A Murine Model of Cutaneous Aspergillosis for Evaluation of Biomaterials-based Local Delivery Therapies

Abstract

Cutaneous fungal infection is a challenging condition to treat that primarily afflicts immunocompromised patients. Local antifungal therapy may permit the delivery of high concentrations of antifungals directly to wounds while minimizing systemic toxicities. However, the field currently lacks suitable in vivo models. Therefore, a large cutaneous wound was created in immunosuppressed mice and inoculated with Aspergillus fumigatus. We fabricated biodegradable polymer microparticles (MPs) that were capable of locally delivering antifungal and characterized in vitro release kinetics. We compared wound bed size, fungal burden, and histological presence of fungi in mice treated with antifungal-loaded MPs. Mice with a cutaneous defect but no infection, mice with infected cutaneous defect but no treatment, and infected mice treated with blank MPs were used as controls. Infection of large wounds inhibited healing and resulted in tissue invasion in an inoculum-dependent manner. MPs were capable of releasing antifungal at concentrations above A. fumigatus MIC for at least six days. Wounds treated with microparticles had significantly decreased size compared to no treatment (64.2% vs 19.4% wound reduction, p=0.002) and were not significantly different than uninfected controls (64.2% vs 58.1%, p=0.497). This murine model may serve to better understand cutaneous fungal infection and evaluate local biomaterials-based therapies.

1. Introduction

While the field of biomaterials has made significant advances in the treatment of wounds featuring bacterial infection, the treatment of tissues with fungal infection has been relatively neglected. While less common than bacterial infection, fungal soft tissue infection poses additional challenges as it often occurs in patients with limited ability to mount an immune response as well as diminished innate healing capacity¹. Furthermore, there are few animal models available that feature an immunocompromised host with a reproducible tissue defect infected by fungi. Therefore, we have created a cutaneous defect infected with Aspergillus fumigatus in a neutropenic mouse model.

Invasive aspergillosis is a serious infection caused by Aspergillus species and predominantly affects immunocompromised patients. Beginning as a cutaneous lesion, the fungi can invade into the adjacent muscle and ultimately disseminate systemically, leading to significant morbidity and mortality^2-4. Due to the expanding population of immunocompromised patients, cutaneous aspergillosis has become a concern within healthcare⁴. In an analysis of patient outcomes comparing surgical treatment to systemic antibiotic treatment, current systemic antifungals were not successful in preventing increased mortality in cutaneous aspergillosis². While systemic antifungals are the gold standard for the treatment of invasive fungal infections, the tendency of Aspergillus to locally invade and destroy local vasculature may prevent effective penetration of systemically administered drugs to the nidus of infection⁵. Given its lethality and current lack of effective treatment options, there is a need for development of a platform to better understand cutaneous aspergillosis pathophysiology and validate new therapeutic strategies, including local drug delivery.

Therefore, new in vivo models are required that recapitulate the challenging conditions associated with disease. Previously, our group developed a murine model of primary cutaneous aspergillosis to study the effect of fungal infection on tissue angiogenesis^5,6. However, the cutaneous ulcer that developed from this subcutaneous inoculation was inconsistent in size and did not consistently evolve to a necrotic tissue defect as is typically observed in clinical cases. In addition, muscle involvement, a poor prognostic sign in the human disease, was not evaluated. By creating an infected wound of reproducible size, the effect of cutaneous aspergillosis infection on tissue regeneration can be investigated. To that effect, we have developed an improved version of our previous murine model.

In addition to establishing this model, we furthermore have developed diol-based biodegradable polyester microparticles to locally deliver large amounts of an antifungal (voriconazole) directly to the infected wound. Microparticle-based systems have been successfully translated and commercialized for clinical use in local antibiotic delivery^7-9. Currently, the most common biodegradable polymer used to fabricate microparticles, poly(lactide-co-glycolide), degrades into biocompatible but relatively inert byproducts (lactic and glycolic acid)⁹. Diols, aliphatic chains with two alcohol groups, have modest antimicrobial properties including activity against fungal species such as Aspergillus and Candida^10,11. Diols have also been found to significantly decrease Staphylococcus aureus burden in skin lesions in a clinical study¹². In addition to possessing inherent antimicrobial activity, diols have also been demonstrated to enhance the efficacy of traditional antimicrobial compounds when used in combination¹¹. We have previously polymerized diols to create large chains with tunable physicochemical properties¹³. By creating microparticles from diol-based polymers, the microparticles themselves may have therapeutic benefit as they degrade into free diols in addition to their payload (traditional antifungal drug) for the treatment of fungal infection. From these diol-based polymers, we have designed a microparticle-based delivery system that 1) locally delivers high concentrations of voriconazole, the preferred drug for treatment of invasive aspergillosis¹⁴ and 2) can biodegrade into diols, which themselves may have an effect on mitigating fungal infection.

In this work, we describe a unique model of cutaneous aspergillosis in an immunocompromised mouse that allows more consistent, sustained, and measurable assessment of healing in a fungal wound infection. Furthermore, we evaluated a new local delivery strategy in this model and found that biodegradable polymer-based microparticles may be leveraged to locally treat cutaneous invasive fungal disease. We hypothesized that increasing inoculum would result in greater inhibition of wound healing and that this inhibition may be at least partly alleviated with local delivery of voriconazole by diol-based microparticles in the wound bed.

2. Materials and Methods

2.1. Effect of Inoculation on Wound Regeneration and Outcome

All animal use was approved by the Animal Welfare Committee of the University of Texas MD Anderson Cancer Center. We immunosuppressed female BALB/c mice of 18-22 g per cyclophosphamide/cortisone acetate as previously described^5,6. Briefly, mice were given 200 μL intraperitoneal injections of cyclophosphamide (100 mg/kg) on Days −4, −1, and +6 and a single subcutaneous dose of 100 μL cortisone acetate (250 mg/kg) on Day −1. Throughout immune suppression, mice were prophylactically given water containing 5% sucrose (Sigma-Aldrich, St. Louis, MO) and 0.5 mg/mL doxycycline (Fisher Scientific, Hampton, NH) to prevent bacterial superinfection. Mice were caged in groups of five and randomly assigned to one of three groups (n = 5 per group) based on level of fungal inoculation: saline (control), 1.75 × 10⁶ conidia, and 1.75 × 10⁸ conidia (labeled as saline, low inoculum, and high inoculum, respectively). On Day 0, the right dorsal flank of mice was shaven by electric trimmer, prepped, and given subcutaneous injections of 200 μL saline or Aspergillus fumigatus Af293 conidia (Fig. 1). On Day +3, a 6 mm biopsy punch was used to create a uniform cutaneous defect over the site of inoculation. For analgesia, mice were prophylactically given subcutaneous injections of 100 μL meloxicam (0.3 mg/kg) (Sigma-Aldrich) and 50 μL 0.25% bupivacaine (Hospira, Lake Forest, IL). Cutaneous biopsy was performed under isoflurane anesthesia. The wound was covered with a sterile strip of Tegaderm™ (3M, St. Paul, MN) surgical tape. The wound was then re-inoculated by injection under the wound dressing with conidia in 100 μL at the same Aspergillus concentration as the original inoculation. For post-surgical analgesia, mice were given daily doses of meloxicam for three days following surgery. Mice were euthanized on Day +9 (nine days after initial infection, six days after surgical excision and re-inoculation). After euthanasia, the wound on each mouse was photographed for analysis of wound surface area by ImageJ (National Institutes of Health, Bethesda, MD). A 10 mm × 10 mm section of tissue around the wound bed was harvested by dissection and fixed in 10% neutral buffered formalin. Tissues were paraffin embedded, sectioned, and stained by hematoxylin and eosin (H&E) and Grocott’s methenamine silver stain (GMS).

Figure 1.

Schematic and timeline of an immunosuppressed murine model of cutaneous aspergillosis in an infected wound bed.

2.2. Microparticle Fabrication and In vitro Release Studies

Prior to fabricating microparticles, an in vitro minimum inhibitory concentration assay was performed on diols of three different lengths (1,6-hexanediol; 1,8-octanediol; and 1,10- decanediol, all purchased from Sigma-Aldrich) against A. fumigatus Af293 following the Clinical Laboratory & Standards Institute (CLSI) M38-A2 broth microdilution antifungal susceptibility testing method to determine the optimal diol for antifungal activity. Concentrations of diols from 1024 μg/mL to 0.0625 μg/mL were analyzed for fungal inhibition. The experiment was performed in triplicate for each diol.

The diol which had the greatest antifungal activity in vitro was selected for polymerization via a Fischer esterification between the terminal diol and fumaric acid via established methods¹³ with all reagents purchased from Sigma-Aldrich.

Diol-based polymer microparticles were prepared by the oil/water method⁹. Briefly, 10 mg of voriconazole was added to 100 mg of purified polymer. For unloaded (“vehicle”) microparticle groups, no voriconazole was added. The powders were then dissolved at room temperature under mild agitation in 0.4 mL dichloromethane (DCM) resulting in a clear solution (oil phase). 0.8 mL of chilled 1 wt% poly(vinyl alcohol) (PVA) was added. The mixture was vortexed for 15s at room temperature, resulting in an opaque white suspension. This suspension was then poured into 25 mL of chilled 1 wt% PVA (water phase) stirring at 400 RPM. After pouring in the 1.2 mL of polymer/voriconazole/DCM/PVA, stirring rate was reduced to 200 RPM. The suspension was stirred for 3 hours to allow evaporation of DCM.

After 3 hours, microparticles were collected by centrifugation (3500 RPM for 3 minutes) and washed and centrifuged 3x in 35 mL of chilled distilled water for removal of excess PVA. The microparticles were flash frozen using liquid nitrogen and lyophilized overnight. Three separate lots of loaded and unloaded microparticles were fabricated to demonstrate reproducibility. Microparticle diameter was characterized by analysis of bright field microscopy images using ImageJ.

20 mg of microparticles (n=3 per drug-loaded and unloaded vehicle group, each from a distinct fabrication lot) were placed in 2 mL PBS at pH = 7.4 at 37°C under mild agitation. At 6 hours, 12 hours, 24 hours, and every 24 hours thereafter, supernatant was collected and replaced with fresh PBS. The collected supernatant was filtered under sterile conditions and frozen until analysis by high performance liquid chromatography (HPLC). HPLC was performed using a 2695 separation module (Waters, Milford, MA), 2996 photodiode array detector (Waters), and a 250 mm x 4.6 mm XTerra RP 18 column (Waters). Recovered supernatant was eluted through the column at a flow rate of 1 mL/min in an isocratic mobile phase (60% acetonitrile/40% 0.1% v/v trifluoroacetic acid) over 5 minutes per sample. Absorbance was measured at λ = 254 using a standard curve with voriconazole concentrations ranging from 0.1-200 μg/mL. Data was analyzed with the software Empower (Waters, Milford, MA).

2.3. Effect of Local Therapy on Wound Regeneration and Outcome

Forty BALB/c mice of 18-22 g were subjected to the same protocol as above with the exception that the biopsy performed was reduced from 6 mm to 5 mm in diameter to minimize surgical complications. Cages were divided randomly into four groups with n=5 mice per group, performed in duplicate on different days, for a total of n=10 per group based on Table 1.

Table 1.

Animal groups to evaluate the efficacy of local diol-based polymer microparticle (MP) delivery for treatment of infected fungal defects (n = 10 per group).

Group	Description	Inoculum	Treatment
No Infection	Positive Control	Saline	None
No Treatment	Negative Control	1.75 × 106 conidia	None
Vehicle Treated	Material Control	1.75 × 106 conidia	Unloaded MPs
Drug Treated	Experimental Group	1.75 × 106 conidia	Voriconazole-loaded MPs

For mice receiving microparticle treatment, 5 mg¹⁵ of microparticles sterilized by ethylene oxide were added to the 100 μL of inoculum immediately preceding injection. To confirm presence of infection at the time of surgery, randomly selected specimens of harvested skin were placed dermis-side down on yeast extract agar glucose (YAG) plates for culture at 37°C.

Twelve hours following surgery, photographs of the wound bed were taken of each mouse (Day +3). These photographs were repeated immediately preceding euthanasia (Day +9). Wound surface area was traced and measured with ImageJ. Mice were euthanized on Day +9 and a 10 mm × 10 mm section of tissue around the wound bed was harvested by dissection under sterile conditions. Three wound beds from each group were randomly selected for histologic analysis. The remaining wound beds were placed in 1.5 mL of sterile saline and homogenized under sterile conditions. 200 μL of a 1:100 dilution of homogenized wound beds was spread on YAG plates and incubated at 37°C for 48 hours for colony-forming unit (CFU) counting and analysis. Similarly, murine kidneys were harvested and either randomly selected for histology (n=3 per group) or for homogenization in 1.5 mL saline (remaining mice per group). 200 μL (no dilution) of homogenized kidney was spread on YAG plates and incubated at 37°C for 48 hours for CFU counting and analysis to assay for potential hematogenous dissemination².

2.4. Statistics

Wound surface area and CFU counts of murine groups were compared by a one-way ANOVA with posthoc analysis via Tukey’s Honestly Significant Difference test (α = 0.05). Kaplan-Meier analysis was performed to determine differences in survival rates with the log- rank test. All statistics were performed using JMP Pro software (version 13.0; SAS Institute, Cary, NC) and conducted with a 95% confidence interval (α = 0.05).

3. Results

3.1. Effect of Inoculation on Outcome

One day after subcutaneous inoculation (Day +1), there were no visible signs of infection. However, by Day +2, mice from inoculated groups demonstrated visible swelling at the site of injection. This gross inflammation persisted at Day +3 at the time of defect creation. In some mice, purulent discharge was observed in the wound bed in the days following surgery. Mice which exhibited abnormal gait 24 hours after surgery (Day +4) were given extended buprenorphine (SR Veterinary Technologies, Windsor, CO) by the veterinary staff for additional analgesia.

While most mice recovered from inoculation and surgery, five mice were euthanized due to persistent right-sided hind leg gait abnormalities (Fig. 2A). Upon autopsy, the four high inoculum animals that demonstrated persistent lameness had necrotic bands of tissue in the wound bed as well as in the underlying muscle of the leg with noted purulence (Supplemental Fig. 1). One mouse in the saline group was euthanized due to complications associated with immunosuppression and surgery. The high inoculum group demonstrated statistically significantly less survival than the low inoculum group (p = 0.014) (Fig. 2B) due to euthanasia as required in the setting of hind leg lameness without improvement. No clinical signs of systemic infection (reduced motor activity, lethargy, shivering, piloerection, and weight loss¹⁶) were observed during the study.

Figure 2.

Effect of inoculum on the model. A) Kaplan-Meier survival curve. B) Statistical comparisons between groups based on survival. C) Representative cutaneous wounds from each group on Day +9. D) Average wound area at Day +9 (n = 4, 5, and 1 for Saline, Low Inoculum, and High Inoculum, respectively). Error bars represent standard deviation. * denotes significant difference compared to Saline animals (p <0.05). Note that as High Inoculum n=l, this group could not be statistically compared to Saline and Low Inoculum for wound size.

3.2. Microparticle Fabrication and In vitro Release Studies

Results from microparticle fabrication and in vitro release studies can be found in Supplementary Data.

3.3. Effect of Inoculation on Wound Healing and Infection

At the time of euthanasia (Day +9), all mice had a persistent wound at different stages of healing. The low inoculum group had significantly greater wound area compared to the saline group (high inoculum could not be compared due to surviving n=l) (Fig. 2C-D).

Fungi were present in all animals in inoculation groups and in no animals in saline groups. In addition, fungal hyphae were present in the tissues (Fig. 3) rather than only the injected conidia. A large number of inflammatory cells was observed in the high inoculum group. Histological sections were also taken from the mice that were euthanized prematurely due to lameness in the high inoculum group on Days +4 and +6, in which also demonstrated active hyphal disease with invasion into the muscle.

Figure 3.

Representative histological sections stained with GMS (top) and H&E (bottom) of Saline, Low Inoculum, and High Inoculum groups. Scale bar =100 μm. White arrows indicate examples of hyphae. No hyphae were observed in Saline sections. A high number of inflammatory cells were observed in H&E sections from High Inoculum groups compared to the other two groups.

3.4. Effect of Local Treatment on Outcome

All plated skin biopsies taken at Day +3 from inoculated groups demonstrated A. fumigatus growth on culture (no growth was observed in the non-infected controls) (Supplemental Fig. 4). During the course of the study, one mouse each from the No Treatment, No Infection, and Drug Treated groups died during surgery on Day +3 (speculated to be due to complications from the immunosuppression protocol and anesthesia) and one mouse had to be euthanized due to an injury sustained during a routine injection on Day +4 (Drug Treated). All other mice (N=36) survived until euthanasia at the terminal time point (Day +9). Mice tolerated the infected lesion with no gait abnormalities or signs of systemic illness. In addition, no aspergilli were detectable by culture of harvested homogenized kidneys for animals in any group.

3.5. Effect of Local Treatment on Wound Healing and Infection Burden

Photographs of the wound taken 12 hours after surgery (Day +3) and immediately before euthanasia (Day +9) were used to calculate the percentage decrease in wound surface area (Fig. 4A-B). The average wound size decreased by 58.1, 19.4, 43.2, and 64.2% for No Infection, No Treatment, Vehicle Treated, and Drug Treated groups, respectively. While all groups demonstrated reduction in wound surface area on average, 2/9 mice in No Treatment had an increase in wound surface area of+18.3% and +21.2% respectively. All other mice demonstrated reduction in wound size. The No Infection and Drug Treated groups both had significantly greater reduction in wound size compared to the No Treatment group.

Figure 4.

Effect of local therapy on the model. A) Average percentage decrease in wound surface area between Days +3 and +9 (n = 9, 9, 10, and 8 for No Infection, No Treatment, Vehicle Treated, and Drug Treated, respectively). Error bars represent standard deviation. Those that do not share the same letter are significantly different (p <0.05). B) Gross photographs of murine cutaneous wounds (scale bar = 5 mm). C) Fungal burden as reflected by CFUxl0³/mL (n = 6, 6, 7, and 5 for No Infection, No Treatment, Vehicle Treated, and Drug Treated, respectively). Error bars represent standard deviation. Those that do not share the same letter are significantly different (p<0.05). D) Level of deepest hyphal tissue penetration observed in histological sections (n=3 per group). – denotes 0/3.

A 10 mm × 10 mm section of tissue surrounding the cutaneous defect was harvested upon euthanasia and homogenized for CFU counting (Fig. 4C). Non-infected animals grew no colonies. The No Treatment group had significantly greater fungal burden than animals from the No Infection group, with no significant differences between the treated groups compared to either control.

Histopathology was conducted on three randomly-selected animals in each group, as both histopathology and CFU counting are destructive studies so cannot be performed simultaneously in the same specimen. In addition to confirming the presence of hyphae in infected animals, the deepest level of tissue (skin, fascia, and muscle) that had been invaded was recorded (Figs. 4D and 5). Non-infected animals had no hyphae. Hyphae were observed in 2/3 and 1/3 skin and underlying muscle in untreated animals, respectively. In animals treated with unloaded microparticles, hyphae were observed as deep as the skin, fascia, and muscle in one animal each. Hyphae were observed in only 2/3 of sections from animals treated with drug-loaded microparticles and no deeper than the dermis. No hyphae were observed in kidney sections.

Figure 5.

Representative histological sections stained with GMS (top) and H&E (bottom) of sections No Infection, No Treatment, Vehicle Treated, and Drug Treated groups from specimens with noted hyphae (except in the No Infection group). Scale bar =100 μm. White arrows indicate examples of hyphae. No hyphae were observed in Saline sections. A high number of inflammatory cells were observed in H&E sections from No Treatment group.

The effect size f was calculated based on the ANOVA analysis for both % decrease in wound size as well as CFU/mL per methodology described by Cohen¹⁷. Assuming intermediate deviation, effect size f for % decrease in wound size was calculated to be 0.60 (or 0.57 for minimum deviation and 0.81 for maximum deviation). Assuming intermediate deviation, effect size f for CFU/mL was calculated to be 0.62 (or 0.59 for minimum deviation and 0.84 for maximum deviation).

4. Discussion

Here we present a model of cutaneous aspergillosis that was created by subcutaneously injecting neutropenic mice with A. fumigatus conidia, allowing the infection to establish for 72 hours, and then surgically creating a standardized large cutaneous defect. As some of the fungal burden may have been alleviated by excision of infected tissue, we re-inoculated the wound at the same concentration at the time of surgery to ensure a robust and persistent infection.

This model (Fig. 1) allows for the development of a standardized and sustained (for at least nine days) local cutaneous Aspergillus infection. There was significantly less murine survival when fungal inoculum was increased between the low and high groups and significantly greater wound area when comparing the saline and low inoculum groups at the terminal time point (statistical comparison could not be made with the high inoculum group as only one animal survived to the end of the study). Mice with the highest level of inoculum had the highest mortality associated with lameness due to local invasion into the musculature underneath the dermis (Supplemental Figure 1), recapitulating features found in human infection^2,18. At lower inoculum, infection lasted through the nine days without evidence of dissemination or extensive tissue invasion (Fig. 2A). On histology, we found that the initial injected spores were capable of maturing into hyphae, the filamentous structures associated with aspergillosis and tissue destruction (Figs. 3 and 5). Aspergillosis in immunocompromised patients has a median time of 26.5 days from the appearance of a cutaneous lesion to systemic dissemination, and dissemination significantly increases patient mortality²; this model therefore could represent this early critical window for intervention.

In addition to creating a sustained local fungal infection, another advantage of this model is the ability to examine the effects of Aspergillus infection on wound healing. In a previous murine model of cutaneous aspergillosis, wounds spontaneously ulcerated from a subcutaneous infection resulting in non-standardized surface areas ranging over several orders of magnitude¹⁹. By creating defects of standardized size, the rate of wound healing can be tracked from similar baseline. As noted in human cases of cutaneous aspergillosis, wound infection significantly affected tissue regeneration in this murine model. As hypothesized, non-infected lesions healed at a faster rate than non-treated wounds infected with Aspergillus (Figs. 2D and 4A). The healing of 5 mm full-thickness cutaneous wounds in murine models have been shown to be delayed in the case of bacterial infection in a previous study using S. aureus in which the mechanism of delayed healing was attributed to collagen destruction by the bacteria¹⁵. While the current study did not seek to determine the mechanism of A. fumigatus interference with cutaneous regeneration, we speculate based on prior work that fungal inhibition of angiogenesis may play a major role in impairment of wound healing^5,20,21. As this model resulted in sterile wounds healing faster than infected wounds, it may be a useful platform for the validation of novel therapeutic strategies. Especially for the field of tissue engineering, there is a paucity of preclinical models of tissue defects featuring fungal infection despite relevance to an increasing susceptible clinical population^1,22.

Next, we studied the local release of antifungals from comprising of diol-based microparticles. Unlike previous biodegradable microparticle systems, the byproducts of these microparticles are terminal diols which themselves have demonstrated modest antifungal activity at high concentration¹⁰. As 1,10-decanediol was found to have the highest efficacy against Aspergillus (Supplemental Table 2), we synthesized decanediol-based polymers and fabricated microparticles capable of releasing voriconazole at physiologically-relevant concentrations for over six days (the duration of in vivo implantation) (Supplemental Fig. 3).

In this model, treatment of infected wounds with voriconazole-loaded microparticles resulted in significantly greater wound healing than non-treated animals (Fig. 4A). By fungal burden and histologic analysis, drug-loaded microparticles did not eradicate fungi from infected wound beds; even though the normal rate of wound healing was restored (not significantly different from uninfected animals), mice treated with drug-loaded microparticles had fungi recovered from homogenized wound beds (Fig. 4C). While the amount of fungi was reduced from 60,000 to 15,600 CFU compared to untreated mice, there were no statistically significant differences between the groups. However, in animals in which histology was performed, those that received local voriconazole treatment had no observed penetration by fungi into the fascia or muscle (whereas the other two infected groups had one animal each with muscle involvement and one with fascial involvement (Fig. 4D)). While this phenomenon was studied in small number, these results are consistent with other work showing that azoles can impair the depth of fungal invasion without affecting fungal viability in models of Candida albicans²³. Voriconazole is fungistatic against most Aspergillus isolates²⁴. It is possible that local treatment with voriconazole inhibited the fungi present from further invasion without causing fungal death, in part explaining why healing occurred at a normal rate while fungi were still present in the wound bed in mice treated with drug-loaded microparticles. Further work will need to be performed to definitively determine the effect of local voriconazole delivery on fungal penetration depth. While lack of statistical difference in CFU/mL could theoretically be attributed to group size (Type II error), the effect sizefbetween groups was calculated to be greater than 0.5 even when making the assumption of minimum deviation, implying a large effect¹⁷ of the dependent variable (different therapeutic arms) on the independent variables studied (decrease in wound size and fungal burden as assayed by CFU/mL). Establishing this effect size allows for a priori power analyses for future work using this immunocompromised murine model of disease.

In addition, it is noted that there were no significant differences in outcome when directly comparing unloaded and loaded microparticles, although unloaded microparticles did not show the same improvement in wound size that loaded demonstrated (Fig. 4A). In addition to diols themselves potentially having antifungal activity, it is possible that other components of the microparticle system may affect wound healing- specifically, fumaric acid esters have been demonstrated to downregulate inflammation in vivo²⁵. Dysregulation of inflammation during acute infection is a major issue in immunocompromised hosts¹ so it is theoretically possible that strategies to decrease inflammation may alone improve healing in an infected wound; while not within the scope of this study, future work could be designed to specifically assay local inflammation by measuring neutrophil activity and concentrations of local cytokines.

Our work had several shortcomings. As the focus was to build a platform to study tissue healing in a fungal infection and demonstrate different biomaterial-based strategies could be compared, the current gold standard therapy of intravenous antifungals was not studied in this model and compared head-to-head with local delivery. This comparison is the focus of future work. In addition, the generalizability of our finding would require further experimentation with different Aspergillus isolates and different non-Aspergillus molds, as well as experiments in mice with different genetic background. As proof of principle, future studies should use different azoles and other classes of antifungals, such as the echinocandins and the polyenes or combinations. While the focus of this work was on the development of an animal model to evaluate biomaterial-based strategies for treatment of wounds with fungal infection, future work will also be performed to further study diol-based microparticles, including characterization of degradation rate/productions/product MIC, effect of different diols on release rate and degradation properties, and release of other molecules beyond voriconazole.

5. Conclusions

In this work, a new murine model of cutaneous aspergillosis was validated. Similar to human disease, animal morbidity was dependent on fungal burden with increasing amounts of inoculum resulting in local tissue invasion into the muscle. Histologically-confirmed active fungal disease persisted for at least nine days following initial inoculation. Infection of the wound significantly inhibited tissue regeneration. Local voriconazole release from diol-based microparticles restored normal wound healing kinetics although fungal burden persisted in the wound bed. This model may function as a platform to further investigate regenerative medicine technologies that either treat fungal infection, stimulate wound healing, or both simultaneously.