Ultrasound-triggered release and activation of drugs and biomacromolecules from nucleic acid scaffolds
Zhao, Pengkun DOI:
10.33612/diss.168542653
IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below.
Document Version
Publisher's PDF, also known as Version of record
Publication date: 2021
Link to publication in University of Groningen/UMCG research database
Citation for published version (APA):
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
Copyright
Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).
Take-down policy
If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.
Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum.
2
Ultrasound promoted drug activation of
aminoglycoside antibiotics from
poly-aptamers
Parts of this chapter have been published:
Huo, S.; # Zhao, P.; # Shi, Z.; # Zou, M.; Yang, X.; Warszawik, E.; Loznik, M.; Göstl, R.; Herrmann, A. Nat. Chem. 13, 131–139 (2021). (#contribute equally)
32
Abstract
Pharmaceutical drug therapy is often hindered by issues caused by poor drug selectivity, including unwanted side effects and drug resistance. Spatial and temporal control over drug activation in response to stimuli is a promising strategy to attenuate and circumvent these problems. Here, we use ultrasound to activate drugs from inactive macromolecules through the controlled scission of mechanochemically labile, weak non-covalent bonds. We show aminoglycoside antibiotics complexed by a multi-aptamer RNA structure that are activated by the mechanochemical opening and scission of the nucleic acid backbone. This work demonstrates the potential of ultrasound to activate mechanoresponsive prodrug systems.
33
2.1 Introduction
The global risk of antimicrobial resistance (AMR) has become one of the world-wide urgent public health issues.[1] The abuse and ineffectiveness of traditional antibiotics lead to severe side effects on patients during immoderate antibiotic treatment.[2] Fortunately, approaches for realizing targeted therapy have been developed in recent years. Noteworthy, smart drug delivery systems that can respond to specific external triggers[3] such as light irradiation, magnetic field, and electric filed, have been constructed to realize targeted and controlled release of therapeutic agents. However, without control over the drug activity, these strategies are still facing the limitations of poor drug selectivity, low drug loading capacity, and potential drug leakage.[4] Meantime, elaborate synthetic propotocols to modify antibiotics are time-consuming and may quickley loose the antibacterial activities.[5] Therefore, dynamic full-control of drug activity is highly desirable, which allows remote activation of drugs at the desired site, thus resulting in more effective and precise treatments.
As a deep penetrating and noninvasive trigger, ultrasound (US) has been widely explored in the field of drug delivery in recent years.[6] However, current ultrasound-mediated drug release systems are mainly based on the cavitation induced collapse of carriers, such as destruction of micelle, liposome, microbubble structures,[7] leading to burst and uncontrollable drug release. Alternatively, the development of mechanochemistry provides a new approach to achieve spatiotemporal control of the activity of drugs by using ultrasound. In general, 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).[8] Mechanophore breakage can 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.[9] Until now, research in polymer mechanochemistry mainly focused on understanding the force-induced chemical transformations and their impact on material properties, but utilization of site-selective bond-scission for the activation of drug molecules remained unexplored.[10]
The most widely synthesized polymer has only one chain-centered mechanophore, in which a single mechanophore is embedded in the central portion of a linear polymer
34
chain.[11] However, several drawbacks are associated to the single mechanophore architecture that limit its utility for applications in stress-responsive mechanical properties.[12] The most important one is the low mechanophore content that makes it
more challenging to quantify reactivity and characterize mechanically generated products.[10] Another challenge with the single chain-centered approach is the difficulty
of accurately controlling the position of the mechanophore in the middle portion of the chain.[13]
Herein, for the first time, we report the selective release and activation of aminoglycoside antibiotics from a poly-aptamer (Poly-APT) with multiple mechanophores along the polymer chain by ultrasonication. Taking the knowledge of mechanochemistry, a long RNA sequence with high molecular weight (MW) was synthesized for drug loading and deactivation without any complicated chemical synthesis by the rolling circle amplification technique (RCA) (Scheme 1).[14] Based on the robust recognition ability of R23 aptamer, the aminoglycoside antibiotics Neomycin B (NeoB) and Paromomycin (Paromo) were captured through forming an aptamer/drug complex. Upon US irradiation, mainly hydrogen bonds and electrostatic bonds were cleaved which resulted in drug dissociation and activation. More importantly, the amount of drug activation can be easily tuned by varying the duration of ultrasonication. This work demonstrated the potential of ultrasound to activate mechanoresponsive prodrug systems.
35
Scheme 1. Schematic representation of Neomycin B/Paromomycin inactivation by R23
poly-aptamer and subsequent activation in response to ultrasound.
2.2 Results and discussion
Figure 1. R23 template ligation and R23 poly-aptamer chain formation. (a) Agarose gel
(4%) characterization of R23 template ligation process. Lane M: ultra-low range DNA marker; lane 1: primer; lane 2: R23 RCT template; lane 3: hybridized template DNA without T4 ligase; lane 4: ligated template DNA with T4 DNA ligase. (b) Agarose gel (0.8%) characterization of R23 poly-APT chain. Lane M: high range RNA marker; lane P: poly-APT RCT product. (c) Representative AFM image of poly-APT chains (height scale: 0-3 nm).
36
We first synthesized a poly-nucleic acid with repeated binding loops for antibiotic loading and deactivation. The poly-aptamers (poly-APT) were obtained by rolling circle transcription (RCT) technique, endowed with tremendous drug loading sites and high payload capacity. In this work, RCT template was designed based on a short R23 RNA aptamer (Table S1). The selective binding and vigorous binding affinity between R23 RNA aptamer and aminoglycoside antibiotics (Neomycin B and Paromomycin) was well-demonstrated in our previous work.[15] The circular template was prepared by ligation of 5’-phosphorylated linear DNA with T4 DNA ligase and the successful synthesis was confirmed by agarose gel electrophoresis with the circularized template exhibiting reduced mobility compared to the linear template (Figure 1a). Fabrication of the poly-APT was verified by gel electrophoresis using a high molar mass RNA ladder (Figure 1b). Most of the product stayed in the well and did not penetrate the gel indicating the high molar mass of the RCT product. Atomic force microscopy (AFM) was carried out estimating the dimensions of possibly coiled or aggregated poly-APT to 0.1-1 µm (Figure 1c).
Figure 2. Binding and inhibition test of R23 poly-APT with aminoglycoside antibiotics. (a)
Agarose gel (0.8%) characterization of the combination of Cy3-Paromo and poly-APT before (top) and after (bottom) SYBR Gold gel staining: fluorescence image (left, green colour indicates the poly-APT and red colour indicates the Cy3-Paromo) and corresponding grayscale image (right, poly-APT and Cy3-Paromo@poly-APT stay at the top of the gels). Lane M: 1 kb plus marker, lane 1: poly-APT; lane 2: Cy3-Paromo@poly-APT; lane 3: Cy3-Paromo only. (b) Agar diffusion test of (1) free NeoB, (2) poly-APT, (3) NeoB@APT and (4) NeoB@poly-APT against S. aureus, respectively.
37
To investigate the binding quality of the antibiotics to the polyaptamer, the RCT product was incubated with cyanine 3 (Cy3) fluorophore-labelled paromomycin (Paromo) (Figure S1-S2). Gel electrophoresis showed a perfect overlap of the Cy3-Paromo fluorescence signal with the SYBR Gold-stained nucleic acid bands highlighting the successful formation of a stable Cy3-Paromo@poly-APT complex (Figure 2a). To investigate whether the complex deactivated the antibiotics, agar diffusion tests were performed on paper discs against the growth of Staphylococcus
aureus (S. aureus) (Figure 2b).[16] After overnight incubation, neither paper discs with
small molecule aptamer, its complex with NeoB, nor NeoB@PAPT showed apparent inhibition zones compared to pure NeoB, underlining the robust binding and deactivation ability of the aptamer and its RCT polymer (Figure S3).[17]
Figure 3. Agarose gel electrophoresis characterization of ultrasound triggered release of Cy3-Paromo from R23 poly-APT. (a) Agarose gel (0.8%) before (top) and after (bottom)
SYBR Gold gel staining: fluorescence image (left) and corresponding grayscale image (right). Lane M: 1 kb plus marker; lane 1: poly-APT; lane 2: Paromo@poly-APT; lane 3: Cy3-Paromo@poly-APT after 1 min US; lane 4: after 5 min US; lane 5: after 10 min US; lane 6: after 20 min US; lane 7: after 30 min US. (b) 3D fluorescence intensity distribution image of the agarose gel. (c) Cumulative release of Cy3-Paromo with ultrasonication for different times, intensity was quantified by Image J from the top grayscale image in a.
To visualize and quantify the drug activation process using US in solution, Cy3-Paromo was used again and the complex analysed by gel electrophoresis over the course of the
38
sonication tracing the remaining quantity of the bound drug. The gel images indicate that the molar mass of Cy3-Paromo-containing components decreased with continuous sonication. SYBR Gold-staining reveals that this observation is most likely caused by covalent bond scission and disaggregation along the poly-APT backbone, as the molar mass of poly-APT decreased equally (Figure 3a-3b). This interpretation is corroborated by ultrasonication of the polyaptamer alone without the bound drug (Figure S4). The remaining Cy3-Paromo bound to poly-APT was quantified by ImageJ revealing that more than 80% of the antibiotic was cumulatively transformed into its active form after 30 min of ultrasonication (Figure 3c).
Figure 4. MIC test in the presence of free NeoB, NeoB@APT, NeoB@poly-APT and
NeoB@poly-APT with in situ ultrasound treatment for different sonication times against S.
aureus. Bacteria were cultured overnight and the experiments were repeated in triplicate. Mean
values ± standard deviation, N = 3.
We investigated whether this US-induced gradual drug activation could be used to ‘turn on’ antibacterial properties by performing minimal inhibitory concentration (MIC) tests against S. aureus using the broth dilution method.[18] Note that the sonication experiments involving NeoB@poly-APT were performed in situ in presence of the bacteria and that within the observed period the application of US alone had no considerable effect on the viability of S. aureus (Figure S5 and Table S2). Moreover, transmission electron microscopy (TEM) confirms the structural integrity of the
39
bacterial membrane after sonication (Figure S6). While we recorded a MIC value of 1 µg∙mL-1 for pristine NeoB, antimicrobial activity could be detected neither when incubated with small-molecule NeoB@APT, with macromolecular NeoB@poly-APT, nor with the sonication-fragmented poly-APT, once again highlighting the strong binding and deactivation of the drug (Figure 4 and S7). However, the MIC of NeoB@poly-APT decreased to 8 µg∙mL-1 after 10 min and reached the MIC of pristine NeoB after 30 min of ultrasonication. Importantly, small molecule NeoB@APT did not show this behaviour indicating that these results originate from the mechanochemical scission of non-covalent host-guest interactions and covalent degradation of the nucleic acid backbone.
Figure 5. Live/dead staining assay of S. aureus in presence of free NeoB, NeoB@poly-APT,
NeoB@poly-APT, and no additions with in situ ultrasound treatment for 30 min. Green fluorescence (DMAO) shows total number of bacteria (live and dead) and red fluorescence (EthD-III) stains exclusively dead cells.
To once more verify this US-triggered drug activation mechanism, we performed bacteria live/dead staining by using DMAO (green, staining all cells) and Ethidium
40
Homodimer III (EthD-Ⅲ, red, staining dead cells). While pristine NeoB killed almost all bacteria, NeoB@poly-APT only showed activity after ultrasonication, but not before (Figure 5).
2.3 Conclusions
In summary, for the first time, we contribute a novel approach for drug activity regulation by combing the advantages of poly-aptamer with mechanochemistry. Long poly-aptamer RNA sequence was successfully synthesized, which has vigorous binding affinity with aminoglycoside antibiotics. The strong hydrogen bonding and electrostatic interactions between binding loops with drugs result in inactive antimicrobial property, which was proved by gel electrophoresis and agar diffusion test. Upon US irradiation, the antibiotic binding loops were selectively unzipped by stretching of poly-APT and, finally, the polynucleic acid chain was cleaved. The consequential release and activation of antibiotics were demonstrated by testing the viability of S.aureus and by a live/dead staining assay. We believe that this proof-of-concept approach provides a blueprint for constructing drug activation systems with external control by ultrasound.
2.4 Experimental sections
2.4.1 Chemicals and Materials
All chemical reagents were of analytical grade and were used without further purification if not stated otherwise. All oligonucleotide sequences were synthesized and HPLC-purified by Biomers Co. Ltd. (Germany). T4 DNA ligase (5 Weiss U∙µL-1), T7 RNA polymerase (200 U∙µL-1), rNTPs (100 mM) and RiboLock RNase inhibitor
(40 U∙µL-1) were purchased from Thermo Fisher Scientific. Bacterial Live/Dead Staining kit was ordered from PromoCell (Heidelberg, Germany). Neomycin B and paromomycin were bought from Sigma (Shanghai, China). Cyanine 3 (Cy3) was obtained from Lumiprobe. S. aureus (ATCC 6538) was obtained from ATCC: The Global Bioresource Center.
41
2.4.2 Design of the template
The DNA template necessary to perform RCT contains three parts: a middle part that is complementary to the desired aptamer sequence and two terminal parts that are complementary to the primer/promotor. Besides, there are spacer parts included between the middle and terminal parts to add distance between each repeating unit after RCT.
Table S1. DNA sequences used in this work.
2.4.3 Ligation reaction of the template
The ligation reaction was performed with 30 µL of the reaction mixture containing 20 µm R23 ligation template, 30 µm short DNA strand containing the T7 promoter sequence, and 1X ligation buffer (40 mm Tris-HCl, 10 mm MgCl2, 10 mm DTT, 0.5 mm ATP). Before adding T4 DNA ligase, the mixture was heated to 95 °C for 5 min and then cooled down to room temperature over 30 min. The circular DNA was synthesized by hybridizing the short DNA strand containing the T7 promoter sequence with R23 ligation template, which encodes one longer (16 bases) and one shorter (6 bases) complementary sequence to the short DNA strand. The nick in the circular DNA was sealed by T4 DNA ligase (5 Weiss Unit∙µL-1) at 22 °C for 5 h. The used sequences are summarized in Table S1. The DNA strand facilitating the circularization of the template was not removed as the initial use of Exonuclease I and Exonuclease III was found not to increase product purity significantly.
2.4.4 In vitro R23 poly-APT formation by RCT
Ligated R23 circular DNA template (3.3 µm) was incubated with T7 RNA polymerase (5 U∙µL-1), Ribolock RNase Inhibitor (1 U∙µL-1) and 16 mm ribonucleotide
tri-42
phosphates at 37 °C for 18 h in the reaction buffer (40 mm Tris-HCl, 6 mm MgCl2, 10 mm DTT, 10 mm NaCl, and 2 mm spermidine) as the final concentrations. T7 RNA Polymerase is an RNA polymerase from the T7 bacteriophage that catalyzes the formation of RNA from DNA in the 5'-3' direction. The reaction was inactivated by heating at 70 °C for 10 min. The concentration of repeated units of R23 poly-APT was measured by Quant-iT RNA Assay Kit.
2.4.5 Modification of paromomycin with cyanine 3 to compound 1
Scheme S1. Reaction of paromomycin with cyanine 3 to compound 1.
Paromomycin sulfate (12 mg, 15.3 µmol, 3 eq.) was dissolved in 10 mM Na3PO4 buffer at pH 7 (1.5 mL) and a solution of Cyanine3-NHS (3.3 mg, 5.1 µmol, 1 eq.) in DMF (0.5 mL) was added. The reaction mixture was stirred overnight protected from light. Solvents were removed under high vacuum and the crude mixture was re-dissolved in water, filtered through cotton and purified by flash automated chromatography system, eluted with a linear gradient 0-100% of buffer B (95% MeCN, 5% H2O, containing 10 mM TFA) in buffer A (95% H2O, 5% MeCN, containing 10 mM TFA). Fractions containing the product were concentrated in vacuo to remove MeCN residues and lyophilized to obtain the product 1 as a pink solid (4× TFA salt). Yield: 3.8 mg (2.5 µmol, 49%). 1H-NMR (600 MHz, D 2O) δ [ppm] = 8.55 (t, J = 13.5 Hz, 1H, -CH-CH=CH-), 7.59 (dd, J = 7.0, 4.1 Hz, 2H, Ar-H), 7.50 (dd, J = 9.0, 5.8 Hz, 2H, Ar-H), 7.38 – 7.33 (m, 4H, Ar-H), 6.34 (dd, J = 18.0, 13.6 Hz, 2H, CH; =CH-CH=CH-), 5.81 (d, J = 4.0 Hz, 1H, 1-H’), 5.44 – 5.39 (m, 1H, 1-H’’), 5.16 (d, J = 1.7 Hz, 1H, 1-H’’’), 4.40 (t, J = 5.7 Hz, 1H, 3-H’’), 4.37 – 4.32 (m, 1H, 2-H’’), 4.28 – 4.18 (m, 2H, 3-H’’’; 4-H’’), 4.17 – 4.12 (m, 2H, (N+)CH2), 4.08 – 3.98 (m, 2H, 5-H’’’;5-H’), 3.97 – 3.86 (m, 4H, 5-H’’a+b; 5-H; 3-H'), 3.83 – 3.70 (m, 4H, 6-H'a+b; 4-H; 6-H),
43 3.67 – 3.62 (m, 4H,N-CH3;4-H’’’), 3.62 – 3.55 (m, 1H, 2-H’’’), 3.55 – 3.48 (m, 2H, 3-H; 4-H’), 3.48 – 3.30 (m, 3H, 2-H’;1-3-H; 6-H’’’b), 3.27 (dd, J = 13.9, 6.1 Hz, 1H, 6-H’’’a), 2.52 (d, J = 12.9 Hz, 1H, 2-Heq), 2.29 (t, J = 7.1 Hz, 2H, CH2), 1.90 (q, J = 11.4, 9.3 Hz, 3H, 2-Hax; CH2), 1.76 (m, 12H, CH3), 1.67 (q, J = 7.2 Hz, 2H, CH2), 1.44 – 1.36 (m, 2H, CH2). 13C-NMR (151 MHz, D2O) signals based on HSQC δ [ppm] = 150.9
(=CH-CH-CH=), 128.6 (Ar), 125.2(Ar), 122.2 (Ar), 111.1 (Ar), 109.5 (C1’’), 102.0 (-CH=CH-), 95.8 (C1’’’), 95.5(C1’), 84.3 (C5), 81.7 (C4’’), 77.4 (C5’’’), 76.1 (3’’), 73.9 (C4) 73.7(C2’’), 72.5(C5’), 72.3 (C6), 69.3 ,69.2 (C4’), 68.8 (C3’), 67.5 (C3’’’), 66.2 (C4’’’), 60.3 (C5’’’), 60.3 (C6’), 53.9 (C2’), 51.1 (C3), 49.7 (C1), 48.9(C2’’’), 48.8, 43.6(N+)-CH2), 39.2(C6’’’), 35.3 (C(O)CH2) 30.7(N-CH3), 28.1 (C2), 27.1 (CH3), 26.4 (CH2), 25.4 (CH2), 24.9 (CH2). (C=O and quaternary C not detectable by HSQC spectroscopy). (Figure S1) ESI-MS: (m/z) calculated for C55H80N7O15+ [M] 1054.5, found: 1054.4. (Figure S2)
44
Figure S1. (a) UPLC traces of the final product 5 showing a single peak at 4.59 min in Total
Ion Current (TIC) and (b) corresponding ESI+ mass spectrum, m/z calculated for C55H80N7O15+ [M] 1054.5; found:1054.4.
45
Figure S2. (a) UPLC elugram of the final product 5 showing a single peak at 4.59 min in Total
Ion Current (TIC) and (b) corresponding ESI+ mass spectrum (m/z calculated for C55H80N7O15+ [MH]+ 1054.5, found:1054.4).
2.4.6 Sonication Experiments
Ultrasonication experiments on poly-APT were performed in a 1 mL ultrasonication vessel (Test tube heavy-walled, 2775/2, Assistant) with a Qsonica Q125 sonicator (USA) equipped with a 3 mm diameter microtip probe (A12628PRB20) at 50% amplitude and f = 20 kHz. The input sonication energy E in J was recorded during sonication and used to calculate sonication power P = E∙t-1 and power intensity IP =
P∙A-1 where t is effective sonication ‘on’ time and A is the area of the probe tip. Pulsed sonication (1.0 s on, 1.0 s off) was used and over the full duration of the sonication this
46
corresponds to t = 1800 s, E = 4551 J, P = 2.53 W, A = 0.07 cm2, and I
P = 36.14 W∙cm -2. The vessel was placed in an ice bath to maintain a temperature inside the vessel of 6-9 °C throughout sonication.
2.4.7 Test of binding by gel electrophoresis
0.8% agarose gel was fabricated to test the binding between R23 poly-APT and Cy3-Paromo (predicted secondary structures in Figure S3). The R23 poly-APT solution in 10 mM Na3PO4 buffer (pH 6.8) was heated to 95 °C for 5 min and then cooled down to room temperature over 30 min to form the secondary structure, which is suitable for binding of antibiotics. Then Cy3-Paromo was incubated with poly-APT in a ratio of 1:1.5 (aptamer unit: Cy3-Paromo) in 10 mM Na3PO4 buffer (pH 7.4) and the mixture was kept at 37 °C for 30 min. Firstly, the gel imager was used to irradiate the R23 poly-APT@Cy3-Paromo and then the images were recorded. Besides, a digital camera was also used to record pictures. Afterwards, the gel was stained with SYBR™ Gold Nucleic Acid Gel Stain solution for 30 min. Finally, the gel imager was operated again to image the gel after staining.
2.4.8 Agar diffusion test
Before the test, isolated colonies of S. aureus were allowed to grow in Müller Hinton Broth (MHB) for 8-10 h to reach their late log phase. After growing, bacteria needed to be diluted back to around 0.5∙109 cfu∙mL-1 (OD600 = 0.5). 200 µL of suspension was plated on Müller Hinton agar plates and left to dry. Then the samples were prepared by pipetting each sample (NeoB only, APT, APT@NeoB complex, poly-APT@NeoB complex) containing 2.5 µg NeoB on each paper disk. Loading rates were 1:1.5 (aptamer unit: NeoB). Once the disks were dry, a flame-sterilized tweezer was used to gently place the disk on top of the agar and lightly pressed. The inhibition zones were measured after overnight incubation.
2.4.9 Ultrasound-controlled antibiotics release
To investigate the release of antibiotics from their complexes with nucleic acids, Cy3-Paromo was incubated with poly-APT (8.5 µM) with a ratio of 1:1.5 according to the method described above. The sonication treatments were performed as described above. The 800 µL of complex suspension was filled into a 1.2 mL cylindrical glass tube and
47
the ultrasonic emitter was immersed into the middle of the solution. At defined times, 20 µL of solution were pipetted and taken out for further gel electrophoresis.
2.4.10 Minimal inhibitory concentration (MIC) test
First, S. aureus was allowed to grow in MHB for 8-10 h to reach its late log phase. Before use, bacteria need to be diluted to around 106 cfu∙mL-1. Then the bacteria were mixed with different amounts of APT/poly-APT@NeoB solution to a final volume of 100 µL. At predefined intervals (0, 5, 10, 20, and 30 min) during in situ ultrasound treatment, 50 µL of treated solution was taken out and mixed with 50 µL MHB in a 96-well plate with a final concentration of 5∙105 cfu∙mL-1 of S. aureus. Then the ultrasonication-treated bacteria were allowed to grow for 8-10 h at 37 ℃ to reach their late log phase. Finally, the optical density of bacterial growth was measured by plate reader at a wavelength of 600 nm. Bacteria samples without APT/poly-APT@NeoB complex were treated similarly as a control. Cell culturing experiments were performed in triplicates with three independent experiments repeated on different days.
2.4.11 Live/dead staining assay
The differentiation of live/dead bacteria after applying ultrasound was performed by a bacteria live/dead staining kit from PromoCell (Heidelberg, Germany). With or without ultrasound, the bacteria were incubated with DMAO (green) and EthD-III (red) mixture for 15 min. 5 μL of the stained bacterial suspension was dropped on a slide with an 18 mm square coverslip and then observed under a fluorescence microscope. The fluorescence from both live and dead bacteria may be viewed simultaneously with any standard FITC long-pass filter set. The live (green fluorescent) and dead (red fluorescent) cells were imaged separately with FITC and Cy3 or Texas Red band-pass filter sets.
2.4.12 Atomic Force Microscopy (AFM) measurements
RNA and mica are both negatively charged, so it is necessary to modify the mica surface or the RNA counter ion to allow binding. The counterion method is carried out by adsorbing the RNA onto the mica in the presence of Mg2+. Mg2+ serves as a counterion on the negatively charged RNA backbone and also provides additional charge to bind the mica. The poly-APT was diluted in MgCl2 (5 mM) solution to a final concentration
48
of 2.5 ng∙μL-1. Thereafter, a piece of mica was glued to a metal support and cleaved with a piece of adhesive tape followed by placing 30 μL of the RNA solution on the center of the mica disk for 1 min. After rinsing with deionized H2O and drying under N2, the prepared sample was loaded onto the AFM scanner. AFM images were taken on an Asylum Research MFP-3D (Santa Barbara, CA) in AC mode. Cantilevers of silicon nitride were purchased from NanoWorld (Neuchatel, Switzerland) with a force constant of 42 N∙m-1. Images were obtained in tapping mode.
Figure S3. Predicted secondary structures of single R23 aptamer sequence (a) and five repeats
49
Figure S4. Ultrasound treatment of poly-APT sequences for different time periods. (a) From
line 1-10: 0 s, 1 min, 3 min, 5 min, 10 min, 20 min, 30 min, 45 min, 1 h, 1.5 h, 2 h. (b) From line 1-5: 30 min, 45 min, 1 h, 1.5 h, 2 h. M represents a molar mass marker (single-stranded RNA transcripts). Numbers indicate nucleotides.
Figure S5. Growth curve of S. aureus after ultrasound treatment for different time. Mean
50
Table S2. Viability of S. aureus with ultrasound treatment for different times.
Figure S6. TEM images of S. aureus before (a) ultrasound treatment and after (b) ultrasound
treatment for 30 min.
Figure S7. MIC test for NeoB only, APT with or without ultrasound treatment and PAPT with or without ultrasound treatment. Mean values ± standard deviation, N = 3.
51
References
[1] R. Sugden, R. Kelly, S. Davies,Nat. Microbiol. 2016, 1.
[2] X. Wang, D. Ryu, R. H. Houtkooper, J. Auwerx, Bioessays 2015, 37, 1045-1053.
[3] (a) H. S. El-Sawy, A. M. Al-Abd, T. A. Ahmed, K. M. El-Say, V. P. Torchilin,
ACS Nano 2018, 12, 10636-10664; (b) M. Qiu, D. Wang, W. Y. Liang, L. P.
Liu, Y. Zhang, X. Chen, D. K. Sang, C. Y. Xing, Z. J. Li, B. Q. Dong, F. Xing, D. Y. Fan, S. Y. Bao, H. Zhang, Y. H. Cao, Proc. Natl. Acad. Sci. U.S.A 2018,
115, 501-506; (c) A. K. Hauser, R. J. Wydra, N. A. Stocke, K. W. Anderson, J.
Z. Hilt, J. Control. Release 2015, 219, 76-94.
[4] (a) M. Karimi, A. Ghasemi, P. S. Zangabad, R. Rahighi, S. M. M. Basri, H. Mirshekari, M. Amiri, Z. S. Pishabad, A. Aslani, M. Bozorgomid, D. Ghosh, A. Beyzavi, A. Vaseghi, A. R. Aref, L. Haghani, S. Bahrami, M. R. Hamblin, Chem.
Soc. Rev. 2016, 45, 1457-1501; (b) S. Mura, J. Nicolas, P. Couvreur, Nat. Mater.
2013, 12, 991-1003.
[5] (a) B. Llano-Sotelo, E. F. Azucena, L. P. Kotra, S. Mobashery, C. S. Chow,
Chemistry & Biology 2002, 9, 455-463; (b) E. Azucena, I. Grapsas, S.
Mobashery, J. Am. Chem. Soc. 1997, 119, 2317-2318.
[6] (a) Z. L. Li, J. Han, L. D. Yu, X. Q. Qian, H. Xing, H. Lin, M. C. Wu, T. Yang, Y. Chen, Adv. Funct. Mater. 2018, 28; (b) J. An, Y. G. Hu, C. Li, X. L. Hou, K. Cheng, B. Zhang, R. Y. Zhang, D. Y. Li, S. J. Liu, B. Liu, D. Zhu, Y. D. Zhao,
Biomaterials 2020, 230.
[7] (a) S. E. Ahmed, A. M. Martins, G. A. Husseini, J. Drug Target 2015, 23, 16-42; (b) C. P. Phenix, M. Togtema, S. Pichardo, I. Zehbe, L. Curiel, J. Pharm.
Pharm. 2014, 17, 136-153; (c) F. Kiessling, S. Fokong, J. Bzyl, W. Lederle, M.
Palmowski, T. Lammers, Adv. Drug Deliv. Rev. 2014, 72, 15-27.
52
[9] (a) C. L. Brown, S. L. Craig, Chem. Sci. 2015, 6, 2158-2165; (b) G. Cravotto, E. C. Gaudino, P. Cintas, Chem. Soc. Rev. 2013, 42, 7521-7534; (c) M. Schaefer, B. Icli, C. Weder, M. Lattuada, A. F. M. Kilbinger, Y. C. Simon,
Macromolecules 2016, 49, 1630-1636.
[10] S. Akbulatov, R. Boulatov, Chemphyschem 2017, 18, 1422-1450.
[11] S. L. Potisek, D. A. Davis, N. R. Sottos, S. R. White, J. S. Moore, J. Am. Chem.
Soc. 2007, 129, 13808-13809.
[12] B. H. Bowser, S. L. Craig, Polym. Chem. 2018, 9, 3583-3593.
[13] Z. S. Kean, G. R. Gossweiler, T. B. Kouznetsova, G. B. Hewage, S. L. Craig,
Chem. Commun. 2015, 51, 9157-9160.
[14] (a) M. G. Mohsen, E. T. Kool, Acc. Chem. Res. 2016, 49, 2540-2550; (b) Z. Y. Li, C. W. Lau, J. Z. Lu, Anal. Chem. 2016, 88, 6078-6083.
[15] A. A. Bastian, A. Marcozzi, A. Herrmann, Nat. Chem. 2012, 4, 789-793. [16] M. Balouiri, M. Sadiki, S. K. Ibnsouda, J. Pharm. Anal. 2016, 6, 71-79.
[17] J. E. Weigand, S. R. Schmidtke, T. J. Will, E. Duchardt-Ferner, C. Hammann, J. Wohnert, B. Suess, Nucleic Acids Res 2011, 39, 3363-3372.
