Elsevier

Biomaterials

Volume 157, March 2018, Pages 98-106
Biomaterials

DNA nanoparticles for ophthalmic drug delivery

https://doi.org/10.1016/j.biomaterials.2017.11.046Get rights and content

Abstract

Nucleic acids represent very appealing building blocks for the construction of nano-scaled objects with great potential applications in the field of drug delivery where multifunctional nanoparticles (NPs) play a pivotal role. One opportunity for DNA nanotechnology lies in the treatment of ophthalmic diseases as the efficacy of eye drops is impaired by the short survival time of the drug on the eye surface. As a consequence, topical administration of ocular therapeutics requires high drug doses and frequent administration, still rarely providing high bioavailability. To overcome these shortcomings we introduce a novel and general carrier system that is based on DNA nanotechnology. Non-toxic, lipid-modified DNA strands (12mers with 4 lipid modified thymines at the 5′ end) form uniform NPs (micelles), which adhere to the corneal surface for extended periods of time. In a single self-assembly step they can be equipped with different drugs by hybridization with an aptamer. The long survival times of DNA NPs can be translated into improved efficacy. Their functionality was demonstrated in several ex-vivo experiments and in an in-vivo animal model. Finally, the NPs were confirmed to be applicable even for human tissue.

Introduction

In the last decades, rapidly advancing research in the field of DNA nanotechnology has resulted in a number of strategies to fabricate nucleic acid nanoarchitectures with well-defined sizes, periodicities and shapes in one-, two- and three dimensions [1], [2], [3], [4]. Therefore, it is not surprising that this type of nano-scaled assemblies has attracted great interest from researchers working in the field of nanomedicine where well-defined multifunctional nanostructures are urgently needed. DNA nanoobjects can be exclusively self-assembled from nucleic acids by Watson-Crick base pairing and functionalization is achieved by hybridization with oligonucleotides. Examples are a DNA icosahedron functionalized with a target cell-recognizing aptamer or a 140 nm-long DNA tube, both loaded with anticancer drugs [5], [6]. On the other hand, DNA strands can be chemically attached to a nanoscopic inorganic template and easily equipped with targeting units and drug molecules by hybridization with complementary oligonucleotide conjugates [7], [8]. A similar type of nanoparticles for drug delivery was introduced by our group where the inorganic material is replaced by a soft matter core consisting of hydrophobic polymer units that are covalently attached to oligonucleotides to form DNA block copolymers [9]. So far, all DNA nanocarriers have been only applied for cancer therapy and proven functionality in vivo is very rare [10].

Currently, the vast majority of chronic and acute ocular diseases that are not managed surgically are treated using eye drops - a non-invasive mode of treatment that can be self-administered without medical supervision. However, only a small percentage of the active compound present in eye drops reaches its target tissue as it is rapidly cleared from the eye by tear fluid and eye lid movement [11], [12]. As such, frequent administration of highly concentrated eye drops is necessary. Aside from the inefficiency of this dosage form, the requirement of frequent administration leads to poor compliance [13], [14], [15]. On the other hand, high concentrations of bioactive compounds produce side-effects that can range from simple irritations to, in extreme cases, a life threatening anaphylactic shock [16], [17]. Thus, increasing the half-life of the drug on the eye is an important goal for more efficient treatment of eye diseases with fewer side effects.

The aim of this study is to overcome the limitations mentioned above, i.e. increase of adherence of the active pharmaceutically ingredient on the ocular surface, which improves drug action, with the help of a novel carrier system based on DNA nanotechnology. More specific, we address the most crucial parameters of a drug-delivery system: biosafety, compatibility with human tissue, the option to load different drugs, the functionality of the drug once released from the carrier and in-vivo efficacy. Therefore, we replaced the hydrophobic polymer unit of our DNA block copolymer delivery system [9] by several alkyl chain-modified 2′-deoxyuridine nucleotides (U) (Fig. 1a). When introduced into an aqueous environment, through microphase separation, DNA amphiphiles self-assemble into micellar nanoparticles (NPs) that exhibit a corona of single stranded DNA surrounding a lipid core [18]. Without any targeting unit, these NP adhere to corneal tissue for extended periods of time and they can be easily functionalized by a hybridization step. For imaging, we hybridized an oligonucleotide functionalized with a fluorophore onto the DNA carrier (Fig. 1b, top). To incorporate drug molecules, a DNA aptamer binding kanamycin B [19] and a RNA aptamer binding neomycin B [20] were extended at the 3′ end with the complementary sequence of the DNA amphiphile. Watson-Crick base pairing of aminoglycoside-complexed aptamers and DNA nanoparticles resulted in two antibiotic-loaded nanocarrier systems (Fig. 1b, bottom), proving the general drug loading strategy. Finally, with this nucleic acid based carrier platform we show that the long in vivo residence time of the nanoparticles on the cornea can be translated into improved efficiency in-vitro and in-vivo compared to the pristine drug, even demonstrating applicability with human tissue. To the best of our knowledge, it is the first time that nucleic acid-based carrier systems have been employed in the field of ophthalmology and that the adherence of DNA amphiphile NPs to corneal tissue has been shown.

Section snippets

Synthesis and characterization of amphiphilic oligonucleotides

The modified 5-(dodec-1-ynyl)uracil phosphoramidite 3 was synthesized in two steps as previously reported in our group (Fig. S6) [21]. The modified uracil phosphoramidite was dissolved in CH3CN to adjust the concentration to 0.15 M, in the presence of 3 Å molecular sieves. The prepared solution was directly connected to the DNA synthesizer. All oligonucleotides were synthesized in 10 μmol scale on a DNA synthesizer using standard β-cyanoethylphosphoramidite coupling chemistry. Deprotection and

Results

First, we investigated the effect of the structural features of the DNA nanoparticle components on the adherence of nanoobjects to the surface of the eye. Therefore, the corneal epithelium of living rats was exposed to different lipid-DNA nanoparticles. The following series of DNA amphiphile constructs was synthesized: U2-12, U4-12, U4-18, U6-12, U6-20, annotated as UX-Y, wherein X and Y represent the number of hydrophobic modified deoxyuridine bases and the total number of nucleotides,

Discussion

Treatment of eye diseases by eye drops is complicated due to several problems and improving efficacy of eye drops has been an important goal for many years [45]. By increasing the exposure time of the target tissue to the active compound in the drops, less frequent administration of less concentrated drops would be required. As a consequence, improved compliance is to be expected alongside lower levels of side effects. Moreover, ocular drug delivery systems provide the opportunity to administer

Conclusion

Here, we introduced a novel, powerful and general approach for treating eye infections using DNA nanotechnology that can be easily extended to treat other ocular indications as aptamers can be evolved against molecules with very diverse structures. We demonstrated functionalization of the DNA carrier with therapeutically active agents, i.e. the aminoglycoside antibiotics neomycin B and kanamycin B, imaging units (two fluorophores) or a combination of the two by simple mixing of components and

Author contributions

Research was conducted by J.W.d.V., S.S., L.S., J.H., M.K. and A.G. under guidance of M.S.S., K.U.B.S. and A.H. The manuscript was written by J.W.d.V., S.S. and A.H.

Funding

This work was supported by an ERC starting grant from the European Commission (A.H.). A.H. also greatly acknowledges financial support from NWO (Vici grant and ChemThem grant) and the Zernike Institute for Advanced Materials. J.H. greatly acknowledges financial support from the Ernst-und-Berta-Grimmke Stiftung.

Acknowledgement

We thank Johanna Wude and Katharina Frößl for their assistance. We thank the cornea bank in Tübingen for providing human corneal tissue. The authors greatly acknowledge the University Eye Hospital Tübingen for their support. We thank Dr. Sandra Schwarz and Dr. Annika Schmidt for providing the Pseudomonas aeruginosa.

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    1

    Present address: DWI-Leibniz Institute for Interactive Materials, Forckenbeckstr. 50, 52056, Aachen, Germany and Institute of Technical and Macromolecular Chemistry, RWTH Aachen University, Worringerweg 2, 52074, Aachen, Germany.

    2

    These authors contributed equally.

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