<p><strong>Introduction</strong></p>
<p>Since antibiotics were discovered, bacteria have demonstrated the ability to develop resistance by many different mechanisms. According to WHO reports from 2014, there has been an alarming increase in the antibiotic resistant bacterial strains in most parts of the world<sup>1</sup>. Our previous results showed that a nanoantibiotic (NAB) design created in our laboratory<sup>2</sup>, composed of a cerium oxide core, mesoporous silica shell loaded with capsaicin, and a chitosan coating, are effective against planktonic E. coli. However, most of the pathogenic bacteria form biofilms during infections. That is why the next stage of studying NAB is to determine whether they are effective against biofilms of different species. Moreover, the results of NAB efficiency against planktonic E. coli did not clearly show the contribution of the antibiotic drug component of NAB &#8211; capsaicin. Hence, the first step of the current study is to determine whether and to what degree, mesoporous silica nanoparticles (MSN) &#8211; serving as NAB model in this case - penetrate biofilms as a function of particle shape and surface coating; as well as finding the efficient concentration of capsaicin against E. coli and S. aureus &#160;to optimize the NAB dosing against biofilms.</p>
<p><strong>&#160;</strong></p>
<p><strong>Aim</strong></p>
<p>To check in vitro penetration of MSN on S. aureus biofilm and antibacterial activity of NAB and pure capsaicin on E. coli and S. aureus biofilms.</p>
<p><strong><br />Methods</strong></p>
<p>To investigate NAB efficiency on biofilms MBEC-high-throughput assay<sup>3</sup> was performed. Equal biofilms formed on peg-lids were incubated with different concentrations of NAB and capsaicin. After different time point biofilms were sonicated and plated on agar plated to perform CFU counting. To determine the efficient concentration of capsaicin, biofilms were formed in 12 well plates and then incubated with different concentrations of capsaicin. To visualize inhibitory effect, plating for CFU counting and Resazurin assay were applied. To evaluate the penetration of particles, labeled and non-labeled particles were added to fully grown St. aureus biofilms, incubated and visualized with confocal microscopy and structured illumination microscopy.</p>
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<p><strong>Results</strong></p>
<ol>
<li>Through two different microscopy techniques penetration of particles into biofilm and their localization next to bacteria cells were observed.</li>
<li>In MBEC-high-throughput assay no inhibitory effect of NAB against E. coli biofilms was detected in comparison with untreated bacteria.</li>
<li>Resazurin assay and CFU counting method allowed us to determine the most efficient concentration of capsaicin against E. coli and St. aureus biofilms.</li>
</ol>
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<p><strong>Conclusion</strong></p>
<ol>
<li>Use of MSN and NAB in particular to deliver active antibacterial agents inside the biofilm is justified.</li>
<li>We cannot claim that NAB does not demonstrate any activity against E. coli biofilms, though we can suggest that the peg-lid set up is not sufficient for the NAB design. Further experiments are required.</li>
<li>The next step is to test different concentrations of NAB against biofilms with more appropriate methods than MBEC-high-throughput assay. These results will allow us to make conclusions about the benefits of NAB in comparison with pure capsaicin.</li>
</ol>
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<p><strong>References</strong></p>
<ol>
<li>Govardhanam, N.P. (2017). Development of nanoantibiotics and evaluation via in vitro and in vivo imaging. University of Turku, Finland.</li>
<li>Ventola, C. Lee.&#160;Pharmacy and Therapeutics&#160;40.4: 277, 2015</li>
<li>Harrison, J. et al., BMC microbiology 5(1), 53, 2005.</li>
</ol>
A High-throughput Assay for mRNA Silencing in Primary Cortical Neurons in vitro with Oligonucleotide Therapeutics
