Antifungal peptide decorated liposomes exhibit enhanced antifungal activity against Candida spp

Candida albicans is an opportunistic fungus that can cause both localized and systemic infections with high mortality rates ( >40%).¹ Unfortunately, there are less than 20 Food and Drug Administration-approved antifungal drugs,² and these drugs are often associated with high toxicity and poor water solubility, which can complicate treatment.³ Liposomes are lipid nanoparticles (NPs) that have been used to enhance the delivery of both toxic and hydrophobic drugs,⁴ making antifungal drugs great candidates for liposomal encapsulation. Liposomes have been shown to accumulate via the enhanced permeability and retention (EPR) effect in areas of inflammation due to leaky vasculature and decreased lymphatic drainage, which can occur in conditions including microbial infections and cancer.^5,6 However, this passive targeting strategy does not promote direct NP interaction with cells, limiting drug delivery to the target cells. The addition of targeting moieties to the NP surface may aid in active targeting, increasing NP-cell interaction, delivery of encapsulated agents to cells of interest, and in the case of C. albicans infections, improved eradication of the infection. Here, we developed liposomes encapsulating the antifungal drug, posaconazole (POS), and decorated with the antifungal peptide penetratin (Pen) as a peptide targeting moiety. We then investigated the antifungal activity of these liposomes against C. albicans.

Pen (RQIKIWFQNRRMKWKK)⁷ was synthesized using microwave solid-phase peptide synthesis (SPPS) with fluorenylmethoxycarbonyl (Fmoc) chemistry; a cysteine residue was added to the N-terminus for conjugation to the liposome surface (Cys-Pen). Liposomes containing POS, 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), cholesterol (CHOL), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] (DSPE-PEG-mal) were fabricated using a thin film hydration method followed by extrusion through a 100 nm polycarbonate membrane. Table 1 details the w/w (%) ratios for each liposome formulation. Cys-Pen was conjugated to the surface of liposomes using maleimide-thiol conjugation chemistry at pH 6.5-7 for 16 hours at 4°C (Figure 1a). The hydrodynamic diameters, polydispersity indices (PDIs), and 𝜁-potentials for all liposome formulations were assessed before and after peptide conjugation using dynamic light scattering. After conjugation, free Cys-Pen was removed via dialysis, and the peptide concentration was quantified using a CBQCA Protein Quantification Kit. The antifungal efficacy of the Pen-decorated POS-loaded liposomes was evaluated using broth microdilution assays against planktonic and biofilm C. albicans SC5314 to obtain liposome concentrations that inhibited growth or biofilm formation, respectively. The interaction between Pen-decorated liposomes and C. albicans was evaluated using flow cytometry with liposome formulations containing 0.1% (w/w) lissamine rhodamine B (LRB). Cell viability following exposure to 10% (v/v) of Pen-decorated POS-loaded liposomes was evaluated against human umbilical vein endothelial cells (HUVECs) using a Cell Counting Kit-8 (CCK-8) assay. For all studies, the Pen-decorated POS-loaded liposomes were compared to POS-loaded liposomes without Pen and POPC:CHOL liposomes without POS and Pen.

Successful synthesis of Cys-Pen was confirmed using liquid chromatography-mass spectrometry (data now shown). Liposomes were fabricated with hydrodynamic diameters from 120 – 145 nm with low polydispersity indices ( < 0.19) and 𝜁- potentials from -10 – -26 mV prior to peptide conjugation. Following conjugation of the cationic peptide, the 𝜁-potentials increased to ~35 mV, confirming successful peptide conjugation. Using the CBQCA Kit, the Pen concentration was calculated to be ~81 and ~84 𝜇g/mL for 1× POS and 5× POS liposomes, respectively (theoretical maximum = 95.3 𝜇g/mL), confirming a high conjugation efficiency. When treated with Pen-decorated POS-loaded liposomes, planktonic C. albicans growth was inhibited at lower liposome concentrations compared to non-Pen-decorated liposomes (1× POS: 25 𝜇g/mL and 6.25 𝜇g/mL for non-Pen and Pen-decorated formulations, respectively; 5× POS: 6.25 𝜇g/mL and 1.56 𝜇g/mL for non-Pen and Pen-decorated formulations, respectively) (Figure 1b). The Pen-decorated liposomes also completely inhibited formation of C. albicans biofilms at 1250 𝜇g/mL and 650 𝜇g/mL for 1× POS and 5× POS liposomes, respectively (Figure 1c). Using flow cytometry, Pen-decorated liposomes increased the percentage of C. albicans cells expressing LRB from ~46% and ~66% to ~85% and ~83% for 1× POS and 5× POS formulations, respectively, demonstrating that the addition of Pen improves liposome-fungal cell interaction (Figure 1d). Finally, no decrease in HUVEC viability was observed after liposome exposure (Figure 1e). Overall, we fabricated Pen-decorated antifungal liposomes that exhibited greater antifungal activity compared to non-Pen-decorated liposomes. This work shows the potential to develop active targeting antifungal liposomes utilizing antifungal peptides for the treatment of C. albicans infections.

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