Tetrahedral DNA nanostructure improves transport efficiency and anti‐fungal effect of histatin 5 against Candida albicans

Abstract Objectives Anti‐microbial peptides (AMPs) have been comprehensively investigated as a novel alternative to traditional antibiotics against microorganisms. Meanwhile, Tetrahedral DNA nanostructures (TDNs) have gained attention in the field of biomedicine for their premium biological effects and transportation efficiency as delivery vehicles. Hence, in this study, TDN/Histatin 5 (His‐5) was synthesized and the transport efficiency and anti‐fungal effect were measured to evaluate the promotion of His‐5 modified by TDNs. Materials and Methods Tetrahedral DNA nanostructures/His‐5 complex was prepared via electrostatic attraction and characterized by transmission electron microscopy (TEM), polyacrylamide gel electrophoresis (PAGE), dynamic light scattering (DLS) and electrophoretic light scattering (ELS). The anti‐fungal effect of the TDN/His‐5 complex was evaluated by determining the growth curve and colony‐forming units of C. albicans. The morphological transformation of C. albicans was observed by light microscope and scanning electron microscope (SEM). Immunofluorescence was performed, and potassium efflux was detected to mechanistically demonstrate the efficacy of TDN/His‐5. Results The results showed that Histatin 5 modified by TDNs had preferable stability in serum and was effectively transported into C. albicans, leading to the increased formation of intracellular reactive oxygen species, higher potassium efflux and enhanced anti‐fungal effect against C. albicans. Conclusions Our study showed that TDN/His‐5 was synthesized successfully. And by the modification of TDNs, His‐5 showed increased transport efficiency and improved anti‐fungal effect.

diseases remains limited because of the gradual development of drug resistance in fungi, which urges aggressive and immediate action for developing novel methods. 4,7,8 Positively charged short anti-microbial peptides (AMPs) found in plants and animals are critical components of the innate immune system and defend against invading microorganisms. 9 Due to its broad-spectrum anti-biofilm activity and ability to modulate the host immune response, AMPs showed clear advantages over conventional anti-microbials. 10 Moreover, AMPs showed distinctive antimicrobial activities like formation of ion channels, transmembrane pores and extensive membrane rupture for extracellular AMPs, and inducing loss of ATP, misfolded proteins and aseptate filaments for intracellular AMPs. Therefore, AMPs presented the great value for conventional drug resistance. 11,12 As for extracellular AMPs, the initial stage of their function depends on the passive combination between their positively charged domain and the negatively charged microbial membrane. 13,14 Therefore, the modification of extracellular AMPs is mainly focused on enhancing its electrostatic property and strengthening the electrostatic and hydrophobic attraction between AMPs and cell membrane including amending the peptide sequence, altering the nature and position of the organometallic group and assembling the characteristic domain. [15][16][17] However, it is more important for intracellular AMPs to be internalized by cells for their intracellular target mechanisms. Moreover, due to their various cellular uptake pathways, like lipid-raft-dependent macropinocytosis for TAT-fusion proteins, permease-mediated translocation for apidaecin and receptor-mediated mechanism for histatin, the delivery carrier that can be internalized by diverse cells actively and loaded with different drugs is required for the modification and improvement for intracellular AMPs. [18][19][20] What is more, distinct from the bacterial cell wall, the fungal cell wall is composed of mannoproteins, chitins and α-,β-glucans, which weakens the initial binding of fungal cell membrane with AMPs, leading to decreased AMPs delivery efficiency and anti-fungal effect. 17,21 Therefore, for the anti-fungal AMPs like His-5, a novel way to optimize them is needed to overcome the obstacles associated with the special cell well structure of fungi.
Recently, tetrahedral DNA nanostructures (TDNs) have attracted increasing interest because of their editability and biocompatibility. [22][23][24][25] Despite the polyanionic nature of DNA, TDNs can be internalized via endocytosis and transported into lysosomes in a microtubuledependent manner. 26,27 Accordingly, TDN-based drug delivery has been proposed and researched extensively at the cellular and bacterial levels over the past decade. 24,28,29 From previous studies, TDNs have yielded satisfactory results as delivery vehicles attached with low-molecularweight drugs like nucleic acids, antibiotics and peptides. 28,30,31 This implied that TDNs can be used to load intracellular anti-fungal AMPs like His-5 to improve their transport efficiency and anti-fungal ability.
In this study, Histatin 5 was selected as the representative of intracellular anti-fungal AMPs and combined with TDNs to construct TDN/His-5 complex, whose characteristics and anti-fungal effects were tested. To our knowledge, this is the first study to design a combination of TDNs and intracellular AMP as anti-fungal agents.
Owing to electrostatic attraction, the cationic AMP His-5 can bind easily to anionic TDNs, leading to more efficient uptake by C. albicans. 32,33 This approach provides a novel alternative for further modification of peptide drugs and exhibits the versatile potential of TDNs in anti-fungal drug delivery.

| Cell culture
Candida albicans SC5314 (wild type) obtained from our laboratory were commercially from the American Type Culture Collection. 34 Candida albicans strains were propagated on liquid or solid YPD medium at 30°C. The yeast cells used in all experiments were in the exponential growth phase (OD 600 = 0.6). For inducing hyphae, C. albicans cells were grown in RPMI 1640 medium containing 2.5% foetal calf serum, 2 mm L-glutamine, 20 mm HEPES and 16 mm sodium hydrogen carbonate (pH 7.0) (RP medium) at 37°C.

| Tetrahedral DNA nanostructures preparation and verification
Tetrahedral DNA nanostructuress were synthesized by four singlestranded DNAs (deoxyribonucleic acids) ( The total mixture was placed into a PCR instrument, and heated to 95°C for 10 minutes then cooled rapidly to 4°C for 20 minutes. 35,36 The primary TDNs were purified by ultrafiltration (Amicon Ultra 10K device) with phosphate-buffered saline (PBS). Transmission electron microscopy (TEM) was applied to analyse the morphology of the TDNs and 8% PAGE was applied to characterize single-stranded DNAs and TDNs.

| Serum stability test of TDN/His-5 complex
To verify stability of TDN/His-5 complex in the serum medium, matrixassisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) was utilized to assess the degradation profile of His-5. His-5 and TDN/His-5 incubated in the 10% foetal calf serum medium for 6h were measured by MALDI-TOF MS. And the proportions of peak areas (mass range from 2900 to 3000) to the total area in spectrum were calculated as the proportions of His-5 in the medium.
The mass percentage of His-5 was calculated with following equation: Initial His-5 and TDN/His-5 in the 10% foetal calf serum medium were measured as control groups. And the proportions were compared between the TDN/His-5 groups and His-5 groups.

| Loading efficiency analysis
The loading efficiency of TDN/His-5 complex was determined using ultrafiltration and spectrophotometry. TDNs and His-5 were incubated for 30 minutes at room temperature in different ratios (1:50, 1:100, 1:200). Then, the TDN/His-5 solutions were ultrafiltered (Amicon Ultra 10K device, USA) at 9 600 g for 10 minutes, and the concentrations of unloaded His-5 in the remaining solutions were measured and calculated by spectrophotometry at the wavelength of 214 nm. The loading efficiency of His-5 onto TDNs was calculated with the following equation: C 1 and C 2 mean the initial and unloaded concentration of Histatin 5, respectively.

| Growth assay
To evaluate the yeast killing ability of the TDN/His-5 complex, exponential-phase C. albicans was used to perform fungicidal activity assays. C. albicans cells were washed with PBS thrice and diluted to 10 6 CFU/mL. Then, the strains were seeded at an initial density of The OD 600 value was detected once an hour, and the plate was shaken every 15 minutes to avoid precipitation. 39,40 Finally, the antifungal rate was calculated with the following equation: in 96-well plates as an density of 5 x 10 5 CFU/mL for 2 hours at 30°C in YPD liquid medium. 41 Then, the cells were rinsed with PBS thrice and diluted with YPD liquid medium. Fungal medium without any interference was used as a positive control. Five hundred cells from each well were plated on YPD agar medium and incubated for 24 hours at 30°C. Finally, the number of colony-forming units (CFUs) was counted andanalysed. 42

| Morphology assay
To determine the morphology of C. albicans under the TDN/His-5 complex conditions, the yeast cells were diluted to a concentration Mass Percentage of Histatin 5 ( % ) = Peak area of Histatin 5 Total area of sample

| Statistical analysis
All the results presented are based on at least three individual experiments. Statistical analyses were executed in GraphPad Prism (GraphPad Inc). Student's t test was used, and the significance level was set to α = 0.05.

| RE SULTS AND D ISCUSS I ON
In this study, we selected His-5 as the representative of intracellular anti-fungal AMPs and combined it with TDNs to construct the TDN/ His-5 complex. By the interaction between TDNs and the fungal cell membrane, cellular uptake and anti-fungal efficacy of His-5 were improved.

| Synthesis and characterization of TDNs and the TDN/Histatin 5 complex
Conventionally, TDNs comprise four different single-stranded DNA (ssDNA) and self-assemble according to the specific sequence of ssDNA based on complementary pairing rules ( Figure 1A). 25 Eight percent PAGE was performed to evaluate the synthesis and characterization of TDNs. Figure 1B showed the successful synthesis of TDNs ranging around 200 bp in size. The morphology of TDNs was observed using transmission electron microscopy, and we found that TDNs comprise some monomers and polymers ( Figure 1D). To verify the optimal mixture ratio, TDNs were combined with a gradient concentration of His-5, and the mixtures were characterized by 8% PAGE. As demonstrated in Figure

| Serum stability and loading efficiency test of TDN/His-5 complex
An obvious difficulty in the application of AMP is its vulnerability to degradation by serum proteases. 29,50,51 Previous studies showed that TDNs protected the 'cargo' they deliver. 49,52,53 For further proving the efficacy of the TDN/His-5 complex, the stability of His-5 protected by TDNs in a medium containing serum was measured.
Firstly, the matrix (the 10% foetal calf serum medium) was detected by MALDI-TOF MS and the results showed that there was no specific peak within the m/z range of 2900-3000, indicating no interference with His-5 ( Figure S1). Moreover, the peak of His-5 of TDN/ His-5 complex after incubation was obviously higher than that of intact His-5 after incubation, within the m/z range of 2900-3000. And the decline of mass percentage of His-5 after degradation in TDNs/ His-5 group (from 36.21% to 20.53%) was much smaller than that in Histatin 5 group (from 35.19% to 9.0.14%), that indicating more stable ability for His-5 with the assistant of TDNs. (Figure 2A,B).
Moreover, at a peptide/TDN ratio of 200:1, the complex showed a significant decrease in payload rate compared with that at 100:1 and 50:1 ratios, indicating that the binding capacity of His-5 and TDN decreased at the ratio of 200:1 ( Figure 2C). Consequently, for achieving both stable combination and optimal anti-fungal effect, 100:1 was selected as the optimal ratio of the TDN/His-5 complex for subsequent experiments.

| Anti-fungal activity assays of the TDN/Histatin 5 complex against Candida albicans
For detecting the anti-fungal activity of drugs against yeast-form C. albicans, the growth curves with TDNs, His-5 and TDN/His-5 complex were measured over 24 hours. As shown in Figure 3A In the morphology assay, bright field images of the control and TDN groups revealed large amounts of hyphae after culturing in the RP medium ( Figure 4A). By contrast, more yeast cells and fewer hyphae were presented in the samples treated with His-5; the yeast bodies shrank, and the hyphae branches frequently formed bundles.

| Cellular uptake of the TDN/Histatin 5 complex
Immunofluorescence and flow cytometry were employed to evaluate the capability of C. albicans to take up TDNs, His-5 and TDN/His-5 complex. Figure 5A showed The uptake efficiency of His-5 was measured quantitatively between the His-5 group and TDN/His-5 group using flow cytometry.
In this study, the surface of His-5 was modified by TDNs, thus allowing the anti-fungal intracellular AMP His-5 to be internalized by

| ROS formation and potassium efflux of Candida albicans
To investigate the intracellular role of TDN/His-5 complex, the early formation of ROS in both yeast and hyphal forms of C. albicans was monitored using the fluorescent probe DCFH-DA. 57,58 As can be seen in Figure 6A,B, intracellular ROS production in the TDN/His-5 group improved markedly compared with His-5 group in both yeast and hyphal C. albicans cells. And the TDN group was not significantly different from the control group. 47,57,59 The same results can be detected by flow cytometer, that the fluorescence intensity of DCF in the TDN/His-5 group in yeast cells was much higher than other groups ( Figure S2).
Potassium efflux caused by His-5, which resulting in volume dysregulation and cell death, was also measured in this experiment ( Figure 6C). 20

| Limitation
There are some limitations to our study. Only one pathogenic fungus was assessed, and detailed in vivo experiments were not conducted in this study, which we plan to perform in the future. In addition, the specific mechanism by which TDNs assist AMP in penetrating the fungal cell wall needs to be explored. We believe that the application of TDNs as the delivery vehicle of anti-fungal AMP can be broadened by overcoming these limitations.

ACK N OWLED G EM ENTS
This study was supported by National Key R&D Program of China

CO N FLI C T O F I NTE R E S T S
There are no conflicts to declare. Data are represented as means ± standard deviations, n = 3; statistical difference: *P < .05, **P < .01, ***P < .001

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.