SREKA-targeted liposomes for highly metastatic breast cancer therapy

Abstract

Chemotherapy is still a leading therapeutic approach in various tumor types that is often accompanied by a poor prognosis because of metastases. PEGylated liposomes with CREKA targeting moiety are well-known therapeutic agents, especially in highly metastatic experimental models. CREKA specifically targets tumor-associated ECM, which is present at the primary, as well as metastatic tumor sites. To better understand the function of the targeting moieties, we decided to design various liposome formulations with different amounts of targeting moiety attached to their DSPE-PEG molecules. Moreover, a new tumor-homing pentapeptide (SREKA) was designed, and a novel conjugation strategy between SREKA and DSPE-PEGs. First, the in vitro proliferation inhibition of drug-loaded liposomes and the cellular uptake of their cargo were investigated. Afterward, liposome stability in murine blood and drug accumulation in different tissues were measured. Furthermore, in vivo tumor growth, and metastasis inhibition potencies of the different liposome formulations were examined. According to our comparative studies, SREKA-liposomes have a uniform phenotype after formulation and have similar characteristics and tumor-homing capabilities to CREKA-liposomes. However, the exchange of the N-terminal cysteine to serine during conjugation results in a higher production yield and better stability upon conjugation to DSPE-PEGs. We also showed that SREKA-liposomes have significant inhibition on primary tumor growth and metastasis incidence; furthermore, increase the survival rate of tumor-bearing mice. Besides, we provide evidence that the amount of targeting moiety attached to DSPE-PEGs is largely responsible for the stability of liposomes, therefore it plays an important role in toxicity and targeting.

Keywords:

• Targeted cancer therapy
• nanocarriers
• SREKA peptide
• tumor metastasis

Acknowledgments

The authors thank the devoted work of all animal caretakers of the Department of Experimental Pharmacology at the National Institute of Oncology, Hungary. The authors acknowledge the contribution of laboratory technicians Anita Hidvégi, Violetta Léner, Anna Mária Tóth, and Ilona Bornemissza in teaching the techniques and protocols to perform animal experiments properly. The authors are thankful to Katalin Parragné Derecskei for the assistance in performing the immune histochemistry experiments.

Disclosure statement

The authors report there are no competing interests to declare.

Data availability

All data generated or analyzed during this study are included in this published article [and its supplementary information files]. Moreover, rare datasets are available on the repository: zenodo.org upon request, under the DOI: 10.5281/zenodo.7075436.

Author contributions

BV designed the experiments, took part in the acquisition, analysis, and interpretation of the data. BV also played a major role in writing the manuscript. LD performed the synthesis of SREKA and CREKA peptides and conjugated the pentapeptides to DSPE-PEG molecules. AB contributed to acquisition, data analysis, and to manuscript writing and revision. AG prepared the liposome formulations under the supervision of NSz and ZV. NSz and ZV also played a role in the writing and revision of the manuscript. DVM took a major part in the acquisition, analysis, and graph design. IR participated in the design of experiments, data interpretation, and revision of the manuscript. GM took part in the experimental design, writing, and revision of the manuscript. JT participated in the experimental design and revision of the article. GM and JT are shared corresponding authors who were responsible for the funding of the project.

Ethical approval

Animals used in our study were cared for according to the “Guiding Principles for the Care, and Use of Animals” based on the Helsinki declaration, and they were approved by the ethical committee of the National Institute of Oncology. Animal housing density was according to the regulations and recommendations from directive 2010/63/EU of the European Parliament and of the Council of the European Union on the protection of animals used for scientific purposes. Permission license for breeding and performing experiments with laboratory animals: PEI/001/1738-3/2015 and PE/EA/1461-7/2020.

Correction Statement

This article has been republished with minor changes. These changes do not impact the academic content of the article.

Additional information

Funding

This work was supported by grants from European Union′s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No 861316. The authors acknowledge financial support from the National Laboratories Excellence program (under the National Tumorbiology Laboratory Project (2022-2.1.1-NL-2022-00010)) and the Hungarian Thematic Excellence Programme (TKP2021-EGA-44). The research within Project No. VEKOP-2.3.3-15-2017-00020 was supported by the European Union and the State of Hungary and was co-financed by the European Regional Development Fund. We also gained funding from the Hungarian Scientific Research Fund (NKFIH-OTKA) grant K119552 and by project no. 2018-1.2.1-NKP-2018-00005 implemented with the support provided by the National Research, Development and Innovation Fund of Hungary, and within the framework of the ELTE Thematic Excellence Programme 2020, National Challenges Subprogramme – TKP2020-NKA-06, supported by the Hungarian Ministry for Innovation and Technology.