Original Article
Metastatic spread inhibition of cancer cells through stimuli-sensitive HPMA copolymer-bound actinonin nanomedicines

https://doi.org/10.1016/j.nano.2022.102578Get rights and content

Abstract

The unresolved task of modern medicine is to prevent metastatic spread during and after the treatment of primary tumors. Here, the design, controlled synthesis, in-depth physicochemical and biological characteristics of a novel panel of well-defined water-soluble copolymer conjugates bearing actinonin intended for advanced drug delivery and inhibition of metastatic spread are described. Three different synthetic approaches were employed to covalently attach actinonin to the N-(2-hydroxypropyl)methacrylamide copolymers to control the release of actinonin to its pharmacologically active form. Actinonin was attached to the polymers via hydroxamate group suppressing its activity during the delivery, with the activity restored after its release in the primary tumors or metastatic foci. Importantly, developed nanosystems with favorable drug release kinetics inhibited the metastatic spread of cancer cells from primary 4T1 tumors into the lungs as well as invasion of B16F10 melanoma cells from circulation into the lungs at the dosage without any sign of toxicity.

Graphical abstract

Well-defined biocompatible and stimuli-sensitive polymer-actinonin conjugates were designed and their anti-metastatic efficacy was proved in two mice models. The silenced actinonin in the form of polymer conjugate is released and activated into the pharmacologically active form in the primary tumors or metastatic foci. The re-activation of the actinonin activity within the tumorous tissue enable to inhibit metastatic spread from the primary tumor.

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Introduction

In recent decades, various polymer-based drug delivery systems (DDS) have been designed, synthesized, and tested for their potential medicinal applications in cancer therapy.1., 2., 3., 4., 5. Copolymers based on N-(2-hydroxypropyl) methacrylamide (pHPMA) are biocompatible and water-soluble polymers, which are often studied as DDS showing excellent solution and non-fouling properties.6., 7., 8. The main advantage of these pHPMA carriers is their non-toxic and non-immunogenic properties and the possibility of exclusion from the organism by renal filtration and controlled chemical attachment with various biologically active molecules, including drugs, dyes, protein inhibitors, radionuclides, and their combinations.4., 9., 10., 11., 12. Generally, the conjugation of LMW drugs with pHPMA leads to prolonged circulation in the blood, higher accumulation in tumors, and more effective therapy with minimal side effects.9., 10., 13.

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases specific for extracellular matrix (ECM) remodeling in various physiological and pathological processes by cleaving ECM proteins such as fibronectin, collagen, laminin, and elastin.14 Their activity is dependent on the presence of metal ions such as zinc or calcium.15 They also play important role in other physiological processes such as organ morphogenesis, embryonic development, apoptosis, wound healing, and angiogenesis.16., 17. MMPs are expressed at a low level physiologically but overexpressed in several diseases such as various types of cancer, central nervous system disorders, cardiovascular diseases, neurodegenerative diseases, and arthritis.17., 18., 19., 20., 21. Their role in carcinogenesis is based on several mechanisms such as (i) the establishment of a microenvironment for primary tumor growth, e.g. by releasing ECM-bound growth factors, (ii) tumor to allow tumor cells to enter and exit the blood-circulation, and (iii) modification of the metastatic site microenvironment, allowing the initial growth and angiogenesis in the metastatic site.21

Generally, inhibitors of MMP can be divided between endogenous, e.g. α2-macroglobulin, or synthetic, e.g. collagen peptidomimetics, non-peptidomimetics, bisphosphonates, or tetracycline derivates. Synthetic MMP inhibitors are predominantly pseudopeptides that mimic the structure of MMP substrates. Several potent synthetic inhibitors have been identified including thiols, carbamoylphosponates, hydroxyureas, hydroxamates, β-lactams, hydrazines, squaric acids, and nitrogenous ligands, which act as competitive reversible inhibitors.21., 22., 23., 24.

Actinonin is a derivative of the naturally occurring tripeptide that was originally isolated in 1962 from the bacterial species Streptomyces cutter. Actinonin has antitumor and antiproliferative effects based on the inhibition of a broad spectrum of MMPs such as MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-12, and MMP-13. The inhibitory activity is due to the strong chelation of Fe2+ or Zn2+ ion located in the MMP enzymes, where the hydroxamate group plays a key role. In vitro studies showed that actinonin inhibits ECM degradation during tumor cell invasion with apoptosis-inducing activity.25., 26., 27., 28., 29., 30.

This work reports the design, synthesis, and preliminary biological evaluation of polymer nanomedicines containing actinonin as potential inhibitors of MMPs in cancer therapy. Three different synthetic approaches were employed for the tailored attachment of actinonin via pH-sensitive linkages. We investigated how actinonin release is affected by detailed structure of the linker between polymer carrier and actinonin.

Section snippets

Materials and methods

Materials and methods are listed in Supplementary material.

Results and discussion

Tumor growth and metastatic spread are based on various mechanisms of tumorigenesis, such as the increased activity of MMP leading to the degradation of the ECM, followed by tumor growth and metastasis. Thus, controlled delivery of MMP inhibitors can at least partly inhibit tumor growth and metastasis. Thus, we have designed, synthesized, and studied in detail polymer-actinonin conjugates as a suitable drug delivery platform for the tailored inhibition of metastatic spread-associated MMP.

Three

Conclusion

Herein, we presented the design, controlled synthesis, comprehensive physicochemical, and preliminary biological evaluation of a novel panel of well-defined water-soluble HPMA copolymer conjugates bearing actinonin intended for advanced drug delivery and inhibition of metastatic spread of cancer cells. Three different synthetic approaches for covalent attachment of the actinonin to the HPMA copolymers with the aim of affecting and controlling the release of the actinonin to its

CRediT authorship contribution statement

Jana Kousalová: Methodology, Investigation, Writing – original draft. Milada Šírová: Methodology, Investigation. Libor Kostka: Methodology, Investigation. Vladimír Šubr: Investigation. Jiřina Kovářová: Investigation. Kateřina Běhalová: Investigation. Martin Studenovský: Investigation. Marek Kovář: Conceptualization, Methodology, Funding acquisition, Supervision, Writing – original draft, Writing – review & editing. Tomáš Etrych: Conceptualization, Methodology, Funding acquisition, Supervision,

Declaration of competing interest

The authors declare no conflict of interest.

Acknowledgments

This work was supported by the Czech Academy of Science (JSPS-22-01), the Ministry of Education, Youth and Sports of the Czech Republic (LTAUSA18083), and the Institutional Research Concept RVO 61388971.

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