Co-delivery of PD-L1- and EGFR-targeting siRNAs by synthetic PEG
        12-KL4 peptide to the lungs as potential strategy against non-small cell lung cancer.

Rico C H Man; Yingshan Qiu; Susan W S Leung; Gilbert O Fruhwirth; Jenny K W Lam

doi:10.1016/j.ejpb.2024.114177

Co-delivery of PD-L1- and EGFR-targeting siRNAs by synthetic PEG 12-KL4 peptide to the lungs as potential strategy against non-small cell lung cancer.

Rico C H Man, Yingshan Qiu, Susan W S Leung, Gilbert O Fruhwirth, Jenny K W Lam

Comprehensive Cancer Centre

The State Key Laboratory of Brain and Cognitive Sciences, The University of Hong Kong, Hong Kong, China. [email protected].
Imaging Therapies and Cancer Group
School of Cancer and Pharmaceutical Sciences
Univ London, Birkbeck University London, University College London, University of London Royal Veterinary College, University of London, St Georges University London, Imperial College London, Kings College London, Royal Holloway University London, Queen Mary University London, Div Populat Hlth Sci & Educ

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

6 Citations (Scopus)

Abstract

BACKGROUND: Small interfering RNA (siRNA) holds great promise for treating various lung diseases, but the lack of safe and efficient pulmonary siRNA delivery systems has hindered its advance into the clinics. The epidermal growth factor receptor (EGFR) which promotes cell proliferation, and the programmed cell death ligand 1 (PD-L1) which plays a crucial role in suppressing cytotoxic T cells activity, are two important targets for treating non-small cell lung cancer (NSCLC). Here, we explored the potential of PEG 12-KL4, a synthetic peptide, to deliver siRNA to various NSCLC cells and to lung tissues in mice.

METHODS: PEG 12-KL4 was used to transfect siRNAs targeted at both EGFR and PD-L1 into NSCLC cells. Immunoblotting was used to evaluate the siRNA silencing effects in HCC827 and NCI-H1975 NSCLC cells. CD8+ T cell-mediated NSCLC cell killing was employed to demonstrate the functional effects of PD-L1 siRNA knock-down. Fluorescent siRNAs were used to visualise siRNA uptake in cells as well as to enable biodistribution studies in BALB/c mice.

RESULTS: Our results showed that PEG 12-KL4 was efficient in mediating siRNA knock-down of EGFR and PD-L1 in various NSCLC cells. Importantly, the PEG 12-KL4 peptide enabled significantly better siRNA delivery than the commercial Lipofectamine 2000 reagent. We hypothesised that PEG 12-KL4 peptide enabled siRNA to either escape from or bypass endosomal degradation as indicated by confocal fluorescence imaging. Notably, combined knock-down of EGFR and PD-L1 in NCI-H1975 cells resulted in better effector T cell-mediated cancer cell killing than knock-down of PD-L1 alone. Moreover, biodistribution of PEG 12-KL4/siRNA complexes following intravenous administration revealed poor lung delivery with the fluorescent siRNA accumulating in the liver. In contrast, intratracheal delivery of PEG 12-KL4/siRNA complexes resulted in the fluorescent siRNA to be detected in the lung with retarded renal excretion.

CONCLUSION: In conclusion, we demonstrated that the co-delivery of siRNAs targeting EGFR and PD-L1 using PEG 12-KL4 is feasible and represents a promising future strategy to treat NSCLC, whereby pulmonary siRNA delivery is favourable to intravenous administration.

Original language	English
Article number	114177
Pages (from-to)	114177
Journal	EUROPEAN JOURNAL OF PHARMACEUTICS AND BIOPHARMACEUTICS
Volume	195
DOIs	https://doi.org/10.1016/j.ejpb.2024.114177
Publication status	Published - Feb 2024

Keywords

Animals
Mice
Carcinoma, Non-Small-Cell Lung/drug therapy
Lung Neoplasms/drug therapy
B7-H1 Antigen/genetics
RNA, Small Interfering/metabolism
Tissue Distribution
Cell Line, Tumor
ErbB Receptors/genetics
Lung/metabolism
Peptides/metabolism

UN SDGs

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

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10.1016/j.ejpb.2024.114177

Cite this

@article{0f6d17e536d04e4384f77153ebc8f421,

title = "Co-delivery of PD-L1- and EGFR-targeting siRNAs by synthetic PEG 12-KL4 peptide to the lungs as potential strategy against non-small cell lung cancer.",

abstract = "BACKGROUND: Small interfering RNA (siRNA) holds great promise for treating various lung diseases, but the lack of safe and efficient pulmonary siRNA delivery systems has hindered its advance into the clinics. The epidermal growth factor receptor (EGFR) which promotes cell proliferation, and the programmed cell death ligand 1 (PD-L1) which plays a crucial role in suppressing cytotoxic T cells activity, are two important targets for treating non-small cell lung cancer (NSCLC). Here, we explored the potential of PEG 12-KL4, a synthetic peptide, to deliver siRNA to various NSCLC cells and to lung tissues in mice. METHODS: PEG 12-KL4 was used to transfect siRNAs targeted at both EGFR and PD-L1 into NSCLC cells. Immunoblotting was used to evaluate the siRNA silencing effects in HCC827 and NCI-H1975 NSCLC cells. CD8+ T cell-mediated NSCLC cell killing was employed to demonstrate the functional effects of PD-L1 siRNA knock-down. Fluorescent siRNAs were used to visualise siRNA uptake in cells as well as to enable biodistribution studies in BALB/c mice. RESULTS: Our results showed that PEG 12-KL4 was efficient in mediating siRNA knock-down of EGFR and PD-L1 in various NSCLC cells. Importantly, the PEG 12-KL4 peptide enabled significantly better siRNA delivery than the commercial Lipofectamine 2000 reagent. We hypothesised that PEG 12-KL4 peptide enabled siRNA to either escape from or bypass endosomal degradation as indicated by confocal fluorescence imaging. Notably, combined knock-down of EGFR and PD-L1 in NCI-H1975 cells resulted in better effector T cell-mediated cancer cell killing than knock-down of PD-L1 alone. Moreover, biodistribution of PEG 12-KL4/siRNA complexes following intravenous administration revealed poor lung delivery with the fluorescent siRNA accumulating in the liver. In contrast, intratracheal delivery of PEG 12-KL4/siRNA complexes resulted in the fluorescent siRNA to be detected in the lung with retarded renal excretion. CONCLUSION: In conclusion, we demonstrated that the co-delivery of siRNAs targeting EGFR and PD-L1 using PEG 12-KL4 is feasible and represents a promising future strategy to treat NSCLC, whereby pulmonary siRNA delivery is favourable to intravenous administration. ",

keywords = "Animals, Mice, Carcinoma, Non-Small-Cell Lung/drug therapy, Lung Neoplasms/drug therapy, B7-H1 Antigen/genetics, RNA, Small Interfering/metabolism, Tissue Distribution, Cell Line, Tumor, ErbB Receptors/genetics, Lung/metabolism, Peptides/metabolism",

author = "Man, {Rico C H} and Yingshan Qiu and Leung, {Susan W S} and Fruhwirth, {Gilbert O} and Lam, {Jenny K W}",

note = "Funding Information: This work was supported by KCL-HKU Postgraduate Scholarships to RM; General Research Fund, Research Grant Council ( GRF 17300319 ) and Seed Fund Programme for Basic Research, The University of Hong Kong ( 201711159172 ) to JL; Cancer Research UK grant ( C48390/A21153 ) to GOF, the National Institute for Health Research (NIHR) Biomedical Research Centre at Guy's and St Thomas' NHS Foundation Trust and King's College London, and the Wellcome/ EPSRC Centre for Medical Engineering at King's College London [ WT 203148/Z/16/Z ]. Views expressed are those of the authors and not necessarily those of the NIHR or the NHS. The authors would like to thank the Centre for PanorOmic Sciences, LKS Faculty of Medicine, The University of Hong Kong for the assistance in live cell confocal imaging, in vivo imaging and flow cytometry studies. Publisher Copyright: {\textcopyright} 2024 The Author(s)",

year = "2024",

month = feb,

doi = "10.1016/j.ejpb.2024.114177",

language = "English",

volume = "195",

pages = "114177",

journal = "EUROPEAN JOURNAL OF PHARMACEUTICS AND BIOPHARMACEUTICS",

issn = "0939-6411",

publisher = "Elsevier",

}

Co-delivery of PD-L1- and EGFR-targeting siRNAs by synthetic PEG 12-KL4 peptide to the lungs as potential strategy against non-small cell lung cancer. / Man, Rico C H; Qiu, Yingshan; Leung, Susan W S et al.
In: EUROPEAN JOURNAL OF PHARMACEUTICS AND BIOPHARMACEUTICS, Vol. 195, 114177, 02.2024, p. 114177.

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

TY - JOUR

T1 - Co-delivery of PD-L1- and EGFR-targeting siRNAs by synthetic PEG 12-KL4 peptide to the lungs as potential strategy against non-small cell lung cancer.

AU - Man, Rico C H

AU - Qiu, Yingshan

AU - Leung, Susan W S

AU - Fruhwirth, Gilbert O

AU - Lam, Jenny K W

N1 - Funding Information: This work was supported by KCL-HKU Postgraduate Scholarships to RM; General Research Fund, Research Grant Council ( GRF 17300319 ) and Seed Fund Programme for Basic Research, The University of Hong Kong ( 201711159172 ) to JL; Cancer Research UK grant ( C48390/A21153 ) to GOF, the National Institute for Health Research (NIHR) Biomedical Research Centre at Guy's and St Thomas' NHS Foundation Trust and King's College London, and the Wellcome/ EPSRC Centre for Medical Engineering at King's College London [ WT 203148/Z/16/Z ]. Views expressed are those of the authors and not necessarily those of the NIHR or the NHS. The authors would like to thank the Centre for PanorOmic Sciences, LKS Faculty of Medicine, The University of Hong Kong for the assistance in live cell confocal imaging, in vivo imaging and flow cytometry studies. Publisher Copyright: © 2024 The Author(s)

PY - 2024/2

Y1 - 2024/2

N2 - BACKGROUND: Small interfering RNA (siRNA) holds great promise for treating various lung diseases, but the lack of safe and efficient pulmonary siRNA delivery systems has hindered its advance into the clinics. The epidermal growth factor receptor (EGFR) which promotes cell proliferation, and the programmed cell death ligand 1 (PD-L1) which plays a crucial role in suppressing cytotoxic T cells activity, are two important targets for treating non-small cell lung cancer (NSCLC). Here, we explored the potential of PEG 12-KL4, a synthetic peptide, to deliver siRNA to various NSCLC cells and to lung tissues in mice. METHODS: PEG 12-KL4 was used to transfect siRNAs targeted at both EGFR and PD-L1 into NSCLC cells. Immunoblotting was used to evaluate the siRNA silencing effects in HCC827 and NCI-H1975 NSCLC cells. CD8+ T cell-mediated NSCLC cell killing was employed to demonstrate the functional effects of PD-L1 siRNA knock-down. Fluorescent siRNAs were used to visualise siRNA uptake in cells as well as to enable biodistribution studies in BALB/c mice. RESULTS: Our results showed that PEG 12-KL4 was efficient in mediating siRNA knock-down of EGFR and PD-L1 in various NSCLC cells. Importantly, the PEG 12-KL4 peptide enabled significantly better siRNA delivery than the commercial Lipofectamine 2000 reagent. We hypothesised that PEG 12-KL4 peptide enabled siRNA to either escape from or bypass endosomal degradation as indicated by confocal fluorescence imaging. Notably, combined knock-down of EGFR and PD-L1 in NCI-H1975 cells resulted in better effector T cell-mediated cancer cell killing than knock-down of PD-L1 alone. Moreover, biodistribution of PEG 12-KL4/siRNA complexes following intravenous administration revealed poor lung delivery with the fluorescent siRNA accumulating in the liver. In contrast, intratracheal delivery of PEG 12-KL4/siRNA complexes resulted in the fluorescent siRNA to be detected in the lung with retarded renal excretion. CONCLUSION: In conclusion, we demonstrated that the co-delivery of siRNAs targeting EGFR and PD-L1 using PEG 12-KL4 is feasible and represents a promising future strategy to treat NSCLC, whereby pulmonary siRNA delivery is favourable to intravenous administration.

AB - BACKGROUND: Small interfering RNA (siRNA) holds great promise for treating various lung diseases, but the lack of safe and efficient pulmonary siRNA delivery systems has hindered its advance into the clinics. The epidermal growth factor receptor (EGFR) which promotes cell proliferation, and the programmed cell death ligand 1 (PD-L1) which plays a crucial role in suppressing cytotoxic T cells activity, are two important targets for treating non-small cell lung cancer (NSCLC). Here, we explored the potential of PEG 12-KL4, a synthetic peptide, to deliver siRNA to various NSCLC cells and to lung tissues in mice. METHODS: PEG 12-KL4 was used to transfect siRNAs targeted at both EGFR and PD-L1 into NSCLC cells. Immunoblotting was used to evaluate the siRNA silencing effects in HCC827 and NCI-H1975 NSCLC cells. CD8+ T cell-mediated NSCLC cell killing was employed to demonstrate the functional effects of PD-L1 siRNA knock-down. Fluorescent siRNAs were used to visualise siRNA uptake in cells as well as to enable biodistribution studies in BALB/c mice. RESULTS: Our results showed that PEG 12-KL4 was efficient in mediating siRNA knock-down of EGFR and PD-L1 in various NSCLC cells. Importantly, the PEG 12-KL4 peptide enabled significantly better siRNA delivery than the commercial Lipofectamine 2000 reagent. We hypothesised that PEG 12-KL4 peptide enabled siRNA to either escape from or bypass endosomal degradation as indicated by confocal fluorescence imaging. Notably, combined knock-down of EGFR and PD-L1 in NCI-H1975 cells resulted in better effector T cell-mediated cancer cell killing than knock-down of PD-L1 alone. Moreover, biodistribution of PEG 12-KL4/siRNA complexes following intravenous administration revealed poor lung delivery with the fluorescent siRNA accumulating in the liver. In contrast, intratracheal delivery of PEG 12-KL4/siRNA complexes resulted in the fluorescent siRNA to be detected in the lung with retarded renal excretion. CONCLUSION: In conclusion, we demonstrated that the co-delivery of siRNAs targeting EGFR and PD-L1 using PEG 12-KL4 is feasible and represents a promising future strategy to treat NSCLC, whereby pulmonary siRNA delivery is favourable to intravenous administration.

KW - Animals

KW - Mice

KW - Carcinoma, Non-Small-Cell Lung/drug therapy

KW - Lung Neoplasms/drug therapy

KW - B7-H1 Antigen/genetics

KW - RNA, Small Interfering/metabolism

KW - Tissue Distribution

KW - Cell Line, Tumor

KW - ErbB Receptors/genetics

KW - Lung/metabolism

KW - Peptides/metabolism

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DO - 10.1016/j.ejpb.2024.114177

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