Development of an extracellular vesicle-based shRNA delivery system to overcome cisplatin resistance in non-small cell lung cancer by targeting STAT3

Mustafa Kotmakçı; Vildan Bozok

doi:10.19161/etd.1952225

TR EN

Küçük hücreli olmayan akciğer kanserinde sisplatin direncini aşmak için hücre dışı vezikül tabanlı STAT3'ü hedefleyen shRNA taşıyıcı sistemin geliştirilmesi

Abstract

Amaç: Sisplatin direnci, genellikle STAT3 aktivasyonu tarafından yönlendirilen, küçük hücreli dışı akciğer kanserinde (KHDAK) önemli bir klinik zorluk olmaya devam etmektedir. Bu çalışma, sisplatine dirençli Calu1 akciğer kanseri hücrelerini yeniden duyarlı hale getirmek için anti-STAT3 shRNA kodlayan plazmit DNA yüklü otolog bir ekstraselüler vezikül (EV) formülasyonu tasarlamayı amaçlamıştır. Gereç ve Yöntem: EV'ler, modifiye edilmiş diferansiyel ultrasantrifügasyon yoluyla atasal Calu1 hücre kültürü süpernatantlarından izole edildi ve elektron mikroskobu, atomik kuvvet mikroskobu ve dinamik ışık saçılımı kullanılarak karakterize edildi. Plazmit DNA, lipofeksiyon aracılı membran füzyonu ile EV'lere yüklendi. Formülasyon bütünlüğü ve koruması, agaroz jel elektroforezi ve bir DNaz I analizi kullanılarak değerlendirildi. İn vitro sitotoksisite, STAT3 susturulması ve sisplatin yeniden duyarlılaştırması, WST-1 canlılık testleri, RT-qPCR ve Western blot analizi kullanılarak sisplatine dirençli bir alt seride (CR-Calu1) değerlendirildi. Bulgular: İzole edilen EV'ler karakteristik küresel morfoloji ve tipik bir boyut dağılımı sergiledi. Biyomimetik formülasyon, pDNA'yı başarıyla enkapsüle ederek DNaz I bozunmasına karşı güçlü koruma sağladı. CR-Calu1 hücrelerinde, pshSTAT3 yüklü EV'ler STAT3 mRNA ekspresyonunu anlamlı ölçüde aşağı regüle etti (%48–58 azalma) ve buna bağlı olarak protein seviyelerini düşürdü. Kritik olarak, bu EV'lerin subletal sisplatin dozlarıyla birleştirilmesi, yalnızca sisplatine kıyasla hücre canlılığını sinerjistik olarak azalttı ve kemosensitiviteyi etkili bir şekilde geri kazandırdı. Stabilite optimizasyonu, liyofilizasyon sonrası EV yapısal bütünlüğünü koruyan üstün kriyoprotektan olarak %7,5 glisini belirledi. Sonuç: Stabil, liyofilize edilebilir bir EV tabanlı gen taşıma platformunu başarıyla tasarladık. Bu otolog sistem aracılığıyla STAT3'ün hedeflenmesi, kazanılmış ilaç direncini etkili bir şekilde tersine çevirmekte olup, dirençli KHDAK için uygulanabilir bir terapötik strateji temsil etmektedir.

Keywords

Supporting Institution

TÜBİTAK

Project Number

115S829

Ethical Statement

Etik kurul Onay belgesi gerekli değildir.

Thanks

Bu çalışma, Türkiye Bilimsel ve Teknolojik Araştırma Kurumu (TÜBİTAK) tarafından desteklenmiştir (Proje No: 115S829, MK). Yazarlar ayrıca, Türkiye Cumhuriyeti Kalkınma Bakanlığı tarafından desteklenen (Altyapı Proje No: 2009K120640) Ege Üniversitesi Eczacılık Fakültesi Farmasötik Bilimler Araştırma Laboratuvarı'nda (FABAL) yer alan analitik cihazların kullanımından dolayı teşekkürlerini sunar.

Development of an extracellular vesicle-based shRNA delivery system to overcome cisplatin resistance in non-small cell lung cancer by targeting STAT3

Abstract

Aim: Cisplatin resistance remains a major clinical challenge in non-small cell lung cancer, frequently driven by STAT3 activation. This study aimed to engineer an autologous extracellular vesicle (EV) formulation loaded with anti-STAT3 shRNA-encoding plasmid DNA to resensitize cisplatin-resistant Calu1 lung cancer cells. Materials and Methods: EVs were isolated from parental Calu1 cell culture supernatants via modified differential ultracentrifugation and characterized using electron microscopy, atomic force microscopy, and dynamic light scattering. Plasmid DNA was loaded into EVs via lipofection-mediated membrane fusion. Formulation integrity and protection were assessed using agarose gel electrophoresis and a DNase I assay. In vitro cytotoxicity, STAT3 knockdown, and cisplatin resensitization were evaluated in a cisplatin-resistant subline (CR-Calu1) using WST-1 viability assays, RT-qPCR, and Western blot analysis. Results: Isolated EVs displayed characteristic spherical morphology and a typical size distribution. The biomimetic formulation successfully encapsulated the pDNA, conferring robust protection against DNase I degradation. In CR-Calu1 cells, pshSTAT3-loaded EVs significantly downregulated STAT3 mRNA expression (48–58% reduction) and correspondingly decreased protein levels. Crucially, combining these EVs with sub-lethal cisplatin doses synergistically reduced cell viability compared to cisplatin alone, effectively restoring chemosensitivity. Stability optimization identified 7.5% glycine as the superior cryoprotectant, maintaining EV structural integrity post-lyophilization. Conclusion: We successfully engineered a stable, lyophilizable EV-based gene delivery platform. Targeting STAT3 via this autologous system effectively reverses acquired drug resistance, representing a viable therapeutic strategy for refractory NSCLC.

Keywords

Supporting Institution

TUBİTAK

Project Number

115S829

Ethical Statement

The study does not require ethical committee approval.

Thanks

This study was funded by the Scientific and Technological Research Council of Türkiye (TÜBİTAK) (Project Number: 115S829 to MK). The authors also acknowledge the use of analytical instrumentation at the Pharmaceutical Sciences Research Laboratory (FABAL), Ege University Faculty of Pharmacy, supported by the Republic of Türkiye Ministry of Development (Infrastructure Project #2009K120640).

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Details

Primary Language

English

Subjects

Chest Diseases, Clinical Sciences (Other)

Journal Section

Research Article

Authors

Mustafa Kotmakçı ^*
0000-0002-5593-8608
Türkiye

Vildan Bozok
0000-0003-3915-6363
Türkiye

Publication Date

September 3, 2026

Submission Date

May 18, 2026

Acceptance Date

June 15, 2026

Published in Issue

Year 2026 Volume: 65 Number: 3

DOI

https://doi.org/10.19161/etd.1952225

IZ

https://izlik.org/JA54NK44EN

Cite

RIS / Bibtex

APA

Kotmakçı, M., & Bozok, V. (2026). Development of an extracellular vesicle-based shRNA delivery system to overcome cisplatin resistance in non-small cell lung cancer by targeting STAT3. Ege Tıp Dergisi, 65(3), 507-518. https://doi.org/10.19161/etd.1952225

AMA

1.Kotmakçı M, Bozok V. Development of an extracellular vesicle-based shRNA delivery system to overcome cisplatin resistance in non-small cell lung cancer by targeting STAT3. EJM. 2026;65(3):507-518. doi:10.19161/etd.1952225

Chicago

Kotmakçı, Mustafa, and Vildan Bozok. 2026. “Development of an Extracellular Vesicle-Based ShRNA Delivery System to Overcome Cisplatin Resistance in Non-Small Cell Lung Cancer by Targeting STAT3”. Ege Tıp Dergisi 65 (3): 507-18. https://doi.org/10.19161/etd.1952225.

EndNote

Kotmakçı M, Bozok V (September 1, 2026) Development of an extracellular vesicle-based shRNA delivery system to overcome cisplatin resistance in non-small cell lung cancer by targeting STAT3. Ege Tıp Dergisi 65 3 507–518.

IEEE

[1]M. Kotmakçı and V. Bozok, “Development of an extracellular vesicle-based shRNA delivery system to overcome cisplatin resistance in non-small cell lung cancer by targeting STAT3”, EJM, vol. 65, no. 3, pp. 507–518, Sept. 2026, doi: 10.19161/etd.1952225.

ISNAD

Kotmakçı, Mustafa - Bozok, Vildan. “Development of an Extracellular Vesicle-Based ShRNA Delivery System to Overcome Cisplatin Resistance in Non-Small Cell Lung Cancer by Targeting STAT3”. Ege Tıp Dergisi 65/3 (September 1, 2026): 507-518. https://doi.org/10.19161/etd.1952225.

JAMA

1.Kotmakçı M, Bozok V. Development of an extracellular vesicle-based shRNA delivery system to overcome cisplatin resistance in non-small cell lung cancer by targeting STAT3. EJM. 2026;65:507–518.

MLA

Kotmakçı, Mustafa, and Vildan Bozok. “Development of an Extracellular Vesicle-Based ShRNA Delivery System to Overcome Cisplatin Resistance in Non-Small Cell Lung Cancer by Targeting STAT3”. Ege Tıp Dergisi, vol. 65, no. 3, Sept. 2026, pp. 507-18, doi:10.19161/etd.1952225.

Vancouver

1.Mustafa Kotmakçı, Vildan Bozok. Development of an extracellular vesicle-based shRNA delivery system to overcome cisplatin resistance in non-small cell lung cancer by targeting STAT3. EJM. 2026 Sep. 1;65(3):507-18. doi:10.19161/etd.1952225