Advances on non-invasive physically triggered nucleic acid delivery from nanocarriers

Abstract

Nucleic acids (NAs) have been considered as promising therapeutic agents for various types of diseases. However, their clinical applications still face many limitations due to their charge, high molecular weight, instability in biological environment and low levels of transfection. To overcome these drawbacks, therapeutic NAs should be carried in a stable nanocarrier, which can be viral or non-viral vectors, and released at specific target site. Various controllable gene release strategies are currently being evaluated with interesting results. Endogenous stimuli-responsive systems, for example pH-, redox reaction-, enzymatic-triggered approaches have been widely studied based on the physiological differences between pathological and normal tissues. Meanwhile, exogenous triggered release strategies require the use of externally non-invasive physical triggering signals such as light, heat, magnetic field and ultrasound. Compared to internal triggered strategies, external triggered gene release is time and site specifically controllable through active management of outside stimuli. The signal induces changes in the stability of the delivery system or some specific reactions which lead to endosomal escape and/or gene release. In the present review, the mechanisms and examples of exogenous triggered gene release approaches are detailed. Challenges and perspectives of such gene delivery systems are also discussed.

Introduction

Nucleic acids (NAs) are among the most important biomolecules. Their main function is to store and to transfer genetic information. Although being discovered very early in 1869, researches involving in NAs have achieved amazing progress in the past few decades. In medicine, NAs are considered as promising therapeutic agents for various types of diseases, from hereditary diseases to acquired diseases such as cancer, degenerative disorders and AIDS. Therapeutic NAs can be categorized into DNA therapeutics (antisense oligonucleotides, DNA aptamers and gene therapy) and RNA therapeutics (micro RNAs (miRNA), short interfering RNAs (siRNA), ribozymes, RNA decoys and circular RNAs) [1]. Since the first successful and accepted nuclear gene transfer in humans in May 1989, a lot of nucleic acid-based treatments have been fabricated and taken into clinical trials [2]. In 2012, Glybera®, an adeno- associated viral vector engineered to express lipoprotein lipase for the treatment of lipoprotein lipase deficiency, was approved as the first gene therapy treatment for sale in the European Union [3]. Until 2016, there are approximately 2600 gene therapies clinical trials [2] and about 20 clinical trials using miRNA and siRNA-based therapies [4]. Moreover, in 2016, the first ex-vivo stem cell gene therapy, Strimvelis™, was accepted in Europe for Severe Combined ImmunoDeficiency due to Adenosine DeAminase deficiency treatment [5].

Efficient cell delivery of NAs is hampered by their charge, high molecular weight and instability in biological environment. Many strategies have been proposed to obtain effective gene delivery to targeted cells [5]. An effective gene delivery system should have the following properties: (i) be able to carry and to protect the therapeutic genes; (ii) accumulate at targeted tissues, and (iii) release the entrapped payload at the targeted tissue. There are two kinds of vector for gene delivery: viral and non-viral vectors. Viral vectors are outstanding candidates for gene delivery due to their high transfection efficiency and ability to incorporate the delivered gene into the host genome [6]. Although serious side-effects of viral vectors leading to patient death and lymphoproliferative disorder were encountered [7], after numerous modifications and innovations, many viral vectors with improved efficiency, specificity and safety have been developed and transferred into clinical trials. Today, viral vectors are used in approximately two thirds of gene therapy trials performed [2]. However, there are still some drawbacks in the use of viral vectors such as: the complexity and high cost of production, and the limited size of transgene inserted in viral vectors [8]. Meanwhile, non-viral vectors are highly interesting delivery systems due to their large versatility: they can be composed of organic materials (for example: polymers, liposomes, peptides…), carbon nanotubes, or inorganic nanoparticles (such as gold NPs, magnetic NPs (MNPs)…) [6]. They can be designed to transfer different and large transgenes [8].

After administration, non-viral vectors are usually uptaken by the cells through endosomal pathway. Because of harsh environment inside endosome (low pH, digestive enzymes), NAs risk to be degraded before reaching their site of action. Endosomal escape and triggered release of the entrapped gene at the target site, therefore, are important requirements for an effective NA-based treatment. For that reason, stimuli-responsive gene delivery systems are under evaluation. Stimuli-responsive vectors should hold off the release function while they are in the blood stream and release entrapped genes inside the cells under exposure to stimuli source which can be internal or external triggers. Internal triggers are based on abnormalities of pathological area such as different pH, redox potential, temperature and over expression of some molecules like enzymes. For instance, in breast and pancreatic cancers, there is a significant increase in the expression of phospholipase A2, or in inflammatory area, there are some soluble extracellular enzymes such as lysozyme, cathepsins and matrix metalloproteinases [9]. A metalloproteinase 2 responsive block copolymer composed of poly(ethylene glycol) (PEG), matrix metalloproteinase 2 (MMP-2)-degradable peptide PLG*LAG, cationic cell penetrating peptide polyarginine r₉ and poly(ε-caprolactone) (PCL) was employed for siRNA against polo-lie kinase 1 (Plk1) delivery. The micelle carrying siRNA showed enhanced gene silencing and tumor growth inhibition. About 49% of cells treated with metalloproteinase responsive siRNA delivery system underwent apoptosis while cells treated with unresponsive system presented only about 24% of death. Furthermore, Plk1 mRNA levels was significantly lower in mice treated with responsive system compared to that with unresponsive system (p < 0.005) [10]. Beside, due to hypoxia, pH may drop to around 6 in the tumor area [11] or inflammation tissues [12]. Taking advantage of this difference, Du et al. designed nanomicelle system based on poly(ethylene glycol)-co-poly[(2, 4, 6-trimethoxybenzylidene-1, 1, 1-tris(hydroxymethyl)] ethane methacrylate-co-poly(dimethylamino glycidyl methacrylate) PEG-PTTMA-P(GMA-S-DMA) (PTMS) for pH responsive siRNA release strategy. In acidic environment, PTTMA polymers undergoes hydrophobic-to-hydrophilic transition leading to the disassembly of the nanomicelle and therefore the siRNA release. Results from in vitro experiments showed that in cells treated with PTMS nanomicelle-based siRNA delivery system there was a highly efficient gene silencing of about 90%, which was even better than that with Lipofectamine 2000. This result was also confirmed through in vivo studies: in mice group treated with PTMS/siRNA complex, tumor growth was significantly inhibited and about 45% gene knockdown efficacy was observed. This enhancing gene silencing effect was attributed to the enhanced siRNA endosomal release [13]. In addition, in cancer cells, the level of glutathione is found to be 100-fold higher than the normal ranges [14]. Glutathione, therefore, can be used as a trigger for stimulus NA release. For example, a DNA delivery nanocarrier called Pluronic-PEI-SS synthesized by conjugating reducible disulfide-linked PEI (PEI-SS) with Pluronic was fabricated. The disulfide link is broken under the action of glutathione. The Pluronic-PEI-SS system showed the highest DNA transfection efficacy into cells of about 4, 3 and 13 times higher than that of Pluronic-PEI, PEI-SS and PEI systems, respectively. Moreover, in vivo experiment, the Pluronic-PEI-SS nanocarrier also exhibited a significantly higher transfection efficacy than the PEI/DNA [15]. Numerous other efforts have been carried out to prompt the release of entrapped gene by internal triggers with promising results and have been recently summarized in many reviews [12,14,16].

However, triggered gene release by intrinsic physical and biological factors faces many limitations. The environment of the disease site is heterogeneous and strongly depends on patient's conditions such as illness or diet, therefore the effects are not easy to predict. In addition, after administration, it is impossible to control or modify the action of the gene delivery systems. Compared to endogenous triggers, exogenous triggered gene release is time and site specifically controllable through active management of external stimuli. Non-invasive external signal like light, heat, magnetic field or ultrasound is applied to the target site from an outside source. The signal induces changes in the stability of the delivery system or some specific reactions which lead to endosomal escape and gene release.

In this review, we will go into details of some external trigger strategies for gene delivery, including ultrasound, magnetic field, light and temperature. In each strategy, mechanism of action and examples are covered. Furthermore, challenges and perspectives of these kinds of smart-gene delivery systems are also discussed.