Aptamer-Functionalized Metal-Organic Frameworks as an Efficient Delivery Vehicle of Antimicrobial Peptides

Infections caused by bacteria are among the most serious physiological conditions that threaten human health. The swift emergence of resistant bacteria is jeopardizing the efficacy of most antibiotics, highlighting the importance of finding non-invasive theragnostic systems to treat bacterial infections. Currently, different antimicrobial systems used in the field include metal-ion nanoparticles (NPs) (<i>e</i>.<i>g</i>., Ag NPs), semiconductor photocatalytic materials (<i>e</i>.<i>g</i>., TiO₂), and antibacterial-nanocarrier composites. However, these systems face several shortcomings such as very low efficiency, short-term effect, and fast release of the antimicrobial agent. To overcome these issues, metal-organic frameworks (MOFs) have emerged as a promising theragnostic system. MOFs are nanoporous crystalline materials made by the self-assembly of metal clusters and organic linkers. They have shown high biocompatibility, high drug loading capacity, slow release of their cargo, and a surface that is easy to functionalize. Despite the potential of these materials as nanocarriers, hardly any research has been carried out using MOFs for antimicrobial delivery.<br/>In this work, a novel MOF named PCN-222 was functionalized with aptamers (apt@PCN-222), short oligonucleotide sequences that selectively bind to bacteria, to deliver doxorubicin and antimicrobial peptides (apt@dox@PCN-222) to bacterial targets. PCN-222 is a photoluminescent Zr-based nanomaterial that has shown great potential for bio-applications due to its large porosity, high stability, and biocompatibility. Growth assays for Gram-positive (<i>Bacillus subtilis</i>) and Gram-negative (<i>Escherichia coli</i>) bacteria were used to determine bacteria growth with bare PCN-222, free doxorubicin, apt@PCN-222, and apt@dox@PCN-222. Different characterization techniques were used to assess the physico-chemical properties of the NPs. High resolution transmission electron microscopy (HR-TEM) was used to confirm the crystallinity NPs. Scanning electron microscopy (SEM) and dynamic light scattering (DLS) were carried out to determine the particle size of the NPs and their colloidal stability, respectively. Ultraviolet-visible spectroscopy (UV-vis) was used to determine the drug loading content and inductively coupled plasma mass spectrometry (ICP-MS) was utilized to quantify the binding of aptamers into the surface of PCN-222. Finally, the binding of apt@PCN-222 NPs to the different types of bacteria was assessed by structured illumination microscopy (SIM).<br/>Crystalline PCN-222 NPs with a size of 145 ± 7 nm and a BET area of 1980 m²/g were successfully synthesized. Doxorubicin was successfully encapsulated into PCN-222, obtaining a drug loading greater than 40%. This result confirms the expected high porosity of this NP. Enhanced binding of PCN-222 to the bacteria was corroborated by measuring the overlapping region by SIM since GFP-expressing <i>E. coli</i> are green and the PCN-222 NPs are red. The increase in binding levels was obtained by exploiting the synergistic effect of two binding mechanisms: electrical binding due to the positive charge of PCN-222 and selective binding by the addition of aptamers to the surface of the MOF. The phosphate ions present in PBS destroyed the porous matrix of PCN-222, thus releasing the doxorubicin in a slower manner than the current antibacterial-nanocarrier composites. The slow release of the drug, together with the selective binding of the apt@PCN-222 conjugate to the bacteria resulted in an increased reduction of bacteria growth when compared to free doxorubicin.<br/>PCN-222 has shown promising physico-chemical properties for the effective delivery of antimicrobial agents to bacteria. Further studies will examine the effect of using larger aptamers or cationic polymers to increase its targeting effects. This study represents the first systematic attempt to target bacteria using aptamer-functionalised PCN-222, promoting the use of nanomaterials against antibiotic resistance.