Christopher} and Jiyuan Yang and Doan, {Mai C.} and Lian Li and Dixon, {Andrew S.} and Owen, {Shawn C.} and Jind{\v r}ich Kope{\v c}ek",

note = "Publisher Copyright: {\textcopyright} 2020 Elsevier B.V.",

year = "2020",

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day = "10",

doi = "10.1016/j.jconrel.2019.12.049",

language = "English",

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T1 - Multivalent HER2-binding polymer conjugates facilitate rapid endocytosis and enhance intracellular drug delivery

AU - Radford, D. Christopher

AU - Yang, Jiyuan

AU - Doan, Mai C.

AU - Li, Lian

AU - Dixon, Andrew S.

AU - Owen, Shawn C.

AU - Kopeček, Jindřich

PY - 2020/3/10

Y1 - 2020/3/10

N2 - Incorporating targeting moieties that recognize cancer-specific cellular markers can enhance specificity of anticancer nanomedicines. The HER2 receptor is overexpressed on numerous cancers, making it an attractive target. However, unlike many receptors that trigger endocytosis upon ligand binding, HER2 is an internalization-resistant receptor. As most chemotherapeutics act on intracellular targets, this presents a significant challenge for exploiting HER2 overexpression for improved tumor killing. However, hyper-crosslinking of HER2 has been shown to override the receptor's native behavior and trigger internalization. This research co-opts this crosslinking-mediated internalization for efficient intracellular delivery of an anticancer nanomedicine – specifically a HPMA copolymer-based drug delivery system. This polymeric carrier was conjugated with a small (7 kDa) HER2-binding affibody peptide to produce a panel of polymer-affibody conjugates with valences from 2 to 10 peptides per polymer chain. The effect of valence on surface binding and uptake was evaluated separately. All conjugates demonstrated similar (nanomolar) binding affinity towards HER2-positive ovarian carcinoma cells, but higher-valence conjugates induced more rapid endocytosis, with over 90% of the surface-bound conjugate internalized within 4 h. Furthermore, this enhancement was sensitive to crowding – high surface loading reduced conjugates' ability to crosslink receptors. Collectively, this evidence strongly supports a crosslinking-mediated endocytosis mechanism. Lead candidates from this panel achieved high intracellular delivery even at picomolar treatment concentrations; untargeted HPMA copolymers required 1000-fold higher treatment concentrations to achieve similar levels of intracellular accumulation. This increased intracellular delivery also translated to a more potent nanomedicine against HER2-positive cells; incorporation of the chemotherapeutic paclitaxel into this targeted carrier enhanced cytotoxicity over untargeted polymer-drug conjugate.

AB - Incorporating targeting moieties that recognize cancer-specific cellular markers can enhance specificity of anticancer nanomedicines. The HER2 receptor is overexpressed on numerous cancers, making it an attractive target. However, unlike many receptors that trigger endocytosis upon ligand binding, HER2 is an internalization-resistant receptor. As most chemotherapeutics act on intracellular targets, this presents a significant challenge for exploiting HER2 overexpression for improved tumor killing. However, hyper-crosslinking of HER2 has been shown to override the receptor's native behavior and trigger internalization. This research co-opts this crosslinking-mediated internalization for efficient intracellular delivery of an anticancer nanomedicine – specifically a HPMA copolymer-based drug delivery system. This polymeric carrier was conjugated with a small (7 kDa) HER2-binding affibody peptide to produce a panel of polymer-affibody conjugates with valences from 2 to 10 peptides per polymer chain. The effect of valence on surface binding and uptake was evaluated separately. All conjugates demonstrated similar (nanomolar) binding affinity towards HER2-positive ovarian carcinoma cells, but higher-valence conjugates induced more rapid endocytosis, with over 90% of the surface-bound conjugate internalized within 4 h. Furthermore, this enhancement was sensitive to crowding – high surface loading reduced conjugates' ability to crosslink receptors. Collectively, this evidence strongly supports a crosslinking-mediated endocytosis mechanism. Lead candidates from this panel achieved high intracellular delivery even at picomolar treatment concentrations; untargeted HPMA copolymers required 1000-fold higher treatment concentrations to achieve similar levels of intracellular accumulation. This increased intracellular delivery also translated to a more potent nanomedicine against HER2-positive cells; incorporation of the chemotherapeutic paclitaxel into this targeted carrier enhanced cytotoxicity over untargeted polymer-drug conjugate.

KW - Affibody

KW - Endocytosis

KW - HER2

KW - HPMA copolymers

KW - Paclitaxel

KW - Receptor crosslinking

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M3 - Article

C2 - 31899273

AN - SCOPUS:85077394507

SN - 0168-3659

VL - 319

SP - 285

EP - 299

JO - Journal of Controlled Release

JF - Journal of Controlled Release

ER -

Multivalent HER2-binding polymer conjugates facilitate rapid endocytosis and enhance intracellular drug delivery

Abstract

Funding

Keywords

UN SDGs

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