Amphotericin B-conjugated polypeptide hydrogels as a novel innovative strategy for fungal infections

The present work is focused on the design and development of novel amphotericin B (AmB)-conjugated biocompatible and biodegradable polypeptide hydrogels to improve the antifungal activity. Using three kinds of promoting self-assembly groups (2-naphthalene acetic acid (Nap), naproxen (Npx) and dexamethasone (Dex)) and polypeptide sequence (Phe-Phe-Asp-Lys-Tyr, FFDKY), we successfully synthesized the Nap-FFDK(AmB)Y gels, Npx-FFDK(AmB)Y gels and Dex-FFDK(AmB)Y gels. The AmB-conjugated hydrogelators are highly soluble in different aqueous solutions. The cryo-transmission electron microscopy and scanning electron microscopy micrographs of hydrogels afford nanofibres with a width of 20–50 nm. Powder X-ray diffraction analyses demonstrate that the crystalline structures of the AmB and Dex are changed into amorphous structures after the formation of hydrogels. Circular dichroism spectra of the solution of blank carriers and the corresponding drug deliveries further help elucidate the molecular arrangement in gel phase, indicating the existence of turn features. The in vitro drug releases suggest that the AmB-conjugated hydrogels are suitable as drug-controlled release vehicles for hydrophobic drugs. The antifungal effect of AmB-conjugated hydrogels significantly exhibits the antifungal activity against Candida albicans. The results of the present study indicated that the AmB-conjugated hydrogels are suitable carriers for poorly water soluble drugs and for enhancement of therapeutic efficacy of antifungal drugs.

The present work is focused on the design and development of novel amphotericin B (AmB)-conjugated biocompatible and biodegradable polypeptide hydrogels to improve the antifungal activity. Using three kinds of promoting selfassembly groups (2-naphthalene acetic acid (Nap), naproxen (Npx) and dexamethasone (Dex)) and polypeptide sequence (Phe-Phe-Asp-Lys-Tyr, FFDKY), we successfully synthesized the Nap-FFDK(AmB)Y gels, Npx-FFDK(AmB)Y gels and Dex-FFDK(AmB)Y gels. The AmB-conjugated hydrogelators are highly soluble in different aqueous solutions. The cryotransmission electron microscopy and scanning electron microscopy micrographs of hydrogels afford nanofibres with a width of 20-50 nm. Powder X-ray diffraction analyses demonstrate that the crystalline structures of the AmB and Dex are changed into amorphous structures after the formation of hydrogels. Circular dichroism spectra of the solution of blank carriers and the corresponding drug deliveries further help elucidate the molecular arrangement in gel phase, indicating the existence of turn features. The in vitro drug releases suggest that the AmB-conjugated hydrogels are suitable as drug-controlled release vehicles for hydrophobic drugs. The antifungal effect of AmB-conjugated hydrogels significantly exhibits the antifungal activity against Candida albicans. The results of the present study indicated that the AmB-conjugated hydrogels are suitable carriers for poorly water soluble drugs and for enhancement of therapeutic efficacy of antifungal drugs.

Introduction
Invasive fungal infection has a significant influence on animal and plant life, which is a major cause of mortality that kills approximately 1.5 million people every year [1]. Amphotericin B (AmB) is a broad-spectrum antifungal macrolide polyene antibiotic [2]. Its antifungal effect is exerted by forming complexes with membrane sterols to allow the increase in the permeability of the fungal membrane [3]. AmB is the most common antifungal agent used in clinical practice despite strong side-effects with low solubility and low bioavailability (less than 0.9%) [4]. Many researchers attempt to develop novel AmB formulations combined with nanoparticles, nanotubes, polymers etc. Current research works have indicated that poly-aggregated AmB formulations could reduce the toxicity and enhance the efficacy [5][6][7].
Peptide hydrogels, a hydrogel type resulting from self-assembly of amino acids in water solution, have emerged as the new class of attractive soft nanomaterials owing to their unique physiochemical characteristics [8]. Because the hydrogelators associate with each other only through non-covalent interactions, hydrogels are inherently biocompatible and biodegradable. So, they are attractive candidates for drug self-delivery [9]. Compared with biodegradable polymers, self-delivery hydrogels based on hydrogelators could change some shortcomings such as adjusting the pore size or networks of polymers, limited loading of drug molecules and functionalizing the polymers with drug molecules [10,11]. Hence, new strategies are needed to conjugate AmB with hydrogels in order to increase its efficacy, reduce its toxicity and to minimize the chance of fungal resistance to AmB.
Considering the problems above, we design and synthesize several AmB-conjugates as molecular hydrogelators. The principles of the design are described as follows: (i) the peptide sequence of hydrogels contains Asp-Lys (DK) dipeptide fragments because it is crucial for antibacterial activity [12]-we hope that it can assist AmB to enhance antifungal activity; (ii) dipeptide of phenylalanine (FF) has been widely used to construct molecular hydrogelators; (iii) 2-naphthalene acetic acid (Nap) and naproxen (Npx) are frequently used to promote self-assembly-in the previous literature, Npx-FF fragments exhibit anti-inflammatory effect [13]; and (iv) dexamethasone (Dex) is an anti-inflammatory which is usually given alone or in combination with other antifungal drugs. Dex could reduce allergic reactions and the cell damage caused by AmB [14]. These are owing to the immunosuppressive effects and the varying permeability of cell membranes of Dex [15]. From the above, the Nap-FFDK(AmB)Y, Npx-FFDK(AmB)Y and Dex-FFDK(AmB) Y hydrogelators (figure 1) are synthesized (hereinafter to be referred as Nap-AmB, Npx-AmB and Dex-AmB hydrogels). Thus, chemical, physical and antifungal properties of AmB-conjugated hydrogels are examined.

Characterizations
Electrospray-ionization mass spectra were recorded using a Triple Quad 6410B instrument (America). Samples, which were presented as 1 mg ml −1 in dimethyl sulfoxide (DMSO), were diluted 100-fold (10 µl sample and 990 µl methanol). The proton spectra nuclear magnetic resonance ( 1 H-NMR) experiments were performed on DMSO-d6 solution of samples at room temperature. The proton spectra were recorded on a Bruker (300 MHz, Germany). Cryo-transmission electron microscopy (TEM) images were taken using a Hitachi transmission electron microscope (Japan). Experiments were carried out using a field emission cryo-electron microscope operating at 80 kV. Thereafter, 5 µl of sample solution at rsos.royalsocietypublishing.org R. Soc. open sci 1.0 wt% concentration was applied on the grid. Grids with vitrified sample solutions were maintained in a liquid nitrogen atmosphere and then cryo-transferred into the microscope. Scanning electron microscopy (SEM) was used to characterize the microstructure of lyophilized hydrogel powder. SEM analysis was performed using SEM-4800 (Japan). Samples were attached to sample stubs and then viewed using an accelerating voltage at the magnification. The images were taken under inert condition with the electron microscope. The crystal structure of the product powder was analysed by a D8 advance powder X-ray diffractometer (PXRD, Bruker, Germany) where Cu radiation was used as an X-ray source. For the analysis, samples were placed in the glass sample holders and scanned from 3°to 40°at 40 kV operating voltage. The diffraction spectra were recorded. Circular dichroism (CD) spectra were recorded using a J-810 Chirascan spectropolarimeter (JASCO, Japan). The sample (0.1-1 mg ml −1 in water) was placed in a coverslip cuvette (0.1 mm thick). Spectra are presented with absorbance A at any measured point with a 1 nm step, 1 nm bandwidth, and 1 s collection time per step at 25°C.

Synthesis
The Nap-FFDKY, Npx-FFDKY and Fmoc-FFDKY hydrogelators were prepared by the solid-phase peptide synthesis (SPPS) using 2-chlorotrityl chloride resin (1.0-1.2 mmol g −1 ) and N-Fmoc-protected amino acids [16]. The resin swelled in dry dichloromethane (DCM) for 20 min, and was washed with dry DCM three times (every time for 2 min). Then the first amino acid (2 equiv.) was loaded onto resin with DIPEA for 2 h in a DCM solution. After being washed with DCM three times (every time for 2 min), the blocking solution (80 : 15 : 5 of DCM/MeOH/DIPEA) was added twice (every time for 10 min) to deactivate the unreacted sites. After being washed with dry N,N-dimethylformamide (DMF) five times (every time for 2 min), the resins were treated with 20% piperidine (in DMF) for 30 min to remove the protecting group. The next Fmoc-protected amino acid (2 equiv.) was coupled using HBTU (2 equiv.) and HOBt (2 equiv.) as the coupling reagent. These two steps were repeated to elongate the peptide chain, which was carried out by the standard SPPS protocol. In the final step, the peptide was cleaved with TFA for 2 h. Ice-cold diethyl ether was added, and then was centrifuged for 10 min at 4000 rpm. The resulting precipitates were dissolved in DMSO and purified by reverse-phase high preparative performance liquid chromatography (HPPLC, Agilent1200, America After the AmB powder was dissolved in the DMF solution, 1.5 equiv. of Nap-FFDKY or Npx-FFDKY compound was added with 2.5 equiv. DIPEA. The resulting reaction mixture was stirred overnight protecting against exposure to light. At last, the product was purified by reverse-phase HPPLC. The pure yellow Nap-AmB hydrogelator compound was obtained with a yield of 58.7%. ESI-MS: The Dex (196.1 mg, 0.5 mmol) and succinic anhydride (117.1 g, 1.5 mmol) were dissolved in 10 ml of pyridine, then the DMAP (183.3 mg, 1.5 mmol) was added. After being stirred overnight at room temperature, the mixture was evaporated under reduced pressure. Ten millilitres of water was added. The mixture was stirred for 10 min and then centrifuged. The resulting precipitate was washed again with H 2 O three times. The white powder (Dex-SA) was obtained with a yield of 75.3%. After the AmB powder was dissolved in DMF solution, 1.5 equiv. of Fmoc-FFDKY compound was added with 2.5 equiv. DIPEA. The reaction mixture was stirred overnight protecting against exposure to light. The product was purified by reverse-phase HPPLC. The product was treated with 20% piperidine (in DMF) for 30 min to remove the protecting group. 1.5 equiv. of Dex-SA was added with 2.5 equiv. DIPEA. The mixture was stirred overnight avoiding light. At last, the result product was purified by reverse-phase HPPLC.

Hydrogel formation
General procedure for the formation of hydrogels: all the compounds dissolved in phosphate-buffered saline (PBS, pH 7.4). 2 equiv. of Na 2 CO 3 (0.1 mol l −1 ) were used to neutralize the carboxyl groups (−COOH) of compounds and adjust the pH to neutral. Then the hydrogels were formed at room temperature (22-25°C) or 37°C within 30 min.

Drug release
The 2.0 wt%, 3.0 wt% and 4.0 wt% of Nap-AmB, Npx-AmB and Dex-AmB hydrogels within the 0.3 ml PBS solution containing trypsin (1 µg ml −1 ) were prepared for drug release study. A quantity of 0.2 ml of the upper buffer solution was taken out and used to run HPLC at every 30 min and 0.2 ml of fresh PBS buffer solution containing trypsin (1 µg ml −1 ) was added back each time. The experiments were conducted at 37°C. The areas of peaks in HPLC spectra were used to determine the percentage of AmB or Dex released from their corresponding hydrogels. The liquid chromatography system (Agilent 1200 series, America) comprised Degasser G1322A, Quaternary pump G1312B, Thermostated auto sampler HiP-ALS G1367C, column oven G1316A and ultraviolet detector G1314A. Separation was achieved using a SepaxGP-C18 reversed-phase column (250 mm × 4.6 mm internal diameter, 5 µm particle size) (America). The mobile phase of AmB analysis was acetonitrile-water (43 : 57, containing 0.05% TFA) at a flow-rate of 1 ml min −1 . Chromatography was carried out at 30°C and the detector wavelengths were set at 383 nm. The mobile phase of Dex analysis was acetonitrile-water (45 : 55, containing 0.05% TFA) at a flow rate of 1 ml min −1 . Chromatography was carried out at 30°C and the detector wavelengths were set at 240 nm. The experiment was conducted in three parallel experiments. The release profiles of AmB and Dex from hydrogels is well described by the following exponential heuristic equation [17]: (

Transmission electron microscopy and scanning electron microscopy micrographs
As shown in TEM micrographs, Nap-AmB hydrogels (figure 3a) afford large, straight and rigid nanofibres with a width of 20-50 nm. Npx-AmB hydrogels (figure 3c) exhibit long, narrow, and flexible nanofibres with a width of 10-30 nm, which tend to form bundles. The Dex-AmB hydrogels (figure 3e) show smaller diameter nanofibres that entangle to form a network with higher density than Nap-AmB and Npx-AmB hydrogels. The morphologies of SEM micrographs of Nap-AmB, Npx-AmB and Dex-AmB hydrogels are similar to those of TEM micrographs. It is noted that Nap-AmB (figure 3b) and Npx-AmB (figure 3d) hydrogels randomly assemble in dendritic clusters, while Dex-AmB hydrogels are observed as aquatic plants or feather structures (figure 3f ). These morphological differences of the hydrogels indicate that the different head group (Nap-, Npx-and Dex-) at the N terminals of hydrogelators probably plays a role in their self-assembly. The self-assembly of Nap-AmB, Npx-AmB and Dex-AmB hydrogels would minimize AmBs molecular chance of self-assembly and enhance its efficacy towards target binding (ergosterol), thus diminishing its toxicity [18].

Powder X-ray diffraction analysis
The PXRD analysis confirmed the crystalline nature of the AmB, Dex and the lyophilized powder of hydrogels ( figure 4a,b). The PXRD of Nap-FFDKY, Npx-FFDKY and Fmoc-FFDKY lyophilized powder of hydrogels shows an amorphous halo ( figure 4c,d,e). The PXRD of AmB exhibits a sharp peak at 2θ scattered angles of 14.06°, 17.29°and 21.65°. The PXRD of Dex exhibits a sharp peak at 2θ scattered angles of 7.59°, 14.28°, 15.21°, 18.63°and 22.88°. However, the characteristic peaks for AmB or Dex are absent in the PXRD spectra of Nap-AmB, Npx-AmB and Dex-AmB formulation hydrogels ( figure 4f,g,h). Therefore, the results demonstrate that the crystalline structures of the AmB and Dex are changed after the formation of hydrogels. In previous reports, AmB had a natural ability to assemble spontaneously into higher order molecular forms that result in its toxic property [19]. The immobilization of AmB on a peptide substrate will minimize its chance of self-assembly. The amorphous hydrogels would enhance AmB efficacy towards binding of fungal ergosterol, diminish drug toxicity and minimize the chance of fungal resistance to AmB [20].

Circular dichroism spectra
The

Drug release
The in vitro drug releases of drugs from the Nap-AmB, Npx-AmB and Dex-AmB hydrogels (2.0, 3.0 and 4.0 wt%) were investigated in the PBS solutions (pH 7.4) at a physiological temperature of 37°C. The cumulative release curves of AmB and Dex from the drug-loaded hydrogels are shown in figure 6. The release behaviour exhibits a sustained release mechanism within 12 h at a physiological temperature without burst releases. After 12 h, the final drug release percentages of AmB are 36.3%, 47. hydrogels, respectively (figure 6c). For Dex-AmB hydrogels, AmB is released from 2.0 wt%, 3.0 wt% and 4.0 wt% hydrogels at a constant rate of about 0.1123, 0.1994 and 0.3517 mg ml −1 per hour throughout the entire measurement period, respectively. As for Dex, the final drug release percentages are 28.5%, 39.6% and 46.8% for 2.0, 3.0 and 4.0 wt% of Dex-AmB hydrogels, respectively (figure 6d). Dex is released from 2.0 wt%, 3.0 wt%, and 4.0 wt% Dex-AmB hydrogels at a constant rate of about 0.08359, 0.1743 and 0.2867 mg ml −1 per hour throughout the entire measurement period, respectively.
According to the empirical equation, the exponential factor (n) has been evaluated to determine the mechanism by fitting the experimental data. For AmB released from hydrogels, the values of n are 0.6874, 0.5722 and 0.5481, for 2.0 wt%, 3.0 wt% and 4.0 wt% of Nap-AmB hydrogels, respectively. The values of n are 0.7428, 0.7784 and 0.7771 for 2.0 wt%, 3.0 wt%, and 4.0 wt% of Npx-AmB hydrogels, respectively. The values of n are 0.8265, 0.6474 and 0.7971, for 2.0 wt%, 3.0 wt% and 4.0 wt% of Dex-AmB hydrogels, respectively. The fitting values of n are between 0.5 and 1, indicating that the release mechanism of AmB is the non-Fickian diffusion. The results indicate the anomalous nature of drug release, to which both diffusion and relaxation processes contribute [21]. It may be due to the breakage of coordinate amide bonds between AmB drug and the peptide chains. For Dex released from Dex-AmB hydrogels, the values of n are 0.4404, 0.3576 and 0.2587 for 2.0 wt%, 3.0 wt% and 4.0 wt% of Dex-AmB hydrogels, respectively. The fitting values of n are lower than 0.5, indicating that the drug release follows the Fickian diffusion. It occurs by the usual molecular diffusion of the drug owing to a chemical potential gradient [22].  For all hydrogels, the rate constant value k approximately increases with increasing the content of hydrogels [23]. These results suggest that the Nap-AmB, Npx-AmB and Dex-AmB hydrogels are suitable as drug-controlled release vehicles for hydrophilic drugs AmB and Dex.

Time-kill curves
Furthermore, we obtained detailed information about antifungal efficacy as a function of both time and concentration by time-killing tests (figure 8) to assess the pharmacodynamics of each antifungal agent. The concentration-dependent actions of AmB and AmB-conjugated hydrogels are confirmed by timekill curves. Moreover, the curves also reveal the time-dependent action between them. The time-kill curves show the AmB and Nap-AmB, Npx-AmB and Dex-AmB hydrogels exhibit antifungal activity for 2 h. The Npx-AmB and Dex-AmB hydrogels exert fungistatic effect more quickly than AmB and Nap-AmB hydrogels. Discernible improvement in the extent of antifungal activity and the slope function of the time-kill curve to each sample is noted as the amount of drug in solution increased. Marked concentration-dependent antifungal activity was also observed, what is more, the rate and extent of antifungal activity varied over time and the time to achieve an antifungal endpoint was shortened as the dose increased. In addition, the AmB-conjugated hydrogels showed a striking improvement in the extent of activity and trend towards a shorter time to the antifungal endpoint versus AmB agents [25].

Conclusion
The present work is focused on the design and development of AmB conjugated to biocompatible and biodegradable polypeptide hydrogels to improve antifungal activity. Amphotericin-conjugated hydrogels are characterized for physicochemical properties and evaluated for in vitro antifungal activity.
The results of the present study indicated that the AmB-conjugated hydrogels are suitable carriers for poorly water soluble drugs and for the enhancement of therapeutic efficacy of antifungal drugs.
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Authors' contributions. C.S., T.L., S.J., D.L. and L.D. carried out the synthesis, characterizations, drug release testing, antifungal effect testing, data analysis and drafted the manuscript. All authors gave final approval for publication.
Competing interests. We declare we have no competing interests.
Funding. This research was funded by National Natural Science Foundation of China (no. 81603072), (no. 81573387) and (no. 81703472).