Smart Nanoparticle Drug Delivery Systems for the Treatment of Bacterial and Fungal Infections

Introduction: : Microbial infections pose significant therapeutic challenges due to rising antimicrobial resistance and limited availability of effective antimicrobials. Microbial species like Staphylococcus bacteria and Candida fungi are particularly problematic, with their ability to form biofilms that are extremely difficult to treat; these biofilms often require significantly higher drug concentrations (>1000×) for effective treatment compared to planktonic microbes. Managing bacterial biofilms is difficult due to persistent resistance mechanisms, while treating fungal biofilms presents additional challenges, including high toxicity and poor aqueous solubility of many antifungal agents. Nanoparticles are promising drug-delivery vehicles for the targeted delivery of antimicrobials into biofilms in addition to reducing toxicity of encapsulated drugs. Here, we will describe several unique nanoparticle drug delivery systems that exhibit excellent antimicrobial activity against infections caused by Staphylococcus aureus and other biofilm forming bacteria, along with Candida spp. First, we developed stimuli-responsive antibiofilm gelatin nanoparticles (R-GNPs) that release encapsulated silver nanoparticles as a broad-spectrum antibiotic alternative, in response to bacterial gelatinases, while disrupting the biofilm matrix. To address Candida infections, we developed a Candida targeting liposomal nanoparticle functionalized with the peptide, penetratin (Pen). We encapsulated the antifungal drug, posaconazole (POS), in these nanoparticles. Overall, we present these two innovative nanoparticle delivery systems as versatile and effective approaches to overcome the current barriers in treating bacterial and fungal biofilm infections.

Materials and

Methods: : For the R-GNPs, deoxyribonuclease I was conjugated to gelatin nanoparticles (GNPs), leveraging its ability to degrade extracellular DNA, a key structural component in Staphylococcus biofilms. Silver was loaded into the GNPs (Ag-GNP) and modified with a pH-degradable poly(ethylene glycol) (PEG) derivative (R-GNP), promoting nanoparticle stability and increased accessibility within the acidic biofilm microenvironment. The minimum biofilm eradication concentration (MBEC) was determined using crystal violet (CV) staining. Treated biofilms were visualized using scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) following staining with a Ruby biofilm matrix stain. In vivo antibiofilm activity was assessed in a murine excisional wound S. aureus biofilm infection model using bioluminescent S. aureus Xen29. The luminescence signal was monitored daily via an In Vivo Imaging System (IVIS) starting 24 hours post-infection.

POS-loaded liposomes containing two POS concentrations (Low Loading (LL): 0.31 % (w/w); High Loading (HL): 1.55 % (w/w) were fabricated with 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), cholesterol (CHOL), and a PEG-maleimide linker using a thin film hydration method. Subsequent surface decoration with Pen was achieved using thiol-maleimide conjugation chemistry. Pen-decorated liposome interaction with Candida cells was measured using flow cytometry and visualized using CLSM with fluorescently labelled liposomes. Antifungal efficacy against planktonic and biofilm Candida was evaluated using broth microdilution assays and CV staining to determine the minimum inhibitory concentrations (MICs) and minimum biofilm inhibitory concentrations (MBICs), respectively. Finally, in vivo prophylactic efficacy of Pen-decorated and POS-loaded liposomes was assessed in a murine model of a localized C. albicans intradermal infection.

Results, Conclusions, and Discussions:: After confirming the dual-stimuli responsive mechanism of R-GNPs, we evaluated their performance in vitro against S. aureus 25932 and in vivo in a murine S. aureus biofilm wound infection. Compared to biofilms treated with the control groups, R-GNP treatment drastically disrupted the Staphylococcus biofilm structure. R-GNP treatment of established S. aureus biofilms led to an 80% reduction in biofilm biomass at a R-GNP concentration of 32 μg/mL (Figure 1a), which was further confirmed via SEM images (Figure 1b). CLSM images revealed substantial disruption of the biofilm extracellular matrix upon overnight treatment with R-GNPs, facilitating the deep penetration of the nanoparticles into the biofilms (Figure 1c). In vivo treatment with R-GNPs resulted in a 99.9% reduction in bacterial burden compared to untreated controls (Figure 1d). Significant biofilm eradication was observed as early as 24 hours post-treatment, with further reduction in bacterial burden noted after subsequent treatments, corroborated via IVIS (Figure 1e-f).

Following fabrication of the antifungal liposomes, we investigated interaction of these nanoparticles with two critical pathogenic Candida spp., C. albicans and C. auris. We observed greater than 80% interaction of Pen-decorated liposomes with these fungi compared to ~50% with non-Pen decorated nanoparticles. CLSM further demonstrated greater Pen-decorated liposome interaction with the fungi compared to non-Pen decorated blank liposomes (Figure 1g). Upon loading with the fungistatic drug POS, planktonic Candida growth was inhibited by 2-8× lower liposome concentrations after Pen conjugation compared to non-Pen-decorated nanoparticles, suggesting increased drug delivery due to fungal targeting (Figure 1h). Further, C. albicans and C. auris biofilms were inhibited by ~90% and ~50% following Pen-decorated liposome treatment, respectively (Figure 1i), requiring up to 1,300× lower POS concentrations to inhibit Candida biofilm formation compared to free POS. Finally, prophylactic treatment with POS-loaded Pen-decorated liposomes in a murine intradermal C. albicans infection model led to a ~60% reduction in fungal burden compared to treatment with non-Pen decorated liposomes (Figure 1j).

Together, these nanoparticle formulations use responsive and targeted drug delivery mechanisms to offer enhanced efficacy against life-threatening microbial infections and have the potential to greatly improve future patient outcomes.

Acknowledgements and/or References (Optional):: AS acknowledges funding support for this work from the Office of Naval Research (N00014-22-1-2336) and the National Science Foundation (CBET-1942418).