A paradigm of endothelium-protective and stent-free anti-restenotic therapy using biomimetic nanoclusters

doi:10.1016/j.biomaterials.2018.06.025

Biomaterials

Volume 178, September 2018, Pages 293-301

https://doi.org/10.1016/j.biomaterials.2018.06.025 Get rights and content

Highlights

•
The standard of care for atherosclerosis and restenosis (vessel re-narrowing) is balloon angioplasty followed by the implantation of a drug-eluting stent; an inherent problem is endothelial damage and associated thrombotic risks imposed by current drugs and stenting.
•
We created a preclinical anti-restenotic system that incorporates the combined merits of an epigenetic modulator, its nanocarrier and their coating with a biomimetic membrane.
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This biomimetic system is intravenously injectable (stent-free) and can home to the injured arterial wall thereby attenuating post-angioplasty restenosis. Of particular significance, it also circumvents the impairment of endothelial recovery that occurs with the status-quo drug rapamycin.
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This system may confer an adjustability of therapeutic regimen (i.e., drugs, doses, and injection timing and frequency) and amenability toward combination therapy and precision medicine.

Abstract

Drug-eluting stents are the most commonly employed method to control post-angioplasty restenosis. Unfortunately, they exacerbate life-threatening stent thrombosis because of endothelium damage caused by both drug and stenting. To solve this major medical problem, an endothelium-protective and stent-free anti-restenotic method is highly desirable. Here we have generated a biomimetic intravenous delivery system using dendritic polymer-based nanoclusters, which were coated with platelet membranes for targeting to the injured arterial wall where restenosis occurs. These nanoclusters were loaded with an endothelium-protective epigenetic inhibitor (JQ1) or an endothelium-toxic status quo drug (rapamycin), and compared for their ability to mitigate restenosis without hindering the process of re-endothelialization. Fluorescence imaging of Cy5-tagged biomimetic nanoclusters indicated their robust homing to injured, but not uninjured arteries. Two weeks after angioplasty, compared to no-drug control, both rapamycin- and JQ1-loaded biomimetic nanoclusters substantially reduced (by >60%) neointimal hyperplasia, the primary cause of restenosis. However, whereas the rapamycin formulation impaired the endothelial re-coverage of the denuded inner arterial wall, the JQ1 formulation preserved endothelial recovery. In summary, we have created an endothelium-protective anti-restenotic system with biomimetic nanoclusters containing an epigenetic inhibitor. This system warrants further development for a non-thrombogenic and stent-free method for clinical applications.

Graphical abstract

Introduction

Drug-eluting stents (DES) represent a major medical advance in reducing restenosis, the re-blockage of angioplastied atherosclerotic arteries. However, stent thrombosis has become a major concern [1]. The problem is two-fold. First, implanting a stent, a foreign object, is pro-inflammatory and thrombogenic, and also triggers neointimal hyperplasia (IH), the chief etiology of restenosis. Second, the anti-proliferative drugs coated on stents to curb IH including paclitaxel and rapamycin also block endothelial cell (EC) re-growth (termed re-endothelialization), thereby exacerbating stent thrombogenicity [2]. The most adverse consequence of stent thrombosis is high rates (up to 50%) of death even though the incidence of stent thrombosis is no more than 1.5% [1]. Anti-coagulant therapies cannot completely prevent stent thrombosis, and they are associated with high costs and bleeding problems. Aside from all of these complications, compromised DES cannot be replaced, and hence a secondary, invasive revascularization such as bypass surgery is often required [1]. Therefore, there is a clear clinical need for the development of an endothelium-protective and stent-free anti-restenotic therapy via innovations in both drug and its delivery method.

The recent discovery of small molecule inhibitors (JQ1 as the first in class) [3] that selectively block the bromo and extraterminal (BET) domain family of epigenetic reader proteins has opened the door to effectively treating previously recalcitrant condition [4]. These inhibitors have shown excellent efficacy in treating proliferative and inflammatory diseases, such as cancer and heart failure, in preclinical tests or clinical trials [4]. Of particular interest, our study using a rat model showed that JQ1 is an effective inhibitor of restenosis [5]. More significantly, JQ1 also exhibited a prominent EC-protective effect in vitro and in vivo [5,6], which is a rare feature among the numerous anti-restenotic agents [7]. Moreover, rapidly growing evidence supports a mechanism whereby JQ1 disrupts molecular complexes formed by BET proteins, transcription factors, super enhancers and/or other regulators that cooperatively define BET protein functional specificity [4]. As such, JQ1 and its analogs appear to be promising EC-protective candidate drugs suitable for next-generation anti-restenotic therapy.

To administer JQ1 in a non-invasive stent-free fashion, targeted intravenous delivery to the injured arterial wall should be the preferable method. Both nanoparticle (NP)-based and cell-based approaches have been extensively explored for targeted delivery [8]. While systemically delivered “alien” NPs can elicit inflammatory responses, effects of cell therapy also are complicated by immune reactions. To reconcile the benefits and drawbacks of these two approaches, biomembrane-coated PLGA NPs have emerged as a novel biomimetic platform for delivery of therapeutics [9]. They have shown excellent homing to injured tissues and minimal immunogenicity in that proof-of-concept study.

In the current study, we applied this biomimicry/nanotechnology hybrid concept in a rat model of balloon angioplasty for targeted intravenous delivery of JQ1, an endothelium-protective BET inhibitor [5,6], in comparison to rapamycin, a status quo drug known to be EC-toxic [2,7]. JQ1 or rapamycin was loaded into nanoclusters formed by multiple PAMAM-polyvalerolactone (PAMAM-PVL) ultrasmall unimolecular NPs. These nanoclusters were then coated with platelet membranes that were deprived of immunogenicity [9] yet retaining the ability to target injured tissues, hereby termed biomimetic nanoclusters. We observed their robust homing to balloon-injured areas in arteries. Remarkably, whereas rapamycin/nanoclusters inhibited IH as well as re-endothelialization, JQ1/nanoclusters preserved the ability of the endothelium to recover while mitigating IH.

Section snippets

Ethics statement

All animal studies conform to the Guide for the Care and Use of Laboratory Animals (National Institutes of Health) and protocols approved by the Institutional Animal Care and Use Committees at the University of Wisconsin and The Ohio State University. Institutional review board (IRB) approval has been obtained for use of human samples.

Reagents and materials

Human platelets and platelet membranes were purchased from Zen-Bio Inc (Research Triangle Park, NC). RNAlater solution, TRIzol, SuperScript IV VILO Master Mix,

Biomimetic nanoclusters effectively home to the balloon-injured carotid artery wall

Biomimetic (platelet membrane-coated) nanoclusters were prepared as indicated in Fig. 1. Detailed characterization of the biomimetic nanoclusters (e.g., size, zeta potential, and drug release profiles) can be found in the supplemental file (Table S1 and Figure S2). Their post-injection homing was visualized via the Cy5 fluorophores that were conjugated to the unimolecular NPs forming the nanoclusters. Immediately after balloon angioplasty in the rat carotid artery, biomimetic nanoclusters

Discussion

DES implantation has become a procedure prevailingly used in the clinic to control post-angioplasty restenosis in the treatment of atherosclerosis. Unfortunately, it has been widely reported that DES exacerbates life-threatening stent thrombosis [1]. This adverse effect is believed to stem from both the EC-toxic drug (e.g., rapamycin) coated on the stent and the stent itself as a non-biological object that intrudes into vascular tissues [1,2]. Here we have created a prototype of stent-free

Conclusions

DES thrombogenicity is a grave concern; the key problem is impaired endothelial recovery caused by both the drugs delivered by DES and stenting [1,2]. Developing an alternative method to DES has been a paramount challenge as it requires innovations in both drug and drug delivery device. The injury site-targeting biomimetic system radically departs from the status quo DES method. It has two prominent features, the use of an endothelium-protective epigenetic inhibitor and the omission of

Author contributions

B.W., G.C., L.-W.G., S.G., and K.C.K. designed the studies; B.W., G.C., G.U., Y.W., and X.S. performed experiments; B.W., G.C., L.-W.G., and S.G. wrote the manuscript; B.W., G.C., L.-W.G., S.G., and K.C.K. critically reviewed the manuscript.

Sources of funding

This work was supported by National Institute of Health (NIH) grants R01 HL129785 (to K.C.K., S.G., and L.-W.G.), R01HL-068673 (to K.C.K.), NIH R01 HL133665 (to L.-W. G.), and EY022678 (to L.-W. G.), NIH K25CA166178 (to S.G.), and an American Heart Association (AHA) Predoctoral Award 16PRE30160010 (to B.W.).

Disclosures

The authors declare no conflicts of interest.

Acknowledgements

We thank Dr. Matthew Stratton for informative discussion and proof reading.

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¹: These authors contributed equally to this work.

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A paradigm of endothelium-protective and stent-free anti-restenotic therapy using biomimetic nanoclusters

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Ethics statement

Reagents and materials

Biomimetic nanoclusters effectively home to the balloon-injured carotid artery wall

Discussion

Conclusions

Author contributions

Sources of funding

Disclosures

Acknowledgements

Trends Biochem. Sci.

EBioMed

Mol. Cell

Nano Today

Biomaterials

J. Contr. Release

Biochem. Biophys. Res. Commun.

Cell Metabol.

J. Contr. Release: Off. J. Cntr. Release Soc.

J. Biol. Chem.

Stent thrombosis and restenosis: what have we learned and where are we going? The Andreas Gruntzig Lecture ESC 2014

Eur. Heart J.

Rapamycin promotes arterial thrombosis in vivo: implications for everolimus and zotarolimus eluting stents

Eur. Heart J.

Selective inhibition of BET bromodomains

Nature

High-throughput screening identifies idarubicin as a preferential inhibitor of smooth muscle versus endothelial cell proliferation

PLoS One

Nanoparticle biointerfacing by platelet membrane cloaking

Nature