Ultrasound-triggered release and activation of drugs and biomacromolecules from nucleic acid scaffolds
Zhao, Pengkun
DOI:
10.33612/diss.168542653
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Publication date: 2021
Link to publication in University of Groningen/UMCG research database
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Zhao, P. (2021). Ultrasound-triggered release and activation of drugs and biomacromolecules from nucleic acid scaffolds. University of Groningen. https://doi.org/10.33612/diss.168542653
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Remotely triggered release and activation
of drugs from nucleic acid-mediated
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1.1 Introduction
Controlled drug delivery refers to the engineered technology for transporting a pharmaceutical compound to the desired site of the body in response to a certain stimulus.[1] It shows tremendous potential for improving the treatment effectiveness of many diseases by reducing nonspecific toxicity and enhancing the efficacy of therapy. The commonly used stimuli can be divided into two categories: endogenous stimuli such as pH, redox potential, ATP, glucose and enzymes and exogenous ones including electric fields, magnetic fields, light and ultrasound.
Endogenous stimuli-responsive drug delivery carriers (also known as self-regulated or closed-loop systems) respond to internal stimuli in the body to regulate drug release. Change in the bio-milieu at the diseased site triggers a chemical or physical deformation of the delivery system, resulting in the release or activation of the therapeutic cargo. In this regard, the physiological status of the site of the disease plays a crucial role in controlling drug release internally. However, the local environment of the disease site can be highly heterogeneous and might vary from patient to patient, therefore the exact effect of the local environment on the release kinetics or drug dosing in most delivery systems may be difficult to predict and cannot be precisely controlled or modified after administration.[2] Contrarily, exogenous stimuli-responsive drug delivery systems, also referred to as open-loop systems, are vectors whose drug delivery capability could be manipulated by a stimulus from outside the body. The activated drug dose and the release kinetics are largely dependent on the duration and intensity of the external forces that are imposed on the system. Compared with various internal stimuli in the microenvironment of cancer, triggers from outside hold better spatial and temporal controllability for precise release of the loaded drugs and activation of prodrugs. Since Watson and Crick discovered the double-helix molecular structure of DNA in 1953, the age of genetics and modern molecular biology started. Over the past four decades the knowledge and understanding of DNA has dramatically expanded. Nowadays DNA is recognized not only as a carrier of genetic information but also as a versatile functional biopolymer. In this context, DNA possess the following advantages that make it particularly attractive for designing stimuli-responsive materials: (1) The Watson-Crick base pairing rules endow DNA-based materials with unique
self-5
recognition capability and high programmability. (2) DNA hybridization is not only predictable but also reversible and thus can be utilized to fabricate dynamic systems. (3) From the synthetic point of view, DNA with well-defined sequences can be synthesized by solid- or liquid- phase oligonucleotide synthesis based on phosphoramidite chemistry. Moreover, DNA can be easily functionalized with other groups or materials such as inorganic nanoparticles, peptides and synthetic polymers to form DNA-based hybrid materials. (4) The intrinsic biocompatibility of DNA makes it a promising building block for in vivo biomedical applications. (5) The wide structural versatility of nucleic acids. Some DNA sequences itself respond to a certain kind of stimuli such as i-motif DNA which is four-stranded quadruplex structures and is formed by cytosine-rich DNA and particularly useful due to their unique sensitivity to changes in acidity near physiological pH. There are also DNA/RNA sequences that specifically recognize and bind to their target molecules or even cells under the proper conditions known as aptamers.
This chapter will highlight the progress of nucleic acid-based drug delivery systems triggered by external stimuli, including electric field, magnetic field, light and ultrasound. The classification of each stimulus based on different mechanisms will also be reviewed by summarizing representative references.
1.2 Electric filed-triggered release
Electric fields (typically＜ 10 V) enable to induce redox reactions or ionization, resulting in bond cleavage or drug carrier deformation. More specifically, most electro-responsive drug delivery systems reported to date are mainly based on intrinsically conducting polymers, such as poly(pyrrole) (PPy), polyaniline (PANi).[3] PPy can be either directly doped with negatively charged drugs or drugs can be bound to it. The release is stimulated by employing a potential that reduces the backbone to the neutral form.[2] In addition, electroporation is also considered one of the most efficient ways for gene transfer by creating transient pores on the cell membrane upon electric pulsing.[4]
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Figure 1. (a) A setup for electroporation on a plasmid-loaded electrode. (A) Silicone frame, (B) counter electrode, (C) electric pulse generator, (D) PBS, (E) cell, (F) plasmid and (G) PEI-adsorbed electrode. (b) In situ SPR R-t monitoring the stepwise disassembly of the (Zr4+/DNA)
11 LbL film. SPR angle was fixed at 62° and kept unchanged. The film was
immersed in sodium citrate solution (10 mM, pH 6.0), and an electric potential of -0.5 V (vs Ag/AgCl) with duration of 10 min was applied to the gold thin film every 15 min. The baseline designates the MUA modified SPR gold thin film and the construction process was omitted for clarity.
1.2.1 Electroporation-induced release
Electroporation is a physical method that allows transferring biomacromolecules such as nucleic acids and proteins into living cells. Due to excellent efficiency, the method has been widely used for studying gene function in a variety of organisms ranging from prokaryotes to eukaryotes.[5] Iwata and coworkers reported electric pulse-triggered gene transfer using a plasmid-loaded electrode. The plasmid was first loaded on a gold electrode surface covered with a layer of cationic polymer, poly(ethyleneimine), to form a thin film. It is expected that this configuration prevents release of plasmids during cell culture before electroporation and protects plasmids from nuclease-mediated degradation. Thereafter, cells were plated directly onto this modified surface. The plasmid was detached from the electrode by applying a short electric pulse and introduced into the cells cultured on the electrode, leading to efficient gene expression. The electroporation setup exploited in this work is schematically depicted in Figure
1a.[5] Similarly, the same group also prepared a transparent indium-tin oxide electrode by layer-by-layer assembly of poly(ethyleneimine) and plasmid DNA for temporally and spatially specific gene transfer.[6] The plasmid DNA was not only treated as a transported cargo but also as a building block for the assembly of this system. The difference here is that they employed a transparent electrode made of electrically conductive indium-tin oxide (ITO) instead of gold as they previously used. It is
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expected that microscopic observation of transfected cells is much easier on a thin layer of ITO than gold using a standard microscope with a transmission light path. The strong quenching of fluorescent molecules in the vicinity of the metallic surfaces, such as silver or gold, can be avoided by using ITO, which is important for detecting small differences in expression of fluorescently active proteins in reporter assays.
1.2.2 Redox reaction induced release
The formation of multilayered polymer films via the layer-by-layer (LbL) deposition method is a commonly used technique. Freund’s group constructed a multilayer film based on poly(anilineboronic acid) (PABA)/ribonucleic acid (RNA) via LbL method. RNA was used both as a polyelectrolyte for multilayer formation as well as dopant for PABA. The PABA interacted with RNA through the formation of a boronate ester, a boron-nitrogen dative bond, as well as electrostatic interactions of anionic phosphates with cationic amines. The PABA/RNA multilayer films are redox-active at neutral pH, consistent with the formation of a self-doped polymer. Electrochemical control of PABA under these conditions allows potential-induced controlled release of RNA from a multilayer film.[7] Instead of using conducting polymers as polyelectrolytes to assemble redox-active multilayer films, Wang et al [8] developed a LbL film composed of DNA and inorganic zirconium ion (Zr4+) on the surface of gold thin film, which serves as the working electrode in a three-electrode setup. As an inorganic tetravalent cation, Zr4+ might be also used as a condensing agent. To verify that DNA release occurs only in the presence of an applied potential, they soaked a (Zr4+/DNA)11 film in a solution identical to that used in the disassembly experiments (10 mM sodium citrate, pH 6.0), and no significant disassembly of the film was observed. Figure 1b shows the disassembly of the film by applying a square wave potential of -0.5 V. A constant potential with a duration of 10 min was applied to the (Zr4+/DNA)11 film and then switched off for 5 min. A stepwise film disintegration was observed upon the application of an on-off step function of potential, as a result of the electro-dissolution of the LbL film. Thus, the disintegration of the film can be precisely controlled electrochemically.
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1.3 Magnetic force-triggered release
Magnetic fields show excellent tissue penetration capability and have been used in whole-body magnetic resonance imaging (MRI). Magnetically triggered release is mainly relying on magnetic particles as the remote control to induce the liberation from a delivery platform, such as a polymer microsphere or liposome. There are two major mechanisms for magnetic controlled release: one is the magnetic particle induced agitation of the carrier vesicle by physically moving; the other is an alternating magnetic field transfers of energy to the magnetic particles leading to the increase of local temperature, and hence triggering release by thermal mechanisms.[2]
1.3.1 Magnetic guidance
It is widely accepted that the main limitations of conventional drug delivery fail to overcome the natural physiological barriers and lack tissue/cell specificity. In order to fill this gap, one could envisage a spatial and temporal control of a cargo delivery through magnetic guidance, provided by an external magnetic field.[9] Yang and colleagues[10] developed magnetic DNA (M-DNA) nanogels as nanovectors for controlled drug delivery. Magnetic Fe3O4 nanoparticles were employed as the core due to their unique superparamagnetic property under an external magnetic field, nanosized particle diameter, and biocompatibility. The shell of the DNA nanogels was constructed via rolling circle amplification (RCA) in situ coating on the surface of magnetic core by physical cross-linking. Doxorubicin (DOX) as a model drug was intercalated in the M-DNA nanogel. M-DNA nanogel could be guided to a tumor site under an external magnetic field and controlled drug release by responding to multi-stimuli, such as temperature, pH, and nuclease. As shown in Figure 2a, the transwell filter was seeded with a compact cell monolayer, and then FITC-labeled DOX-loaded M-DNA nanogels in the filter were guided to penetrate the cell monolayer by a magnet. The penetrated M-DNA nanogels were internalized by the cells on the underlayer plate and imaged at different time intervals of 2 h and 4 h by fluorescence microscopy (Figure 2b).It was observed that stronger green and red fluorescence signals appeared in cells with an external magnetic field than that of control group without a magnet at the same incubation time. In addition, with the extension of incubation time, green and red fluorescence signals increased, which verified the efficient penetration and enhanced
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accumulation of the nanogels within tumor cells guided by a magnetic field.
1.3.2 Magnetic hyperthermia induced release
An intrinsic property of magnetic materials is their ability to generate heat under an alternating magnetic field (AMF). The heat generally comes from hysteresis losses (ferromagnetic particles) or oscillation of their magnetic moment due to Néel and Brownian relaxations (superparamagnetic particles). The size of nanoparticles smaller than 30 nm can be treated as single magnetic domains exhibiting superparamagnetism.[11] This means that the spin moments of these nanoparticles align into a single, large overall magnetic moment in the presence of an external magnetic field. AMF causes the magnetic moments to rotate and return to equilibrium by dissipating thermal energy through Néel and Brownian relaxations.[12] Mesoporous particles specifically mesoporous SiO2 nanoparticles have long been used as drug delivery carriers[13] due to their high surface area and loading capacity, biocompatibility, chemical stability, and surface functionalities. Vallet-Regi and coworkers[14] demonstrated oligonucleotide-modified mesoporous silica, encapsulating iron oxide superparamagnetic nanoparticles, was loaded with fluorescein (as a model drug) and subsequently capped with the complementary strand. The selected DNA duplex displayed a melting temperature of 47℃, which corresponds to the upper limit of therapeutic magnetic hyperthermia. Once a temperature of 42-47℃ was reached under an AMF, the double-stranded DNA denatured and the pores in the mesoporous silica nanoparticles were uncapped, leading to release of fluorescein (Figure 2c). Similarly, Tao’s group[15] reported DNA-capped Fe
3O4/SiO2 magnetic mesoporous silica (MMS) nanoparticles as nanocarriers for AMF controlled doxorubicin release and hyperthermia.
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Figure 2. (a) Illustration of device to stimulate the M-DNA nanogel, penetrating tumor cells under an external magnetic field with human glioma U87MG cells in culture medium. (b) Fluorescence microscope images of human glioma U87MG cells in a 24-well plate after 2 and 4 h of incubation with FITC-labeled M-DNA nanogel/DOX added in the cell culture insert with or without a magnetic field. Scale bar represents 50 μm. (c) Schematic representation of reversible magnetic nanogates enabling drug release from magnetic mesoporous silica particles through DNA hybridization/dehybridization.
1.4 Light-triggered release
The mechanisms of light-controlled release systems can be divided into three categories: physical destruction of the carrier by a photothermal effect; chemical degradation of the carriers by a photochemical effect; and molecular structure change of the carriers by a photoisomerization effect.[16]
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Figure 3. Gold-DNA nanosunflowers for efficient gene silencing. (a) Assembly and disassembly of the large-sized nanostructure (200-nm gold-DNA nanosunflowers) from/to ultrasmall nanoparticles. (b) Representative TEM image of the nanosunflowers. (c) Masterpiece: Sunflowers (Vincent van Gogh,1889). Design and generation of light-triggered, cargo releasing nanocages. (d) Scheme of the chemical activation of a cargo molecule with a photolabile cross-linker and an oligonucleotide. (e) Schematic depiction of the encapsulation of cargo, the photocleavage reaction, and subsequent cargo release.
1.4.1 Photothermal activation
The ability of photothermal agents such as gold nanorods and graphene nanosheets to convert light to heat (or convert photo energy to vibrational energy) has long been used to activate drug release via NIR irradiation, triggering a local hyperthermia that can disrupt the carrier of drugs.[17] Huo et al[18] constructed sunflower-like nanostructures as shown in Figure 3a-3c based on self-assembly of ultrasmall gold nanoparticles conjugated with c-myc oncogene silencing sequence and triplex-forming oligonucleotide sequence for gene therapy. The sunflower-like nanostructures exhibited strong near-infrared (NIR) absorption and photothermal conversion ability. Upon NIR irradiation, the large-sized nanostructure could disassemble and generate ultrasmall nanoparticles modified with c-myc oncogene silencing sequence, which could directly target the cell nucleus. By synergistically controlling the preincubation time in vitro, circulation time in vivo, and the time point of irradiation, they could achieve increased cellular uptake, tunable gene silencing efficacy, and controlled tumor inhibition effect. In addition to gold nanoparticles, gold nanorods have also been
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intensively studied as electron-rich photothermal agents for the light-controlled release. Li and colleagues[19] developed DNA-wrapped gold nanorods with doxorubicin (DOX) loaded (GNR@DOX) for treatment of metastatic breast cancer via a combination of chemotherapy and photothermal ablation. Dox was released from the GNRs upon NIR laser irradiation due to thermally triggered DNA melting and lysosomal hydrolase-induced DNA degradation. Similarly, Zhou’s group[20] reported photothermally triggered controlled drug release from mesoporous silica nanoparticles based on base-pairing rules. More specifically, poly adenine (poly A) was exploited as gatekeeper of thymine (T)-modified mesoporous silica nanoparticles (MSN-T), which was loaded with the model drug DOX. The gate keeper maintains stability to avoid drug release during the delivery process but is effectively removed by a photothermal effect to trigger drug release at the tumor tissue by NIR.
1.4.2 Photochemical activation
The mechanisms of photochemical activation are based on photodynamic effects of photosensitizers for remotely controlled drug delivery. Photodynamic effects require three key components: photosensitizer, oxygen, and a light source. Light irradiation (wavelength, 400-690 nm) excites the photosensitizer to a short-lived singlet state. Through intersystem crossing, the photosensitizer then relaxes to a triplet state that has a longer lifetime and triggers chemical reactions that generate ROS in the irradiated area.[21]
1.4.2.1 Light labile bond-mediated release
Photo-labile molecules have been widely used not only in organic synthesis but also in biological studies. Conjugation of a photo-labile protecting group with the functional group of a bio-active molecule is a straightforward way to generate caged biomolecules, temporarily deactivating specific functions. Upon photo irradiation, the photo-labile group can be removed from the caged compound, thereby uncaging the compound with respect to its activity or function.[22] Most of the photo-labile protecting groups such as 2-nitrobenzyl, 3-nitrophenyl, benzyloxycarbonyl, phenacyl, benzoinyl and related groups have been used as photoremovable protecting groups in the synthesis of peptides, polysaccharides and nucleotides. Han and coworker[23] developed a general approach that uses light to activate encapsulated cargoes from DNA nanostructures with high
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spatiotemporal precision. A photolabile linker was introduced to incorporate cargo molecules into the cavities of DNA nanostructures, so that light irradiation-induced breakage of the linker would allow the molecules to diffuse away from the protective cavity. This photolabile linker (Figure 3d and 3e) consists of an o-nitrobenzyl (o-NB) motif for photocleavage, an azido group for attachment to alkyne functionalized oligonucleotides, and an activated carbonate group for attachment to cargo molecules containing a free amino functional group. The linker was designed to release cargo upon photo cleavage in its original state without chemical remnants remaining. Given the fact that most peptides, proteins, and bioactive compounds contain exposed amino residues, the cross-linker design is capable to trap many molecules into this DNA nanostructures. In addition to that, Willner’s group[24] reported a method to assemble light-responsive microcapsules loaded with different cargos based on the layer-by-layer deposition of photocleavable o-nitrobenzyl-phosphate-modified nucleic acids on poly(allylamine hydrochloride)-functionalized CaCO3 core microparticles. The core of the microparticles could be dissolved by EDTA. Irradiating the microcapsules at 365 nm resulted in the cleavage of the o-nitrobenzyl groups, destruction of microcapsule shells and the release of the loaded DOX.
1.4.2.2 ROS-sensitive cleavage
The concept of photodynamic therapy (PDT) dates back to 1990.[25] PDT has since become a well-studied therapy for cancer and various non-malignant diseases including infections. PDT utilizes photosensitizers that are activated by absorption of visible light to initially form the excited singlet state, followed by transition to the long-lived excited triplet state. This triplet state can undergo photochemical reactions in the presence of oxygen to form reactive oxygen species (including singlet oxygen) that can destroy cancer cells, pathogenic microbes and unwanted tissue.[26] Various types of ROS responsive linkers are well-summarized in this review.[16a]
In order to take advantage of the increased tissue penetration of NIR light as compared to UV or visible light, upconverting nanoparticles (UCNPs) have intensively been exploited to excite the photosensitizers due to their ability to absorb in the NIR and emit visible light.[27] He et al[28] reported DNA-mediated assembly of core–satellite structures composed of Zr (IV) based porphyrinic metal-organic framework (MOF) and NaYF4, Yb, Er UCNPs for PDT. They incorporated the porphyrins directly into MOFs
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to yield structures that can effectively generate 1O2 without relying on small molecule photosensitizers. Using DNA as a templating agent, well-defined MOF-UCNP clusters were produced where UCNPs were spatially organized around a centrally located MOF. Under NIR irradiation, visible light emitted from the UCNPs was absorbed by the core MOF to produce 1O
2. The MOF–UCNP core–satellite superstructures induced strong cell cytotoxicity against cancer cells, which was further enhanced by attaching epidermal growth factor receptor targeting affibodies to the PDT clusters, highlighting their promise as theranostic photodynamic agents. Instead of employing inorganic nanoparticles, programmable DNA nanostructure such as DNA origami can be exploited as carrier for loading photosensitizer. Liang and coworkers[29] developed a 3, 6-bis[2-(1-methylpyridinium) ethynyl]-9-pentylcarbazole diiodide (BMEPC)-functionalized delivery nanosystem using a two-dimensional triangular-shaped DNA origami as the nanocarrier. BMEPC has a low solubility and quantum yield in aqueous environment until binding to DNA macromolecules by intercalating into the base pairs, which induces much higher fluorescence. Hence the complex could play the roles of both imaging and photosensitizing agents inside cells. After the BMEPC-loaded DNA origami were taken up by tumor cells, upon irradiation, BMEPC generated free radicals and was released due to DNA photocleavage as well as the following degradation. Apoptosis was then induced by the generation of free radicals.
The Herrmann group also developed a light-triggered release system based on DNA block copolymer-lipid vesicles. The vesicles were fabricated by amphiphilic DNA block copolymers (DBCs) and phospholipids. Hybridization of photosensitizer units and light irradiation induced selective cargo release. It was proven that DBCs can be stably anchored on the phospholipid membrane of liposomes. The oligonucleotide-photosensitizer conjugates were hybridized with protruding oligonucleotide via Watson-Crick base pairing. Treated by light irradiation, selective cargo release was achieved relying on the DNA code on the surface of the vesicles.
More recently, Liu and colleagues[30] constructed DNA nanosponges by the rolling circle amplification (RCA) technique as depicted in Figure 4. The DNA nanostructures were rationally designed by encoding specific sequences and integrating of diverse functions for effective loading of photosensitizer, precisely targeting tumor sites through incorporating sgc8c aptamer, and overcoming tumor hypoxia or resistance to
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PDT by introducing catalase. The loaded catalase resulted in O2 generation, and the replicated hypoxia-inducible factor 1a (HIF-1a) antisense DNA could down-regulate HIF-1a to improve sensitization of PDT.
Figure 4. Schematic representation of designer DNA nanoassemblies for safe and effective photodynamic therapy (PDT). (a) Design and synthesis of DNA nanosponges by rolling circle amplification (RCA). (b) Proposed strategy for enhanced PDT with programmable nanoassemblies.
1.4.3 Photoisomerization activation
Photo-isomerization is a common strategy to achieve light-triggered release of macromolecules. Isomerization of chromophores generally can switch the polarity of polymers and induce the release of cargo.[31] The typical molecules undergoing photoisomerization are coumarin[32], azobenzene and its derivatives,[33] as well as spiropyran and its derivatives.[34]
Tan’s group[35] prepared photo-responsive DNA hydrogels by a cross-linking method. Specifically, photosensitive azobenzene (Azo) moieties were incorporated into DNA strands as cross-linkers, such that their hybridization to complementary DNAs (cDNAs) responds differently to various wavelengths of light. Upon UV-Vis light irradiation, Azo isomerizes between the trans and cis state and also determines the hybridization between Azo-incorporated DNA and its cDNAs. Besides the photo-regulated reversible
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sol-gel transition, they tested the encapsulation and release capabilities using small molecules, proteins, nanoparticles and DOX. The results showed stable and controllable release processes. The same group[36] designed a mesoporous nanocontainer by azobenzene-modified nucleic acid as a carrier for photo-controlled drug release. In this system, the azobenzene-incorporated DNA double strands were immobilized at the pore mouth of mesoporous silica nanoparticles. The photoisomerization of azobenzene induced dehybridization/hybridization switch of complementary DNA, resulting in uncapping/capping of pore gates of mesoporous silica. This nanoplatform can keep the guest molecules within the nanopores under visible light but releases them when light wavelength turns to the UV range. These DNA/mesoporous silica hybrid nanostructures were exploited as carriers for cancer cell chemotherapy employing DOX as drug. It was found that the drug release behavior is sensitive to different light wavelengths.
Figure 5. Photoresponsive DNA nanocapsule (PR‐NC) with open/close system. (a) Schematic presentation of the PR‐NC showing configurational changes between the open and closed states by UV and visible light (Vis) irradiation, respectively. (b) AFM images (300 nm×300 nm) of the closed and open form NC. (c) AFM images (300 nm×300nm) of PR‐NC in the initial state (closed) and after UV irradiation (open).
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In addition, the DNA origami technique was utilized to construct nanocarriers for light-controlled release. Sugiyama and coworkers[37] developed a novel square pyramidal DNA nanocapsule (NC) with a photo-responsive opening and closing system. A pair of photo-responsive oligonucleotides (ODNs) named PR-ODN 1 and PR-ODN 2 can hybridize in the trans form of azobenzene and dissociates in the cis form by photoirradiation at different wavelengths as depicted in Figure 5. Correspondingly, the photo-responsive NC can be opened by ultraviolet (UV) light irradiation and is closed by subsequent visible light irradiation. They also demonstrated the capture and release of a gold nanoparticle (AuNP) by the PR-NC. Moreover, a DNA-conjugated AuNP was incorporated into the opened PR-NC with the help of complementary strands inside. Consequently, the PR-NC was opened by UV irradiation, and then the AuNP was released using strand displacement through a toehold system.
1.5 Ultrasound-triggered release
Ultrasound is a sound wave with frequencies above 20 kHz.[38] It can generate longitudinal pressure that induces mechanical force or/and local heating in a noninvasive manner.[39] It has been widely employed in medicine as an imaging modality to observe internal tissues, therapeutically to massage muscles and ligaments, and also to ablate tissue and kidney stones.[2] The force produced by ultrasound can change the permeability or absorption of tissues and “push” the drug into the cells or across a tissue membrane. Moreover, it can change the chemical properties of carriers.[40]
1.5.1 Sonoporation
Ultrasound with frequency between 20 and 100 kHz is defined as low-frequency ultrasound (LFUS), which is usually exploited to induce sonophoresis and affect drug carriers.[41] Gene therapy holds great promise for the treatment of many pathologies of the central nervous system (CNS), including brain tumors and neurodegenerative diseases. However, the delivery of systemically administered gene carriers to the CNS is hindered by both the blood-brain barrier (BBB) and electrostatically charged brain extracellular matrix (ECM), which acts as a steric and adhesive barrier. Mead et al[42] formulated highly PEGylated DNA-brain-penetrating nanoparticles (DNA-BPN) based
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on the cationic polymer, polyethylenimine (PEI), which allowed the formulation of highly compact and colloidally stable nanoparticles with a PEG/PEI (w/w) ratio of 5. They demonstrated that BBB opening with focused ultrasound and microbubbles can facilitate the delivery of “brain penetrating” nanoparticles (BPN) across the BBB resulting in sustained transgene expression in the CNS.
1.5.2 Cavitation
The chemical effects of acoustic fields on the chain scission of polymers in solution have been extensively studied over the past several decades.[43] It is generally accepted that the chain scission of polymers is the result of solvodynamic shear created by cavitation, which involves the nucleation, growth, and collapse of microbubbles in solution. The type of cavitation relies highly on the power intensity of the ultrasound wave, the size and material properties of the bubble and also molecular weight of sonicated polymer, viscosity and concentration of solutions.[44] Tang’s group[45] synthesized multifunctional anti-tumor targeted FoxM1 siRNA-loaded cationic nanobubbles (CNBs) conjugated with an A10-3.2 aptamer (siFoxM1-Apt-CNBs), which demonstrated high specificity when binding to prostate-specific membrane antigen (PSMA) positive LNCaP cells. siFoxM1-Apt-CNBs combined with ultrasound-mediated nanobubble destruction (UMND) by cavitation significantly improved transfection efficiency, cell apoptosis, and cell cycle arrest in vitro while reducing FoxM1 expression. In vivo xenograft tumors in a nude-mouse model were treated with siFoxM1-Apt-CNBs combined with UMND and as a result significant inhibition of tumor growth and prolonged survival of the mice was observed. At the same time, low toxicity, an obvious reduction in FoxM1 expression and a higher apoptosis index was detected.
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Figure 6. Aptamer-conjugated and DOX-loaded droplets for highly specific targeted therapy.
1.5.3 Phase transition
The size of commonly used ultrasound microbubbles in the clinic is in the micrometer-size range, which is associated to some intrinsic disadvantages such as the poor stability and the short imaging duration, limiting application in lymph nodes diagnosis. The design of phase-transition nanodroplets can potentially solve this critical issue. Phase-transitional nanodroplets can enter the tumor tissues and cells due to their small nanometer size. The post phase transition by external irradiation can effectively respond to ultrasound and provide excellent contrast-enhanced US imaging, which is controllable, stable and continuous.[46] Wang et al[47] developed aptamer conjugated and doxorubicin (DOX)-loaded acoustic droplets comprising cores of a liquid perfluoropentane compound and lipid-based shell materials (Figure 6). Conjugation of sgc8c aptamers is a favorable solution to specifically target CCRF-CEM cells for both imaging and therapy. High-intensity focused ultrasound (HIFU) was introduced to trigger targeted acoustic droplet vaporization (ADV), resulting in both mechanical cancer cell destruction by inertial cavitation and chemical treatment through localized drug release.
1.6 Multiple stimuli-triggered release
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respond to dual and multiple external stimuli, which assures drug release under complex pathological conditions with a fine-tuned drug release profile to augment therapeutic efficacy.[48] Xu et al. reported ordered DNA-surfactant hybrid nanospheres triggered by magnetic cationic surfactants for photo- and magneto-manipulated drug release.[49] More specifically, they developed a strategy for creating highly ordered functional nucleic acid-surfactant hybrid nanospheres by dropwise adding double-stranded (ds) DNA into a solution containing a light-responsive cationic surfactant with magnetic counterions, [FeCl3Br]-. DNA was also employed as an electrostatic scaffold to help loading the anticancer drug doxorubicin (Dox). The advantages of employing light- and magneto-responsive nanospheres as drug vehicles are not only their rapid response and external controllability but also their slow release properties and potential in targeted drug delivery, point-to-point drug release and more complicated on-demand cargo delivery.
Figure 7. Schematic illustration of the synthesis process of Fe3O4@Au@mSiO2-dsDNA/DOX
nanoparticles for therapy combining chemotherapy and photothermal treatment of cancer cells in vivo in a magnetic targeting manner.
Similarly, Yeh’s group[50] designed a magnetically targeted and near-infrared light responsive theranostic platform based on oligonucleotide-gated silica shell-coated Fe3O4-Au core-shell nanotrisoctahedra (Figure 7). Fe3O4@Au was fabricated by the formation of Fe3O4@polymer@Au with the assistance of a layer of polymer providing a platform for Au seeds grafting and growth. The core-shell Fe3O4@Au nanoparticles
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were further embedded within a mesoporous silica shell, yielding Fe3O4@Au@mSiO2. The pores on the mesoporous silica shell were sealed by oligonucleotides (dsDNA) loaded with Dox. Taking advantage of the magnetism, remotely triggered drug release was facilitated by magnetic attraction accompanied with the introduction of NIR radiation. DNA-gated Fe3O4@Au@mSiO2 served as a drug control and release carrier that features functions such as magnetic targeting, MRI diagnosis and combination therapy through the manipulation by magnetic fields and NIR light.
1.7 Conclusion
In this chapter, we summarized various external stimuli-controlled release systems relying on nucleic acids as main building blocks. Compared with other biocompatible systems, a variety of DNA-based materials, including aptamers, hydrogels, origami structures, and tetrahedra, possess unique merits. Firstly, the DNA offers unprecedented recognition properties and programmability due to Watson-Crick base pairing. Secondly, excellent predictability and reversibility of DNA hybridization renders it a promising candidate for building dynamic systems. Thirdly, DNA can be easily functionalized with different groups, which further promotes the possibilities to conjugate the nucleic acid structures with other molecules. Fourthly, inherent biocompatibility of DNA is highly desirable in biomedical fields. Last but not least, special DNA structures such as aptamers themselves have unique properties, including specific target recognition ability and enzyme-like properties (DNAzymes).
In the field of controlled drug delivery, systems based on external stimuli have been intensively explored with more and more enthusiasm. The responsiveness to light, ultrasound, electrical and magnetic fields, and combinations of these triggers qualified these DNA-based materials for the release of therapeutics to be triggered at a desired time, location and dose on demand.
Aside from the impressive advancements in the field of controlled drug delivery from DNA-based materials, there are still many concerns that remain to be addressed. A better understanding of the pharmacokinetics of DNA nanostructures in vivo is highly needed to be developed, especially when compared to alternative delivery technologies based on liposomes and polymers. Additionally, nuclease degradation is common to all
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DNA-based structures, so that chemical modifications such as fluoro-, amino-,O-methyl bases were exploited to enhance the circulation duration. Finally, the relatively expensive synthesis of the starting materials, especially when the building block nucleosides need to be modified to enable advanced applications, will inevitably limit its scale-up.
1.8 Thesis motivation and overview
Treatment using pharmaceutical drugs is arguably the most important medical therapy warranting the health of every human. However, the systemic application of drugs is a compromise between desirable treatment and side effects due to the intrinsic lack of drug selectivity. In addition, systemic use and overuse of antibiotics are factors leading to the emergence of antimicrobial resistance (AMR) and putting millions of lives per year at risk due to the increase of drug-resistant bacterial infections by 2050. This can be attenuated or circumvented either by increasing the drug selectivity, e.g. by endowing it with auxiliaries that deliver it to a specific target, or by controlling the activation and release of drugs in response to external and internal stimuli. Among the latter, systems addressed by physicochemical stimuli, such as light, electromagnetic fields, pH, or redox reactions, enable the control over release and activation of therapeutic agents. However, many of these seminal approaches suffer from poor selectivity, low loading capacity, leakage, or systemic side effects. It is thus desirable to develop new concepts, which resolve these complications while offering remote control over local drug activity, regardless of the selected target.
In the field of photopharmacology, spatiotemporal control over drug activity is exerted by illumination with light. Yet, this technique is limited by the toxicity of UV light, photothermal damage to healthy cells, or low tissue penetration depths. Concurrently, ultrasound (US) is used in clinical applications as it allows the spatiotemporally controlled release of drugs from carriers, such as micelles, liposomes, or microbubbles, or to synergistically increase drug efficacy. Polymer mechanochemistry exploits US to control transformations on the molecular level by rearranging or cleaving bonds at predetermined breaking sites. Generally, this is achieved by applying mechanical force to a macromolecular framework that transduces it to the mechanochemically labile bond of the latent molecular motif (the mechanophore). Mechanophore breakage can
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occur in bulk material by exposure to mechanical stress and strain but also in solution via the collapse of US-induced cavitation bubbles generating shear stress. Until now, research in polymer mechanochemistry mainly focused on understanding the force-induced chemical transformations and their impact on material properties. However, the utilization of mechanochemical bond scission for the activation of drug molecules has remained unexplored.
In Chapter 1, we briefly introduced DNA-based materials as drug carriers for controlled release by external stimuli, including electrical fields, magnetic fields, light, ultrasound, and combined stimuli. The most representative examples were explained according to different mechanisms behind these systems. In the end, we pointed out what problems still need to be solved and concerns remain unexplored to further boost the development of DNA-based externally controlled systems.
Without control over the drug activity, current drug delivery strategies are still facing limitations during disease treatment. Fortunately, approaches for realizing targeted therapy have been developed in recent years. In Chapter 2, we reported the selective release and activation of aminoglycoside antibiotics from poly-aptamers (poly-APT) with multiple mechanophores along the polynucleic acid chain by ultrasonication. Taking the knowledge of mechanochemistry, long RNA sequences with high molecular weight were synthesized for drug loading and deactivation without any complicated chemical reactions by the rolling circle transcription (RCT) technique. Based on the robust recognition and drug-loading ability of R23 aptamer, the aminoglycoside antibiotics (Neomycin B (NeoB) and Paromomycin (Paromo)) were captured through forming aptamer/drug complexes. Upon US irradiation, the hydrogen bonds within the loop structures were cleaved selectively, which resulted in drug dissociation and activation.
Proteins display a complex set of functions in the body and are involved in nearly all biological processes. Thus, precise regulation of protein activity at the single-molecule
24
level is of vital importance to understand complex biological signalling networks or identifying new therapeutic targets. The release and activation of proteins from polynucleotides with multi
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mechanophores by ultrasonication was presented inChapter 3. Due to the robust recognition and protein-loading ability of TBA15 aptamer, the target thrombin was bound and inhibited through forming aptamer/protein complexes. Upon US irradiation, the proteins were released and further activated.
In a similar vein, nanoparticles (NPs) functionalized on the surface with nucleic acids were employed for protein activation. Nanoparticle assemblies exhibit physical and chemical properties, which are different from those of both individual NPs and their bulk aggregates. Controlling the assembly of nanometer-sized objects precisely with external stimuli is essential for applications in many fields, especially in nanofabrication and controlled drug delivery. In Chapter 4, a new strategy to manipulate the assembly/disassembly of protein-loaded NPs reversibly using ultrasonication was developed. In the presence of the target protein-thrombin, two split aptamers, conjugated on the surface of AuNPs, can assemble into the intact aptamer tertiary structure and induce AuNP aggregation. When the external stimulus US was employed, the aggregated AuNPs were disassembled and the loaded thrombin was activated consequently.
In Chapter 5, we developed a common method to detect the association/dissociation between aptamers and payloads by a colorimetric assay based on 30 nm AuNPs with positive charge. The electrostatic interaction between the positively charged AuNPs and polyanionic DNA leads to the aggregation of cationic AuNPs accompanied with a rapid red-to-blue color change. The negative charge of lysozyme binding aptamer (LBA) was reduced when the lysozyme binds to it, resulting in the remaining of the red color
25
of AuNPs. Besides, the process could be monitored by UV-Vis spectroscopy. From the changes of solution color and the shift of absorbance spectra of AuNPs, a convenient and fast method was established to study the interaction of proteins and nucleic acids.
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