A Nanoemulsion as an Effective Treatment Against Human Pathogenic Fungi

The emergence of immunocompromising diseases such as HIV/AIDS or other immunosuppressive medical conditions have opened an opportunity for fungal infections to afflict patients globally. An increase antifungal drug resistant fungi have posed a serious threat to patients. Combining these circumstances with a limited variety of antifungal drugs available to treat patients has left us in a situation where we need to develop new therapeutic approaches that are less prone to development of resistance by pathogenic fungi. In this study we present the utilization of the nanoemulsion NB-201 to control human pathogenic fungi. We found that the NB-201 exhibited in vitro activity against C. albicans, including both planktonic growth and biofilms. Furthermore, treatments with NB-201 significantly reduced the fungal burden at the infection site and presented enhanced healing process after subcutaneous infections by multidrug resistant C. albicans in a murine host system. NB-201 also exhibited in vitro growth inhibition activity against other fungal pathogens, including Cryptococcus spp, Aspergillus fumigatus, and Mucorales. Due to the nature of the activity of this nanoemulsion, there is a minimized chance of drug resistance to develop, thus presents a novel treatment to control fungal wound or skin infections.


Introduction 54
During the past decade there has been an exponential growth in discoveries and 55 medical advances for the treatment of human disease. This has led to better treatment for 56 patients, and as a result we have been able to prolong human life. While these recent medical 57 advances have certainly been beneficial overall, procedures such as solid organ transplants and 58 cancer treatments have left many patients in an immunocompromised state. The emergence of 59 immunocompromising diseases such as HIV/AIDS or other immunosuppressive medical 60 conditions have opened an opportunity for fungal infections to plague patients globally (1)(2)(3)(4). 61 Candida albicans is a human commensal fungus found on the skin, mucosal 62 membranes, and the normal gut flora (5, 6). C. albicans is known to be an opportunistic fungus 63 and the most common fungal pathogen which typically infects immunocompromised patients (3, 64 4). Treatment for candidiasis currently relies on three major classes of antifungal drugs including 65 echinocandins, azoles, and polyenes (7,8). Prior to the introduction of echinocandins, 66 fluconazole was the most common drug used to treat C. albicans infections (7). Recently there 67 has been an increase in cases of drug resistant C. albicans infections resulting in an increase in 68 morbidity and mortality of patients (4,(9)(10)(11). One explanation for drug resistance is the 69 development of mutations in the target genes of the antifungal drug (9). Secondly the 70 overexpression of efflux pumps and multi-drug resistance genes could also lead to antifungal 71 drug resistance (9). In addition, pathogenic fungi can also form biofilms that are resistant to 72 antifungal drugs (12,13). Thus, it is of upmost importance to develop new therapeutic 73 approaches that are less prone to the development of resistance by pathogenic fungi. 74 Membrane disruptive nanoemulsions have been developed to control pathogenic 75 bacteria (14, 15). One example is the nanoemulsion NB-201, which is an emulsification of 76 refined soybean oil, water, glycerol, EDTA, Tween 20, and the surfactant benzalkonium chloride 77 (BZK), which is commonly used as an antimicrobial preservative in drugs, topical antiseptic, 78 determined by using a 100% killing point of the C. albicans planktonic cells collected at 1, 24, 103 48, and 72-hour post addition of NB-210 to the media (Table 2). We observed within 1 hour, a 104 concentration of 1:512 of the NE was able to kill all the planktonic cells plated. As the incubation 105 time was increased, we observed a lower MIC was required. At 24 hours, a concentration of 106 1:1024 was able to kill all the strains plated. Within 48 hours the concentration of NB-201 107 required to kill all ten strains remained at an MIC of 1:1024. At 72 hours the MIC for 100% killing 108 of the strains plated was lowered to a concentration of 1:2048 (Table 2). 109 The ability of C. albicans to form biofilms, which increases antifungal drug resistance, is 110 a major virulence factor observed in the clinical setting (12,13). To test the efficacy of NB-201 111 on C. albicans biofilms, two multi-drug resistant clinical isolates TW1 and TW17 (21) were 112 chosen. The C. albicans clinical isolates TW1 and TW17 were plated on 96-well plates and 113 allowed to form a biofilm over the course of 24 hours (PFB). We then treated these PFBs with 114 the NE added in various ratios ranging from 1:1-1:2048 followed by a second-generation 115 tetrazolium (XTT) metabolic assay (Sigma-Aldrich) (22) to measure ratio of the metabolism, 116 indicative of disruptions of the biofilms, after NB-201 treatments. Within 2 hours a NE 117 concentration of 1:32 was able to inhibit 100% of the metabolism in the TW1 clinical isolate 118  Within 2 hours post addition of NB-201, a concentration of 1:16 was required for 100% 126 metabolism inhibition, while we find it important to note that a concentration of 1:32 inhibited 127 95% of the metabolism in the PFBs (Figure 1 G). Within 4 hours of exposure to the NE the 128 PFBs presented 100% metabolism inhibition at a 1:32 concentration with >50% inhibition being 129 observed at a concentration of 1:64 ( Figure 1H). After 6 hours of exposure, a 1:64 concentration 130 of NB-201 inhibited 80% of the metabolism in the PFBs ( Figure 1I). A 24-hour exposure to the 131 NE at a concentration of 1:128 presented a 70% inhibition of the metabolism in the TW17 PFBs 132 while a concentration of 1:64 inhibited 100% of the metabolism ( Figure 1J). At 48 hours of 133 exposure a concentration of 1:256 presented 85% inhibition of metabolism ( Figure 1K) while a 134 72-hour exposure increased that to 100% inhibition at the same concentration ( Figure 1L The formulation of the NB-201 was further tested to examine the ability to kill other pathogenic 181 fungi (Table 1) (Table 2). 186

Aspergillus fumigatus 187
We performed a checkerboard assay with ten different strains of Aspergillus fumigatus including 188 drug resistant strains, all of which are known clinical isolates (Table 1). Within one hour we 189 observed a concentration of 1:16 showed complete killing of all clinical isolates. We would like 190 to note that a concentration of 1:128 was able to kill seven out of the ten A. fumigatus clinical 191 isolates within the same timepoint ( Table 2). As incubation time with the NE progressed, we 192 observed a reduction in the MIC required to kill all of the A. fumigatus clinical isolates. Within 24 193 hours a concentration of 1:128 showed 100% killing of these clinical isolates (Table 2). Finally,194 at 48 and 72 hours all ten of the A. fumigatus clinical isolates were killed at a concentration of 195 1:512 (Table 2). 196

Mucorales 197
We tested ten clinical isolates of varying Mucorales species (Table 1) hour an MIC of 1:1024 was able to kill all four serotypes of C. neoformans (Table 2). This was 212 followed by 24, 48, and 72 hours showing an MIC of 1:2048 (Table 2) The stains used in this study are listed in Table 1. C. albicans and C. neoformans strains were 221 grown in liquid or solid yeast extract peptone dextrose [YPD, 10 g/L yeast extract, 20 g peptone, 222 20 g dextrose, 20 g agar (for plates only)] at 30C. Mucorales were grown in potato dextrose 223 agar (PDA, potato starch 4 g/L, dextrose 20 g/L, agar 15 g/L) or yeast extract peptone glucose 224 agar (YPG, 3 g/L yeast extract, 10 g/L peptone, 20 g/L glucose, 2% agar, pH = 4.5) at 30C in 225 the light for four days. A. fumigatus strains were grown PDA at 30 C for 4 days. To collect 226 spores of Mucorales and A. fumigatus, sterile water (2 ml per plate) was added to the plate and 227 spores were collected by gently scrapping the fungal mycelial mats.

In vitro efficacy of NB-201 against Mucorales Spp., C. neoformans, and A. fumigatus 259
The respective fungal strains were inoculated at a concentration of 1x10 6 in a 96-well plate 260 containing NB-201 serially diluted in RPMI (100 l per well). Dilution concentrations ranged from 261 1:1 to 1:2048. 10µl samples taken at 1, 24, 48, and 72 hours from each well and plated on PDA 262 agar plates, which were incubated for 48 hours. After incubation, every plate was examined for 263 growth on the site of inoculation. 264 265

In vivo efficacy of NB-201 in a murine subcutaneous infection model 266
CD-4 mice weighing between 19-23 g were housed together. C. albicans strains 267 SC5314, TW1, and TW17 were grown in YPD liquid media, washed in PBS, and suspended in 268 PBS at a concentration of 1x10 6 . Under anesthesia, the dorsal fur of the mice was shaved. The 269 exposed skin was washed with 70% ethanol and mice were infected with 1x10 6 CFUs via 270 subcutaneous injection on the shaved dorsal side. Subsequent subcutaneous injections of NB-271 201, PBS, or fluconazole followed at 6, 24, and 48 hours. The mice were euthanized at 72 272 hours, and the skin of the infected area was collected immediately for analysis. 273 The collected mouse tissue was placed in PBS on ice then homogenized with a tissue 274 homogenizer. The homogenized tissue was then diluted 1:10 and plated on YPD agar plates 275 treated with antibiotics to prevent unwanted bacterial growth. The plates were incubated at 32°C 276 for 48 hours. The CFUs were then counted and quantified.  Table 1). The in vitro susceptibility test with NB-201 301 was observed on every tested fungus, with an exceptional killing efficacy observed in all four 302 serotypes of C. neoformans. 303 During our in vitro test we observed a similar trend in the efficacy of NB-201. 304 Interestingly, we observed that longer incubation times with NB-201 resulted in a lowered MIC 305 regardless of the fungal organism that was being tested. The top etymological agent of 306 candidiasis, C. albicans, still ranks amongst the leading fungal organisms to cause infection in 307 immunocompromised patients around the world and causes >50% of bloodstream infections in 308 the US (3). The biofilms produced by this fungal organism makes it intrinsically harder to treat 309 and is a growing problem and concern in the clinical setting (12,13,26,27). We found that NB-310 201 has in vitro antifungal activity against planktonic form and biofilms of C. albicans. 311 Furthermore, the in vitro activity was also observed against drug-resistant clinical isolates. In an 312 animal subcutaneous infection model, NB-201 also exhibited antifungal activity against two 313 azole-resistant strains, TW1 and TW17 (Figures 2, 3, and 4). These results demonstrate that 314 NB-201 has anti-C. albicans activity both in in vitro and in vivo regardless of drug resistance. 315 And C. albicans is less likely to develop resistant to NB-201 ( Figure 2).