Understanding the Uptake Mechanism and Interaction Potential of the Designed Peptide and Preparation of Composite Fiber Matrix for Fungal Keratitis.

The conventional use of antibiotics for the treatment of infectious keratitis currently faces two major challenges: poor drug penetration and the emergence of antibiotic resistance in microbial strains. Cell-penetrating peptides (CPPs) with antimicrobial properties have the potential to address these challenges. However, their mode of action, mechanism of uptake, and interaction potential have not been explored in detail. In this study, we probed the mechanism of uptake and interaction potential of our previously designed peptides (VRF005 and VRF007). Our results showed that VRF005 undergoes direct translocation and induces a rough membrane surface, whereas VRF007 undergoes clathrin-mediated endocytic uptake. The gel shift assay showed that VRF005 is bound to genomic DNA, whereas VRF007 is bound to chitin and β-d-glucan. Gene expression studies revealed the effect of peptide VRF005 on Candida albicans transcription. Molecular docking and simulations showed that VRF005 forms noncovalent interactions (such as H-bonding and water bridges) with natamycin. It exhibited synergistic antifungal activity in the colony-forming assay. VRF005, functionalized in the polycaprolactone fiber matrix, showed sustained delivery and antifungal activity.

Introduction

The delivery of therapeutic molecules to their intended site of action is crucial for effective treatment. Cell-penetrating peptides (CPPs) have gained popularity as being effective nonviral transmembrane delivery vehicles.[1] CPPs have been used for the delivery of plasmid DNA, short interfering RNA, and various drugs.[2] CPPs have been conjugated with cytotoxic agents (drugs, short interfering RNA, etc.) using a linker to form peptide–drug complexes (PDCs).The peptide and conjugation site need to be specific and carefully selected to avoid perturbations that make the PDC complex nonfunctional. Recent attention has been drawn to noncovalent drug delivery systems that do not have drawbacks and are easy to prepare.[3] These noncovalent bonds include hydrophobic, electrostatic, and hydrogen bondings and are currently favored as drug delivery systems.[4]

CPP or CPP conjugates enter the cell by direct penetration or through endocytosis.[5] Endocytic uptake can however lead to their lysosomal degradation. The translocation process sustains the delivery and improves the efficacy. Antimicrobial peptides are another class of CPPs that share similar physicochemical properties and membrane interaction.[6] However, they could also serve as therapeutic molecules in addition to their role as delivery vehicles.[7] Despite their great therapeutic potential, antimicrobial peptides find limited clinical application.[8] Around 2000 antimicrobial peptides have been reported in worldwide databases (http://aps.unmc.edu/AP/), and very few of them have been approved by FDA, such as bacitracin, daptomycin, etc.[9] One of the major issues that these antimicrobial peptides face is their instability in physiological conditions. This has been addressed using various delivery systems such as hydrogels, nanoparticles, liposomes, and fibers.[10]

Electrospun fibers find important applications in the field of drug delivery.[11] These fibers have large surface area, easy functionalization with therapeutics, and good adsorption/release properties.[12] Electrospun fibers are functionalized with therapeutic molecules such as peptides, antibiotics, anti-inflammatory drugs, or anticancer agents.[13] For functionalization of polymers such as polycaprolactone (PCL), PCL–keratin composite nanofibers have been used as synthetic scaffolds for biomedical applications.[14] The incorporation of therapeutic molecules in the nanofibers by simple co-electrospinning or physical binding provides sustained delivery and also improves wound healing with antimicrobial capacities.[15] However, there are very limited studies on therapeutic antifungal peptide–PCL composite nanofibers for drug delivery.

Fungal infections of the eye can be vision-threatening, and their treatment is challenging. Currently available antifungal drugs have serious limitations including inadequate drug penetration, low bioavailability, poor efficacy, high toxicity, and the recalcitrant nature of some fungi to treatment. The range of antifungal drugs suitable for ocular use is also limited. Recently, we have designed two cell-penetrating peptides VRF005 and VRF007 using a subtractive proteomic approach and reported them to have antifungal activity.[16] In this study, we deciphered the uptake mechanism of these previously designed peptides (VRF005 and VRF007) in both corneal epithelium and fungal species (Candida albicans and Fusarium solani). Further, we also identified the noncovalent interaction potential of our designed antifungal peptides (VRF005 and VRF007) with natamycin, genomic DNA, and fungal cell wall components such as chitin and β-d-glucan. VRF005 showing interaction with both DNA and natamycin was functionalized in polycaprolactone fibers to be used as a matrix for fungal keratitis.

Results and Discussion

Peptide Uptake Mechanism

Two major mechanisms through which CPPs have been reported to penetrate the cell membrane include energy-dependent endocytosis, which is temperature-dependent, and energy-independent direct translocation, which is temperature-independent.[17,18] The entry mechanism of the designed peptides VRF005 and VRF007 is not known. We used C. albicans, F. solani, and primary corneal epithelium to investigate the mode of penetration at 37 and 4 °C. The results (Figure A,B) showed that the uptake of VRF005 peptide was not affected by lower temperature in both C. albicans and F. solani at 4 °C. Hence, we conclude that VRF005 did not use endocytic uptake. Cells after VRF005 peptide incubation were treated with trypsin to further confirm if VRF005 is membrane-bound or not. The cellular peptide uptake results post trypsin treatment did not show any difference in both F. solani and C. albicans, suggesting complete internalization. Interestingly, VRF007 showed drastic reduction in uptake at 4 °C (Figure A). This indicated the involvement of endocytic vesicles in VRF007 uptake. Further, we evaluated the membrane binding affinity of VRF007 with trypsin treatment. Our data showed that after trypsin treatment there was a reduction in the uptake of VRF007 (Figure A). The results conclude that VRF005 uptake was temperature-independent and that VRF005 entered cells through direct translocation, whereas VRF007 uptake was temperature-dependent and mediated through endocytic machinery. We further investigated their localization since both the peptides showed uptake in C. albicans, as a model organism. The peptide charge determines its cellular localization. Peptide VRF005 has a net charge of +5, and VRF007 has a net charge of 0.[16] The histatin 5 peptide having a similar charge was reported to have antifungal activity and is in clinical trials. The cationic peptides such as histatin 5 have been reported to be localized in mitochondria.[19] Thus, we performed colocalization studies for VRF005 with mitotracker. The results (Figure B) showed that VRF005 was colocalized with mitotracker (brown), in keeping with the earlier report that the charge of the peptides determines their localization.[20]

Figure 1

VRF005 peptide uptake studies at 37, 4, and 37 °C with 0.25% trypsin treatment: (A) C. albicans and (B) F. solani (scale = 10 μm).

Figure 2

(A) VRF007 peptide uptake studies in C. albicans at 37, 4, and 37 °C with 0.25% trypsin treatment (scale = 10 μm). (B) Peptide localization along with mitotracker in C. albicans (scale = 10 μm).

As our results clearly suggested that VRF007 undergoes endocytic uptake, we next investigated the type of endocytosis involved as there are different types of endocytosis known to exist. We used primary corneal epithelial cells collected from patients undergoing refractive surgery as a model to study the mode of endocytic uptake of VRF007. As already reported, the peptide showed uptake in primary corneal epithelial cells, and we also performed colocalization with clathrin heavy chain and caveolin1. Our data (Figure ) showed that VRF007 was completely localized with clathrin heavy chain but not with caveolin1, suggesting that the endocytic uptake was clathrin-mediated.

Figure 3

(A) Colocalization of peptide VRF007 with clathrin heavy chain and caveolin1 (scale = 10 μm).

Surface Morphology of C. albicans after Treatment with Peptide

The results show that VRF005 undergoes direct translocation and VRF007 undergoes clathrin-mediated uptake. We further investigated the surface changes in C. albicans by scanning electron microscopy after the peptide treatment. The results (Figure A) showed that the control cells were round and dome-shaped and had a smooth surface, whereas VRF005-treated cells showed increased roughness on the cell surface, implying a direct translocation. On the other hand, VRF007 peptide treatment did not elicit changes in cell surface morphology or additional pore formation. We further validated the observation by performing the propidium iodide influx assay. Propidium iodide (PI) uptake is only seen when there is a membrane damage because it is a membrane-impermeable dye.[21] Our results showed VRF005 had more PI staining with the appearance of more yellow-stained cells (merged), whereas VRF007 had comparatively less yellow-stained cells (merged) (Figure B). Hence, this corroborated with our scanning electron microscopy results, and the data for peptide uptake at 4 °C confirm that VRF005 undergoes direct translocation via pore formation.

Figure 4

(A) SEM images of C. albicans: control and treated with VRF005 and VRF007 (scale = 5 μm). (B) VRF005 and VRF007 treatment followed by propidium iodide staining for analyzing the membrane integrity of C. albicans (scale = 20 μm).

Interaction Potential of the Designed Peptides

Both peptides use different modes of cellular uptake, i.e., VRF005 undergoes direct translocation and VRF007 undergoes clathrin-mediated endocytosis. We next examined the binding affinity of VRF005 and VRF007 with insoluble polysaccharide chitin and β-d-glucan. Chitin and β-d-glucan are major fungal cell wall components.[22] As reported earlier, on performing coprecipitation, we found that the VRF005 peptide did not coprecipitate with either chitin or β-d-glucan. Our data (Figure A,B) showed that there was no loss of intensity of the VRF005 band with or without chitin or β-d-glucan, whereas VRF007 showed drastic reduction in the intensity of the VRF007 band with chitin and β-d-glucan, suggesting the binding of VRF007 with both chitin and β-d-glucan. The binding studies agreed with the VRF007 uptake studies where trypsin treatment showed drastic reduction in the fluorescence intensity of VRF007 peptide uptake. Hence, we summarize that VRF007 binds to the fungal cell wall component, whereas VRF005 does not show any binding.

Figure 5

(A) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showing lack of affinity of VRF005 and binding affinity of VRF007 for insoluble chitin. (B) SDS-PAGE showing lack of affinity of VRF005 and binding affinity of VRF007 for β-d-glycan. (C) Gel retardation assay to show the interaction of FITC-labeled peptide VRF005 and VRF007 with fungal genomic DNA: lane 1, 300 ng of DNA alone; lane 2, 300 ng of DNA + 1 μM VRF005; lane 3, 300 ng of DNA + 10 μM VRF005; lane 4, 300 ng of DNA + 50 μM VRF005; lane 5, VRF005 alone; lane 7, 300 ng of DNA alone; lane 8, 300 ng of DNA + 1 μM VRF007; lane 9, 300 ng of DNA + 10 μM VRF007; lane 10, 300 ng of DNA + 50 μM VRF007; and lane 11, VRF007 alone.

After elucidating the mechanism of uptake of the designed peptides (VRF005 and VRF007) and their nature of interaction with fungal cell wall components, we further investigated their interaction potential with genomic DNA and natamycin. We performed the gel shift assay to assess the changes in the DNA mobility after the peptide complex had been prepared since VRF005 is cationic and VRF007 is charge-neutral. Peptide–DNA complexes were prepared by keeping the concentration of the genomic DNA constant, and four different concentrations of peptides (1, 10, and 50 μM VRF005 and VRF007) were used for incubation. More than 1 μM peptide showed binding to DNA as a significant shift was observed in the migration of DNA (Figure C). Hence, our results suggested that peptide VRF005 has the potential to interact with C. albicans genomic DNA, whereas VRF007 does not have. Our observation supported the earlier report of a short CPP with antifungal activity derived from bovine lactoferrin forming a stable and noncovalent complex with the plasmid DNA and efficiently entering human lung cancer A549 cells.[23] Thus, our results confirmed that VRF005 undergoes direct translocation into the cell via pores, localizes in the mitochondria, and has the ability to form a complex with genomic DNA. These properties can be efficiently exploited in the future for delivering nucleic acid in host cells.

Further, the transcriptional role of VRF005 in C. albicans was analyzed using gene expression studies. Agreeing to previously reported cationic peptide MAF-1A (charge is +2), which acted as a transcriptional regulator in C. albicans,(24) we also observed genes such as ERG11 (ergosterol biosynthesis), CTA1 (catalase1), and YHB1 (yeast flavo-hemoglobin1). FAS2 (fatty acid synthetase 2), ALS1 (agglutin-like protein), and KRE1 (killer toxin resistance) were significantly downregulated compared to control (Figure ), thus suggesting the impact of peptide VRF005 in regulating the transcription of C. albicans.

Figure 6

Comparison of gene expression between control and VRF005-treated C. albicans. Student’s “t” tests were performed to find the statistical significance.

Docking and Molecular Simulation of Peptide and Natamycin

Next, we determined the binding affinity of the peptides (VRF005 and VRF007) with natamycin, a frequently used antifungal drug, using docking and molecular simulation. They were docked using Glide suite, and the docked complexes (peptide–natamycin) were further analyzed for their interactions. The best docking poses were determined based on the glide score. For the VRF005–natamycin complex, the glide score was −2.4 kJ/mol, whereas for VRF007–natamycin, the score was −3.07 kJ/mol. Based on the glide score analysis, it was inferred that the VRF007 peptide had higher binding affinity to natamycin compared to VRF005.

Interaction analysis revealed that residues K3, E4, and W8 of VRF005 (Figure A,C) and residues K1, W13, and E14 of VRF007 (Figure B,D) formed hydrogen bonds with natamycin. Further, the stability of the docked complexes was analyzed through molecular dynamics using Desmond of Schrodinger suite. Comparative root-mean-square deviation (RMSD) from trajectory analysis (Figure A) revealed that the VRF005–natamycin complex has attained convergence after ∼100 000–200 000 ps. The observed maximum mean value for VRF005–natamycin was ∼8 Ǻ with a standard deviation of 1.5 Ǻ. The plot of root-mean-square fluctuation (RMSF, Figure B) indicated that the loop conferring residues of VRF005 complex showed minimum range of fluctuations within 4–6 Ǻ. The overall radius of gyration (Figure C) plot showed that VRF005 maintained its structural compactness without any variation. Moreover, the secondary structure analysis of the peptide in complex with natamycin (Figure S1a,b) showed that VRF005 has lost its helical structure after 50 ns and completely transformed as a loop. Interhydrogen bond analysis of the peptide–natamycin complexes (Figure D) revealed that the VRF005–natamycin complex has constantly maintained two hydrogen bonds between with natamycin throughout the simulation; however, after a few picoseconds, the interactions were increased to three hydrogen bonds. Only Trp8 and Trp12 residues of peptide VRF005 have majorly contributed to the stable hydrophobic and hydrogen bond interaction with natamycin throughout the simulation (Figure A); however, only Lys17 has shown strong hydrogen bond formation with natamycin at the final frame of the molecular dynamics production run (Figure A,C).

Figure 7

Molecular docking analysis of peptides (VRF005 and VRF007) with natamycin; Peptides (VRF005 and VRF007) are shown in surface representation colored based on their residue charge, while natamycin is represented as sticks (green color): (A) VRF005–natamycin and (B) VRF007–natamycin. Two-dimensional (2D) representation of the docked complexes, (C) VRF005–natamycin and (D) VRF007–natamycin, where the hydrogen bonds are represented as pink arrows.

Figure 8

Comparative molecular dynamics simulation analysis of peptide VRF005 (blue) and VRF007 (green) complexes with natamycin: (A) backbone RMSD convergence, (B) residue fluctuation, (C) radius of gyration plot representing the compactness of the complex, (D) number of hydrogen bonds in the peptide–natamycin complexes maintained throughout the simulation.

Figure 9

Residues of peptides interacting with natamycin throughout the simulation: (A) VRF005 and (B) VRF007.

Figure 10

Interaction analysis of the peptide–natamycin complexes post molecular dynamics simulation; peptides (VRF005 and VRF007) are shown in surface representation colored based on their residue charge, while natamycin is represented as sticks (green): (a) VRF005–natamycin and (b) VRF007–natamycin. 2D representation of the complexes (c) VRF005–natamycin and (d) VRF007–natamycin, where the hydrogen bonds are represented as pink arrows.

RMSD trajectory analysis (Figure A) of the VRF007–natamycin complex has shown higher deviations initially and has converged after 130 000–180 000 ps with a mean value of ∼4.5 Ǻ. The RMSF plot (Figure B) of VRF007–natamycin complex C-terminal residues fluctuates around 5–6 Ǻ. The overall radius of gyration (Figure C) result showed that VRF007 has a higher degree of compactness in complex with natamycin. VRF007 has maintained its loop structure (Figure S2a,b) and did not show any partial secondary structure deformation throughout the simulation. The VRF007–natamycin complex had 4 hydrogen bonds constantly throughout the simulation and attained maximum 5–6 hydrogen bonds at ∼50 000–200 000 ps (Figure D). In both the complexes, natamycin was well bound to the peptides throughout the simulation, and this correlated with the interhydrogen bond analysis (Figure D). The hydrophobic interaction by residue Leu8 and hydrogen-bond-forming residues Tyr12 and Glu14 has contributed to the stable interaction of VRF007–natamycin complex throughout MD simulation (Figure B). While, the final frame complex showed hydrogen bond interaction with Lys1 residue (Figure B,D).

The VRF007–natamycin complex has attained convergence, with the maximum number of hydrogen bonds favoring the stability of the VRF007–natamycin complex throughout the simulation. Hence, these data clearly indicated that the VRF007–natamycin complex was more stable than the VRF005–natamycin complex.

Biophysical Validation of the Peptide–Drug Complex

We performed Fourier transform infrared (FTIR) spectroscopy for the complex to confirm that the peptide interacts with natamycin. The data (Figure A) indicated characteristic peaks such as 1715, 1300–1000, and 1600–1700 cm–1 of natamycin, as reported earlier.[25] The signature peak of natamycin at 1715 cm–1 corresponds to the stretching band of the carbonyls from the lactone ring of natamycin. C–O vibrations correspond to 1300–1000 cm–1, whereas the vibration of N–H groups corresponds to 1600–1700 cm–1. The absorption peak of O–H was observed at 3500–3000 cm–1. Interestingly, the peptide–natamycin complex retained the characteristic peak of natamycin at 1715 cm–1. However, there was a drastic change in the peak at 1000–1500 cm–1 in peptide–drug complexes compared to control natamycin. CH2 bending and strong C–O–C stretching were observed at 1000–1500 cm–1. The vibrational changes suggested a possible interaction of the peptide and natamycin. Our data showed that the peptide–natamycin complex exhibited the characteristic spectra of natamycin, and due to noncovalent interactions, there were certain changes like stretching and bending of the groups, which were also observed. Further, X-ray diffraction studies were performed to validate the FTIR data. Diffraction data (Figure B) showed pure natamycin in a crystalline pattern, and the peak was observed between the 2-theta values of 25 and 20°, as previously reported.[26] Further, the physical mixing of peptides (VRF005 and VRF007) and natamycin followed by freeze-drying showed diffraction peaks between the 2θ values of 20 and 35°. The presence of natamycin was clearly detectable in the physical mixture of peptide and natamycin, clearly indicating that the compound may exist in an amorphous or molecularly dispersed state within the peptide–drug mixture, as reported by others.[27]

Figure 11

(A) FTIR spectra of natamycin and the peptide–natamycin complex. (B) X-ray diffraction of natamycin, peptides, and the peptide–natamycin complex.

Functional Validation of the Peptide–Drug Complex

The abovementioned data indicated that the peptides interact noncovalently with natamycin and the complex exists in an amorphous form. Hence, we validated their function through the colony-forming assay. Our earlier studies had shown that the peptides had antifungal activity.[16] We first determined the minimum inhibitory concentration (MIC) of natamycin using the colony-forming assay. Different concentrations of natamycin such as 8, 16, 32, 64, and 128 μg/mL were tested. The results demonstrated that there was no colony formation at 32 μg/mL, whereas 8 and 16 μg/mL concentrations showed colony formation. Hence, we took 8-fold lower concentrations of natamycin and peptide (VRF005 and VRF007) and incubated for 1 h for the formation of the complex. The complex was used to treat C. albicans along with peptide and natamycin alone. Our data (Figure ) clearly showed that 5 μM peptide with 4 μg/mL natamycin resulted in significant reduction in the number of colonies compared to the peptide or natamycin alone. Similar to VRF005, other peptides such as PGLA and magainin 2 from frog skin have been reported to have synergistic antimicrobial activity.[28] Due to increased use of antifungal agents, the C. albicans species has become resistant to conventional antifungal drugs, thus to sensitize various peptide and drug molecules are in use such as d-penicillamine (PCA) in combination with antifungals such as fluconazole.[29] Thus, we summarized that the designed peptides not only interact with the drug but also exert synergistic activity with the drug.

Figure 12

Colony forming assay of peptides and the peptide–natamycin complex.

Preparation and Characterization of PCL–Peptide Composite Fibers as a Matrix for Fungal Keratitis

VRF007 was found to be membrane-bound, as its uptake was significantly reduced at lower temperature and also upon trypsin treatment. VRF007 did not show any interaction with DNA. Peptide VRF005 showed direct translocation, pore forming ability, and interaction with DNA and natamycin. Thus, we decided to functionalize VRF005 in 5% polycaprolactone fibers for sustained delivery and stable activity. Two different concentrations of the VRF005 peptide (100 and 200 μM) were functionalized in 5% PCL. The obtained fibers were analyzed through SEM (Figure A). Our results showed that there was no significant difference in the fiber diameter between PCL and the PCL–peptide composite. Hence, functionalization of the peptides with the fiber did not alter the structure or size. The functionalization of the peptide on the PCL scaffold was confirmed by FTIR spectroscopy (Figure B), where it showed a peak between 2361 and 600 cm–1. Both the peaks correspond to the C=O stretching and N–H (amide I) bending of proteins. Hence, FTIR spectroscopy data confirmed that the functionalization of peptide (Figure B) in PCL had been done without affecting the diameter of PCL fibers.

Figure 13

(A) SEM image of polycaprolactone fibers alone and after forming the composite nanofiber with VRF005 (scale = 10 μm). (B) Fourier infrared spectroscopy for fiber characterization. (C) CD spectral analysis of the peptides released from 5% PCL fibers. (D) Time killing kinetics of PCL composite fibers in C. albicans for 48 h.

Peptide release kinetics were then evaluated from the fibers made up of different concentrations of VRF005 (5, 10, 56.5, and 565.6 μg) peptides as well as different concentrations of PCL as composite fibers. The release of the peptides was measured at different time points. However, cumulative release of the VRF005 peptide from PCL composite fibers up to 24 h is represented in Table . Our results showed concentration-dependent release. The secondary structure of the released peptides was analyzed using circular dichroism spectroscopy and compared to that of the native peptide as reported earlier.[16] We did not find structural difference between the released peptide and the native peptides (Figure C). However, VRF005 released from PCL fibers showed high amplitude of the negative band at nearly 205 nm compared to the native peptides retaining the α helical structure. This confirmed that functionalizing PCL with the peptides did not affect either PCL or the peptide structures.

Table 1

Release Kinetics Showing the Release of the Peptide from PCL When Different Concentrations of the Peptide were Loaded

peptide	% PCL	total amount of peptide loaded (μg)	peptide release up to 24 h (μg)
VRF005	2.5	56.5	25.4
	5	56.5	41.15
	5	565.6	134.5

Antifungal Activity of PCL–Peptide Nanofiber Composites

The peptide-functionalized PCL fibers were tested for the antifungal activity. We performed time killing kinetics of the peptide fibers against C. albicans. PCL–natamycin composite fibers were used as positive controls. PCL–VRF005 showed antifungal activity for 48 h. The PCL–VRF005 composites showed antifungal activity similar to PCL–natamycin composite fibers (Figure D). Thus, taken together, our data shows that PCL–VRF005 composite fibers have similar activity to that of PCL–natamycin fibers and have the potential to act as a matrix for fungal keratitis.

Conclusions

In this study, we established the mechanism of VRF005 and VRF007 uptake. Our results showed that VRF005 undergoes direct translocation, whereas VRF007 undergoes Clathrin-mediated endocytic uptake and binds to the membrane of the cells. Interaction studies showed that VRF005 specifically interacts with DNA, whereas VRF007 does not. Molecular docking, simulation, and biophysical studies showed that VRF005 and VRF007 both interact with natamycin. XRD data showed the peptide complex to be amorphous or in a molecularly dispersed state within the peptide–drug mixture. The functional validation studies were also performed using the colony-forming assay to show synergistic antifungal activity. Thus, VRF005 has interaction with both DNA and natamycin, so it was functionalized in PCL fibers. PCL–VRF005 composite fibers showed similar antifungal activity to that of PCL–natamycin composite fibers, suggesting that it could be used as a matrix to enhance the treatment of fungal keratitis.

Experimental Section

Ethical Approval

The primary corneal epithelium cells used in the study were collected from patients who underwent laser refractive surgery for correction of their refractive error, after obtaining informed signed consent from the patient (Ethics no. 489-2015-P). The study was approved and reviewed by the local ethics committee at Vision Research Foundation, Sankara Nethralaya, Chennai, India, and the committee deemed that it conformed to the principles of research in accordance with the Declaration of Helsinki.

Peptide Synthesis

Using solid-state synthesis, peptides labeled as VRF005 (KKKWFETWFTEWPKKKK) and VRF007 (KDRPIFQLNTSYWEMGA) were synthesized as reported earlier.[16] The HPLC purity was more than 95%. Peptides were labeled with fluorescein isothiocyanate (FITC) at the N terminal. Peptides were procured from M/S Genescript.

Peptide Uptake Studies and Its Localization

C. albicans (ATCC) and F. solani (ATCC) cultures were grown for 12 h in yeast nitrogen broth (YNB) for peptide uptake studies. The final cell concentration was adjusted to 1.5 × 105 colony-forming unit (CFU)/mL. A concentration of 10 μM of both peptides was used for a period of 1 h. Uptake was studied at two different temperatures 37 and 4 °C for evaluating the effect of temperature on the peptide uptake in C. albicans and F. solani. Cells were treated with 0.25% trypsin to elucidate whether the peptide is localized in the plasma membrane or in cytosol. The cells were visualized using fluorescence microscopy to monitor the localization of peptides (Axio Zeiss, Carl Zeiss, Oberkochen, Germany). The mitochondria of C. albicans (ATCC) were stained with 150 nM mitotracker (Thermo Fisher Cat. No. M7512) for 30 min at 37 °C. After the staining, cells were washed with phosphate-buffered saline (PBS) thrice. Colocalization studies were done with 10 μM peptides. Further, the cells were washed thrice with PBS and were visualized using fluorescence microscopy for colocalization.

Peptide Uptake Studies in Primary Corneal Epithelial Cells

Primary corneal epithelial cells were cultured in defined keratinocyte serum-free medium (SFM) (Thermo Fisher Cat. No. 10744019) for studying colocalization of the peptide. The tissues were adhered by a gravitational force from viscoelastic solution added on top of the tissues (HEALONOVD, Abbott Medical Optics) as reported earlier.[30] The tissues collected from control patients were cultivated in type 1 collagen-coated plates. Adherence of the tissues to culture plates was assured. VRF007 (10 μM) was incubated for 1 h followed by fixation and immunofluorescence with clathrin heavy chain (CST-D3C6) and caveolin1 (CST-D46G3) for colocalization studies.

Scanning Electron Microscopy

Scanning electron microscopy was used for identifying the structural changes induced by the peptides in C. albicans cells. They were incubated with 10 μM VRF005 and VRF007 peptides for 24 h. The suspension was centrifuged at 2000 rpm for 2 min; the pellet was washed twice with phosphate buffer. The cells were fixed with 2% glutaraldehyde. After the fixation, the cells were washed and then dehydrated in a graded series of ethanol. After the samples were dry, sputter-coating was performed with a 10 nm layer of gold. All samples were viewed and imaged on a scanning electron microscope (SEC Co., Ltd., Korea).

Polycaprolactone nanofibers (5%) alone or with peptides (VRF005) were electrospun for obtaining composite fibers. The samples were sputter-coated with a 10 nm layer of gold. All samples were viewed and imaged through scanning electron microscopy (SEC Co., Ltd., Korea)

Propidium Iodide Influx Assay

Permeabilization in C. albicans after treatment with peptides (VRF005 and VRF007) was detected using the propidium iodide influx assay. VRF005 and VRF007 (10 μM) were incubated with C. albicans (1 × 106 cells/mL YPD) for 24 h at 37 °C. After the incubation, cells were washed and resuspended in PBS. Subsequently, the cells were treated with 6 μM propidium iodide and incubated for 5 min at room temperature. The permeabilization of propidium iodide (red) into C. albicans cells was visualized using fluorescence microscopy (Axio Zeiss, Carl Zeiss, Oberkochen, Germany).

SDS-PAGE Pull-Down Assay

This coprecipitation assay was modified as reported earlier for peptide interaction with fungal cell wall components.[31] Different concentrations of VRF005 and VRF007 (2.5–10 μM) were incubated with fungal cell wall components such as chitin and β-d-glucan (Sigma) for 2 h at 37 °C. For determining the binding of the peptides with the complex, it was centrifuged at 10 000g. Further, the supernatant was analyzed using SDS-PAGE (15%). To quantify the binding of the peptides, we ran the peptides alone in the gel along with the supernatant and compared the fluorescence intensity.

Gel Shift Retardation Assay

C. albicans cells were harvested by centrifuging at 10 000 rpm for 30 s. Genomic DNA was isolated using the Trizol method as mentioned in the manufacturer protocol. The purity of the extracted genomic DNA was evaluated with spectrophotometry ratio of 260 and 280 nm. Different concentrations of the peptide were incubated with the genomic DNA (500 ng). The mixtures were incubated at room temperature for 10 min. A well was created in the middle of the agarose gel so that the shift in the migration can be observed using 0.5% agarose gel. The gel was run in 1× TAE buffer. The gel was visualized under UV illumination and FITC filter using a Gel Imaging System (Bio-Rad).

RNA Extraction and qPCR

C. albicans was treated with VRF005 (10 μM), and total RNA was extracted using the Trizol method following the manufacturer’s protocol (Sigma Aldrich). The total RNA concentration was determined using a Nanodrop ND-1000 spectrophotometer (Thermo Scientific). cDNA was synthesized using the iScript cDNA conversion kit (Bio-Rad).

Quantitative real-time PCR was performed using Applied Biosystems 7300 with SYBR Green chemistry (Applied Biosystem). The primers and the thermal cycler condition used were reported earlier.[24]

Molecular Docking

The plausible structures of VRF005 and VRF007 were processed toward stereochemistry optimization and energy minimization with OPLS2005. Protein preparation wizard of Schrödinger suite was used to obtain optimal geometry, which was favorable for the docking studies.

Receptor Grid Generation and Ligand Preparation

The entire peptide was considered for grid generation with van der Waals radius of 1.0 and a partial cutoff of 0.25 Ǻ. Prior to docking, the ligand molecule natamycin was optimized by fixing the van der Waals radii at 0.80 Ǻ and partial charge cutoff of 0.15 Ǻ and was further energy-minimized (OPLS2005 as force field) using Schrodinger Ligprep module.

Peptide–Drug Docking

The optimized peptides and ligand were subjected to molecular docking using Glide. The peptide was set rigid, and the ligand was flexible. In this docking study, 10 000 poses were generated, and among those, the best binding pose was selected based on the binding affinity determined by the Glide XP docking score.

Molecular Dynamics Simulation Study Using Desmond

The docked complexes VRF005–natamycin and VRF007–natamycin were subjected to molecular dynamics simulation studies toward analyzing the stability of the docked complexes. The molecular dynamics simulation of the docked complexes was commenced with the OPLS_2005 force field and by solvating the cubic system with the TIP3P water model. The entire system was further neutralized by adding respective counterions followed by energy minimization using the OPLS_2005 force field. To restrain geometry of water molecules and bond lengths and bond angles of heavy atoms, the SHAKE algorithm was used, while the periodic boundary conditions were set to stimulate a continuous system. Furthermore, the system was equilibrated by an NPT ensemble in terms of temperature and pressure parameters set to 300 K and 1.0 bar, respectively. Therefore, the Nose–Hoover chain and Martyna–Tobias–Klein were considered as a coupling algorithm to set temperature and pressure, respectively. Finally, the equilibrated system was subjected to a molecular dynamics (MD) production run of 300 ns (nanosecond), while the trajectories were recorded at every 4.8 ps (picosecond). Afterward, the resulting MD trajectories were analyzed for backbone deviations (i.e., RMSD), residue fluctuations (RMSF), and number of interhydrogen bonds of docked complexes throughout the production run of 300 ns for insights into the stability of the docked complexes.

X-ray Diffraction and Fourier Transform Infrared Spectroscopy of the Peptide–Drug Complex

X-ray diffraction (XRD) and Fourier transform infrared (FTIR) spectroscopy were used to evaluate the interaction between the peptide (VRF005 and VRF007) and natamycin. The peptide (VRF005 and VRF007) and natamycin were dissolved at a molar ratio of 1:1 and then stirred for 12 h. The resulting solution was freeze-dried to obtain the peptide (VRF005 and VRF007) and natamycin complex powder. X-ray scattering measurements were carried out by XRD (the radiation source used was Cu Kα, and scanning was performed at a rate of (2y/min) of 4/min in the range of 0–50°). For FTIR, the freeze-dried samples were mixed with potassium bromide (KBr) for FTIR analysis using infrared spectrometry (Bruker Tensor 27).

Antifungal Activity of the Peptide–Drug Complex

C. albicans (ATCC) (0.5 OD) was treated with different concentrations of natamycin such as 8, 16, 32, 64, and 128 μg/mL. The colony-forming assay was performed using 100 μL from each concentration, which was spread on Sabouraud’s dextrose (SD) agar plate followed by incubation for 48 h at 37 °C. After this time point, the colonies were calculated using the viable cell count, with an appropriate dilution factor. Similarly, for peptide–drug synergistic activity, we have taken two concentrations of peptides, that is, 1 and 5 μM, along with 4 μg/mL natamycin. The complex was spread on SD agar plates. The plates were incubated at 37 °C for 48 h, and the colonies were viewed.

Preparation of PCL–Peptide Composite Fibers

Polycaprolactone (5%) was prepared in hexafluoroisopropanol (HFIP, Sigma). Different concentrations of peptides were dissolved in 5% PCL in HFIP for preparing composite PCL–peptide fibers, and a control solution of PCL (5% wt/wt) was used. The fibers were produced using an instrument from the E-Spin Nanotech, India. A 5 mL syringe with a 27 G needle was used. The flow rate was kept at 10 μL/min. The applied voltage used was 12 kV. The fibers were collected on aluminum foil (7 cm × 8 cm) at a distance of 20 cm from the needle. After deposition, all of the samples were dried under vacuum.

Fourier Transform Infrared Spectroscopy of Composite Fibers

The prepared fiber samples were mixed using potassium bromide (KBr) to form a pellet and placed on a sample holder. The IR spectra were recorded in absorbance mode in the region between 400 and 4000 cm–1 using a deuterated triglycine sulfate detector in a Tensor 27 Bruker FTIR instrument. The analysis was done using OPUS software. Background corrections were performed.

Release Kinetics of Peptide from PCL–Peptide Composite Nanofibers and Structural Analysis

PCL–VRF005 composite nanofibers with different concentrations of PCL and peptide (VRF005) were prepared. Quantification of peptide release was done using a standard graph prepared as reported earlier.[16] The released peptides were quantified using a UV–visible spectrophotometer at 488 nm.

Circular Dichroism Spectra of Released Peptides

The secondary structure of the released peptides was analyzed using CD spectroscopy. The instrument used was a spectropolarimeter (J810; Jasco International Co., Ltd., Tokyo, Japan) using a 0.1 cm path length quartz cuvette at 37 °C. Spectra of VRF005 and VRF007 were recorded from 260 to 190 nm at a scan rate of 50 nm/min.

Killing Kinetics of PCL–Peptide Composite Fibers against Fungal Pathogen

The antifungal activity of PCL–VRF007 composite fibers, PCL–VRF005 composite fibers, and PCL–natamycin composite fibers were assessed against a fresh culture of C. albicans (1–5 × 107 cells/mL). The PCL–natamycin composite fiber was used as a positive control. The experiments were performed in duplicate and were repeated at least three times. The fibers were incubated with the fresh culture of C. albicans. Spectrometry readings were taken in a spectrophotometer (600 nm) at predetermined intervals: (30 min and 1, 2, 4, 24, and 48 h). The graph was plotted using GraphPad Prism 6.0 (GraphPad Software, Inc., La Jolla, CA).