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Stability of ssDNA micelles and PMB micelles upon dilution with water, showcasing the enhanced stability upon cation depletion. (A) Schematic representation of interactions in the (i) ssDNA micelles and (ii) PMB loaded DNA micelles (PMB micelles). Size and PDI changes upon serial dilution of (B) ssDNA micelles and (C) PMB micelles.
Source publication
Nucleic acid-based materials showcase an increasing potential for antimicrobial drug delivery. Although numerous reports on drug-loaded DNA nanoparticles outline their pivotal antibacterial activities, their potential as drug delivery systems against bacterial biofilms awaits further studies. Among different oligonucleotide structures, micellar nan...
Contexts in source publication
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... work by Liu et al has shown that the presence of counterions (e.g., sodium and potassium) enhanced the thermal stability of DNA micelles 33 . We hypothesized that the blank ssDNA micelles would not withstand ion depletion whilst the incorporation of cationic PMB would contribute to an increased stability (Figure 4a&b, ...
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... test the stability of the structure, we made serial dilutions of the ssDNA and PMB micelles in water and assessed changes in nanoparticle size and PDI with the depletion of counterions. As expected, ssDNA micelles exhibited a large size increase and broad PDI variation upon serial dilutions (Figure 4b), indicating their lack of stability against dilution. Similar findings showed the critical micelle concentration of these structures to be 1 µM (observed with TEM images) 24 . ...
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... the other hand, a highly stable complex formation for the PMB micelles was observed with no changes in size or PDI of the micelles up to a concentration of 0.63 µM (after 32x dilution) (Figure 4c). ...
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... obtained 2D and 3D projection images of the micelle-treated biofilms to illustrate the influence of time on the penetration as well as the overall depth of penetration attainable by the micelles. Control images obtained after treatment with only the buffer confirmed the formation of mature biofilms with dense clusters of P. aeruginosa as shown in Figure S4). In contrast, higher intensity was obtained from the red channel on the 2D images after 2 h (Figure 6a), which demonstrates that biofilm penetration was highly dependent on incubation time. ...
Citations
... Other studies have advanced our understanding of biofilm-NP dynamics [31][32][33][34][35], however, a significant gap in research remains, especially regarding the use of DNA-based nanostructures. Our previous work has demonstrated the biofilm penetration capabilities of polymyxin-B loaded DNA micelles [36], yet this promising area remains largely under-researched. This study seeks to fill this gap by exploring the interaction, diffusion and retention of a range of drug-free DNA NPs distinct in their physicochemical properties with or within P. aeruginosa biofilms. ...
... It is important to note that even though no drug was loaded into the NPs in this study, we believe all three carriers would be suitable candidates as drug delivery systems. For instance, we have previously shown that ssDNA micelles can be loaded with different concentrations of polymyxin B and showed antibiofilm effect [36]. Other plain DNA based structures have shown the capacity to load antimicrobial peptides [62], including TDNs [63], with enhanced antimicrobial action. ...
... Lastly, we would like to note that despite the fact that toxicity evaluations were not presented in this work, we have strong reason to believe that these particles would not present immediate signs of toxicity. Namely, previous work conducted in our group evaluated the cytotoxicity of ssDNA micelles at the same concentration were evaluated against macrophages and fibroblasts that showed no impact on cell viability, both plain and loaded with different concentrations of polymyxin B [36]. Those findings were in agreement with work conducted by other research groups using DNA based micelles with no signs of toxicity [21,66]. ...
Biofilms present a great challenge in antimicrobial therapy due to their inherent tolerance to conventional antibiotics, promoting the need for advanced drug delivery strategies that improve therapy. While various nanoparticles (NPs) have been reported for this purpose, DNA-based NPs remain a largely unexploited resource against biofilm-associated infections. To fill this gap and to lay the groundwork for their potential therapeutic exploitation, we investigated the diffusion, penetration, and retention behaviors of three DNA-based nanocarriers —plain or modified—within P. aeruginosa biofilms. Watson-Crick base pairing or hydrophobic interactions mediated the formation of the plain NPs whilst electrostatic interaction enabled optimization of coated NPs via microfluidic mixing. We assessed the interactions of the nanocarriers with biofilm structures via Single Plane Illumination Microscopy – Fluorescence Correlation Spectroscopy (SPIM-FCS) and Confocal Laser Scanning Microscopy (CLSM). We demonstrate the impact of microfluidic parameters on the physicochemical properties of the modified DNA NPs and their subsequent distinct behaviors in the biofilm. Our results show that single stranded DNA micelles (ssDNA micelle) and tetrahedral DNA nanostructures (TDN) had similar diffusion and penetration profiles, whereas chitosan-coated TDN (TDN-Chit) showed reduced diffusion and increased biofilm retention. This is attributable to the relatively larger size and positive surface charge of the TDN-Chit NPs. The study shows first and foremost that DNA can be used as building block in drug delivery for antibiofilm therapeutics. Moreover, the overall behavioral findings are pivotal for the strategic selection of therapeutic agents to be encapsulated within these structures, possibly affecting the treatment efficacy. This research not only highlights the underexplored potential of DNA-based NPs in antibiofilm therapy but also advocates for further studies using different optimization strategies to refine these nanocarrier systems for targeted treatments in biofilm-related infections.
... To prepare the blank DNA micelles, 20 µM of a cholesterol modified DNA sequence (DNA amphiphile) was diluted in 1× Micelle buffer (2.5 mM NaCl, 1.25 mM MgCl 2 and 0.05 M sodium acetate, pH 4.5), hybridized at 95°C and slowly cooled to 4°C. For the polymyxin B (PMB) loaded DNA micelles, PMB stock solution was prepared in 1× MB and added to 40 µM suspension of the DNA amphiphile to achieve final concentrations of 64 µg/mL PMB as previously reported [30]. ...
... The FESEM images suggest that the sputtering deposition of Ag led to the formation of nanoparticles instead of a continuous film, and the increasing deposition reduced the nano-bowl curvature, decreasing the effective surface area. It has previously been established creating a higher intensity of hotspots compared to the flat surface [30]. The shape anisotropy of the Ag-PDMS film also induces a higher density of hotspots compared to a flat film due to its tightly spaced discontinuous Ag nanoparticles that exhibit strong LSPR for a robust enhancement in the Raman signal. ...
... The SERS enhancement also depends on the shape of the optimized Ag-coated nano-bowl structure to produce an enhanced electric field [29]. In brief, the curved structure, acting as a lens focuses the laser light spot tightly, creating a higher intensity of hotspots compared to the flat surface [30]. The shape anisotropy of the Ag-PDMS film also induces a higher density of hotspots compared to a flat film due to its tightly spaced discontinuous Ag nanoparticles that exhibit strong LSPR for a robust enhancement in the Raman signal. ...
Programmable nanoscale carriers, such as liposomes and DNA, are readily being explored for personalized medicine or disease prediction and diagnostics. The characterization of these nanocarriers is limited and challenging due to their complex chemical composition. Here, we demonstrate the utilization of surface-enhanced Raman spectroscopy (SERS), which provides a unique molecular fingerprint of the analytes while reducing the detection limit. In this paper, we utilize a silver coated nano-bowl shaped polydimethylsiloxane (PDMS) SERS substrate. The utilization of nano-bowl surface topology enabled the passive trapping of particles by reducing mobility, which results in reproducible SERS signal enhancement. The biological nanoparticles’ dwell time in the nano-trap was in the order of minutes, thus allowing SERS spectra to remain in their natural aqueous medium without the need for drying. First, the geometry of the nano-traps was designed considering nanosized bioparticles of 50-150 nm diameter. Further, the systematic investigation of maximum SERS activity was performed using rhodamine 6 G as a probe molecule. The potential of the optimized SERS nano-bowl is shown through distinct spectral features following surface- (polyethylene glycol) and bilayer- (cholesterol) modification of empty liposomes of around 140 nm diameter. Apart from liposomes, the characterization of the highly crosslinked DNA specimens of only 60 nm in diameter was performed. The modification of DNA gel by liposome coating exhibited unique signatures for nitrogenous bases, sugar, and phosphate groups. Further, the unique sensitivity of the proposed SERS substrate displayed distinct spectral signatures for DNA micelles and drug-loaded DNA micelles, carrying valuable information to monitor drug release. In conclusion, the findings of the spectral signatures of a wide range of molecular complexes and chemical morphology of intra-membranes in their natural state highlight the possibilities of using SERS as a sensitive and instantaneous characterization alternative.
Introduction
Resistance of intracellular pathogens is a challenge in microbial therapy. Methicillin-resistant Staphylococcus aureus (MRSA), which is able to persist inside the cells of infected tissues, is protected from attack by the immune system and many antimicrobial agents. To overcome these limitations, nano-delivery systems can be used for targeted therapy of intracellular MRSA.
Methods
Hyaluronic acid-modified azithromycin/quercetin micelles (HA-AZI/Qe-M) were synthesized by thin film hydration. The micelles were characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS) and Fourier transform infrared spectroscopy (FTIR), and the drug loading (DL) and encapsulation efficiency (EE) were detected by high performance liquid chromatography (HPLC). The uptake ability of RAW264.7 cells was investigated, and its distribution in mice was evaluated by in vivo imaging. The inhibitory effect of the micelles against MRSA in vitro and its ability to eliminate intracellular bacteria were evaluated. Bacterial muscle-infected mice were constructed to evaluate the therapeutic effect of the micelles on bacterial infections in vivo and the biocompatibility of the micelles was investigated.
Results
HA-AZI/Qe-M had suitable physical and chemical properties and characterization. In vitro antibacterial experiments showed that HA-AZI/Qe-M could effectively inhibit the growth of MRSA, inhibit and eliminate the biofilm formed by MRSA, and have an excellent therapeutic effect on intracellular bacterial infection. The results of RAW264.7 cells uptake and in vivo imaging showed that HA-AZI/Qe-M could increase the cellular uptake, target the infection site, and prolong the treatment time. The results of in vivo antibacterial infection experiments showed that HA-AZI/Qe-M was able to ameliorate the extent of thigh muscle infections in mice and reduce the expression of inflammatory factors.
Conclusion
HA-AZI/Qe-M is a novel and effective nano-drug delivery system that can target intracellular bacterial infection, and it is expected to be safely used for the treatment of MRSA infection.
Antibiotic resistance requires alternatives to fight multi-drug resistant strains. Antimicrobial peptides (AMPs) act by disrupting or solubilizing microbial cell walls or membranes in accordance with mechanisms difficult to counteract from the microbe’s point of view. In this review, structure–activity relationships for AMPs and their assemblies are discussed, considering not only their self-assembly but also their interactions with their carriers for optimal delivery or their combinations with other complementary antimicrobials or moieties covalently bound to their chemical structure. The effect of the formulations on AMP activity is also evaluated, revealing a myriad of possibilities. Depending on the interaction forces between the AMP, the carrier, or the elements added to the formulations, AMP activity can be reduced, enhanced, or remain unaffected. Approaches protecting AMPs against proteolysis may also reduce their activity.
