One-pot synthesis of chitosan–dehydropregnenolone acetate ketimine nanoparticles and their antifungal bioevaluation

Abstract

This paper presents a new method for fabricating biodegradable bio-polymeric nanoparticles via a convenient one-pot strategy at room temperature under stirring conditions for application to communicable diseases. The simultaneous synthesis and assembly of chitosan–16-dehydropregnenolone acetate (CHDPA) nanoparticles were characterized using Fourier transform infrared (FT-IR) spectroscopy, and ¹H NMR and UV vis analyses and the morphology along-with the particle size were identified using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). In this study, the antifungal properties of the CHDPA nanoparticles were tested by measuring their antifungal activity against the fungus Colletotrichum gloeosporioides. The drug loading capacity (LC), encapsulation efficiency (EE) and drug release were investigated using UV spectrophotometry.

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1. Introduction

Nanostructured materials have recently gained much attention in many biology related applications and advanced nanodevices in the field of bio-nanotechnology. The assembly of nanostructures across several length-scales is of paramount importance in the synthesis of organized materials with advanced functions. Chitosan, a natural-based polymer obtained by alkaline deacetylation of chitin, is nontoxic, biocompatible, and biodegradable. Owing to its properties, chitosan can be used in medicine, pharmacy, biotechnology, agriculture, the food industry as well as for biodegradable and biocompatible materials, and antimicrobial compounds.^1–4 The polycationic biopolymer receives a great deal of attention for biosensing, medical, and pharmaceutical applications^5–7 and it is the most commonly used natural polymer in regenerative medicine and tissue engineering.⁸ Chitosan micro- or nanofibers have been widely accepted as biomedical scaffolding materials to restore, maintain, or improve the functions of various tissues.^9,10 Therefore, the development of chitosan nanostructures with controllable morphologies is highly desirable, but has had limited success as yet. However, one elegant method, electrospinning, has been reported for producing chitosan nanofibers. Because of their biocompatibility and biodegradability, the resulting chitosan nanostructures can potentially be tailored to mimic a natural extracellular matrix, achieve controlled drug delivery, and develop tissue-compatible scaffolds for tissue cultures. Thus, chitosan derivatives possess many useful biological properties such as biocompatibility, biodegradation, wound healing and anti-bacterial action.^1,11–13 Therefore, much attention has been paid to developing chitosan-based biomedical materials. Chemical modifications of chitosan are useful for the association of bioactive molecules to the polymer and for controlling the drug release profile.

In this study, the nanostructural mechanical properties and biocompatibility of novel chitosan–16-DPA particles were evaluated. They have been widely used in pharmaceutical research and industry as a carrier for drug delivery and as a biomedical material.¹⁴ Chitosan was selected for the nanoparticles because of its recognized mucoadhesivity and ability to enhance the penetration of large molecules across mucosal surfaces.¹⁵

Our efforts have been concentrated on synthesizing hybrid nanoparticles using a convenient one-pot method and a literature survey showed that no work has been reported on this topic, particularly using 16-dehydropregnenolone acetate. Significantly, our results show that a polymer of single composition and short length could contribute to the growth of highly anisotropic structures. Such an anisotropic aggregation is most likely due to the nonuniform distribution of the capping agents on the amorphous steroid nanoparticle surfaces. The aim of the present investigation is the synthesis and characterization of novel biodegradable nanoparticles based on chitosan for the encapsulation of 16-DPA, and the product has been screened for antifungal activity against the fungus Colletotrichum gloeosporioides. The drug loading capacity (LC), encapsulation efficiency (EE) and drug release were investigated using UV spectrophotometry.

2. Materials and methods

2.1. Materials and chemicals

Chitosan (CH) (MW 2.4 × 10⁶), 16-dehydropregnenolone acetate (16-DPA), N,N-dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP) were purchased from Sigma-Aldrich and used directly without purification. Acetonitrile, ethyl acetate, methanol, NaCl, Na₂HPO₄ and KH₂PO₄ were purchased from CDH with analytical grade and only distilled solvents were used for the reactions. Triple distilled H₂O was used for the preparation of the solutions.

2.2. Methodology

2.2.1. Synthesis of chitosan–16-DPA nanoparticles. A solution of 16-DPA (100 mg) in MeOH (60 ml) was stirred for 10 min at room temperature and 25 ml of 1% chitosan solution was added slowly. A solution of DCC (100 mg) in MeOH (5 ml) was added and after that DMAP (300 mg) in MeOH (5 ml) was added to catalyze the reaction.¹⁶ The reaction mixture was stirred for 24 h at room temperature. In order to enhance precipitation of the by-products the resulting suspension was treated with acetonitrile (30 ml). The resulting solution was filtered and the filtrate was evaporated under reduced pressure to get the product. After that, the product was redissolved in ethyl acetate (50 ml) and the undissolved by-products were discarded by filtration. Ethyl acetate was evaporated under reduced pressure to get the final required product. The product was purified by washing with MeOH, and finally the product was formed as a white crystalline solid (Scheme 1).

Scheme 1 Schematic representation of reaction mechanism for chitosan–16-DPA.

2.2.2. General methods. Melting points were measured with a Buchi B-540 melting point apparatus and are uncorrected. The progress of each reaction was monitored using Merck thin layer chromatography silica gel 60 F254 plates. IR spectra were recorded with a Perkin-Elmer model 2000 series FT-IR spectrometer using solutions in chloroform. Infrared absorbance is reported in reciprocal centimeters (cm⁻¹). ¹H and ¹³C NMR spectra were recorded using a Bruker DPX (300 MHz) spectrometer using CDCl₃ or DMSO-d₆ solvent with tetramethylsilane (TMS) as the internal standard on a ppm scale (δ). Multiplicities of the resonance peaks are indicated as singlet (s), broad singlet (bs), doublet (d), triplet (t), quartet (q) and multiplet (m). The surface morphologies were examined using scanning electron microscopy (SEM; JSM-35, JEOL, Japan). The particles were made conductive by sputter-coating with palladium prior to SEM analysis. The particle sizes of the polymeric aggregates were observed using TEM (JEM-2000 FX II, JEOL, Japan). A drop of the product suspension was placed on a copper grid coated with carbon film, dried at 25 °C and the measurement was performed at 80 kV.

2.2.3. In vitro antifungal activity.
2.2.3.1. Fungus and media. The antifungal activity of the new modified chitosan (CHDPA) was studied against the fungus Colletotrichum gloeosporioides.

The inhibitory effects of the samples were tested in vitro on the mycelia growth of Alternaria alternata. The poisoned food technique^17,18 was used to test the antifungal activities of the samples. Samples at concentrations of 100, 200, 300, 400 and 500 ppm were used. Petri plates (90 mm dia.), each containing 20 ml of potato dextrose agar (PDA) medium amended to the desired concentrations of the samples, were inoculated with test fungus. A 5 mm diameter disc of the test fungus, cut with a cork borer from the periphery of an actively growing 8 days old culture on a PDA plate, was placed at the center of each treated PDA Petri plate, containing 100, 200, 300, 400 and 500 ppm of the solvent, while a plate without any sample served as a control. The experiments were conducted with three replications. Then, the plates were kept in an incubator at a temperature of 25 ± 1 °C. Fungal growth was observed at every 24 h interval. At the end of the incubation period after 72 h, the minimum inhibitory concentration (MIC) that caused complete inhibition of the mycelia growth was measured. The percentage inhibition of the mycelial growth was calculated from mean values of the colony diameters in the treated and control Petri dishes using the following formula:¹⁸

Inhibition% = 100(control − treatment)/control.

2.2.4. Phosphate buffer saline pH 7.4. Phosphate buffered saline is a buffer solution commonly used in biological research.¹⁹ A 1 liter stock of 10× PBS was prepared by dissolving 8 g NaCl, 1.44 g Na₂HPO₄ and 0.24 g KH₂PO₄ in 800 ml of distilled water, and topping up to 1 liter. The pH is ∼6.8, but when diluted to 1× PBS it should change to 7.4.

2.2.5. Standard graph for drug content. A stock solution was prepared¹⁹ by dissolving DPA (10 mg) in suitable solvent or buffer (1 ml solvent + 9 ml buffer) and 1 ml of the mixture from the stock solution was diluted with 9 ml of phosphate buffer saline pH 7.4 which is used as a standard solution. Aliquots of the standard solution were further diluted with phosphate buffer saline pH 7.4 to get working solutions of 5, 10, 15, 20 and 25 μg ml⁻¹. The working standards were scanned using UV spectrophotometry which shows the maximum absorbance at λ max. The same λ max was used for the further measurements of the drug. Finally, the prepared standards were measured, in each case against a solvent blank similarly prepared, and calibration graphs of the absorbance versus the concentration of the drug were plotted.

2.2.6. Determination of DPA loading capacity (LC) & encapsulation efficiency (EE). Proper amounts of the product were mechanically ground in a mortar with a pestle. About 5 mg of the product was placed into a 10 ml standard flask with a cap and 10 ml of a suitable solvent was added. After that the mixture in the flask was dispersed using ultrasonication for 30 min, the flask was capped and shaken for 24 h at room temperature. Finally, the mixture was transferred into a 25 ml flask and diluted to 25 ml with solvent. The suspension was filtered through a 0.45 μm membrane. The concentration of the drug in the filtered solution was measured at the wavelength of λ max.¹⁹

The recovery of the product (final product amount) was defined as the weight ratio of the dried product to the initial loadings of polymer and drug. The dried product was dissolved in acetonitrile and sonicated for 5 min, and then distilled water was added to precipitate the polymer preferentially. The drug content in the supernatant after centrifugation (15 000 for 15 min) was measured spectrophotometrically at the particular λ max for each drug using Ultraviolet spectroscopy (UV). The product recovery, drug loading capacity and encapsulation in the product were calculated using the following equations.¹⁹

2.2.7. DPA release study. For DPA release, the weighed polymeric nanoparticles were suspended in 2 ml phosphate buffer saline pH 7.4 and placed in Sigma dialysis tubing. The tube containing the dispersion of the polymeric nanoparticles was then introduced into a 200 ml beaker containing 100 ml release medium (phosphate buffer pH 7.4), and the mixture was stirred at 400 ± 20 rpm using a magnetic stirrer. Drug release was assessed by intermittently sampling the receptor media (5 ml) at predetermined time intervals, and each time the taken sample was replaced with 5 ml of fresh phosphate buffer saline pH 7.4. The amount of drug release in the buffer solution was quantified using UV spectrophotometry at λ max.¹⁹

3. Results and discussion

3.1. Chemistry

3.1.1. UV studies. The UV spectrum of chitosan shows a sharp prominent peak at 190 nm and the spectrum of 16-DPA shows a prominent band at 246 nm. In the case of the CHDPA product, the spectrum shows a peak at 259 nm. After the conjugation reaction, the signal shifted indicating the formation of the new ketimine bond, as shown in Fig. 1.

Fig. 1 UV-vis spectra of (A) chitosan and (B) (a) 16-DPA and (b) chitosan–16-DPA.

3.1.2. Chitosan (CH). The FT-IR spectrum of chitosan (CH) shows a characteristic band from 2920 to 3378 cm⁻¹ corresponding to the N–H stretching of the hydrogen bonded amino groups; the peak at 1726 cm⁻¹ is assigned to the C–O stretching (C=O in carboxylic acid), 1612 cm⁻¹ appears due to the amide I group (C–O stretching along with N–H deformation mode), the peak at 1560 cm⁻¹ is attributed to the NH₂ group due to N–H deformation, 1460 cm⁻¹ is assigned to the symmetrical deformation of CH₃ and CH₂ group, the peak at 1425 cm⁻¹ is due to C–N axial deformation (amine group band), the peak at 1384 cm⁻¹ is due to the COO– groups in the carboxylic acid salt, 1196 cm⁻¹ is assigned to the special broad peak of the β(1–4) glycoside band in the polysaccharide units, 1108 cm⁻¹ is attributed to the stretching vibration mode of the hydroxyl group, 1020 cm⁻¹ is assigned to the stretching vibration of C–O–C in the glucose ring and the bands at 1060–1015 cm⁻¹ correspond to CH–OH in the cyclic compounds.^20,21 The band between 590 and 770 cm⁻¹ appears due to O–C=O in the carboxylic acids because of the preparation of the CH solution using acetate buffer solution. The absorption band at 1650 cm⁻¹ was attributed to the carbonyl groups O=C–NHR of chitosan²² and the absorption band at 1599 cm⁻¹ was assigned to the amino groups of chitosan with a high deacetylation degree. This signal shifted to 1528 cm⁻¹ after the conjugation reaction, indicating the formation of imine bonds by condensation of the ketone of 16-DPA with the amino groups of chitosan. A weak shoulder peak occurred at 1738 cm⁻¹ which was assigned to the carbonyl group of the ester bond of CH, as shown in Fig. 2.

Fig. 2 FT-IR spectra of (A) chitosan and (B) (a) 16-DPA and (b) chitosan–16-DPA.

The ¹H NMR spectrum of chitosan shows chemical shifts of the protons appearing at 4.58 ppm for the acetal proton (–CH) of the glucosamine, 3.01 ppm for –CH–NH₂, 3.75 ppm for –CH–OH, 3.59 ppm for –CH₂–OH and 1.94 ppm for the acetamido protons (–NH–CO–CH₃).

The ¹³C NMR spectrum of chitosan shows the following chemical shifts: δ (ppm) = 98.3 (C-1), 56.9 (C-2), 70.9 (C-3), 78.0 (C-4), 75.7 (C-5) and 61.4 (C-6).

3.1.3. 16-Dehydropregnenolone acetate (16-DPA). ¹H NMR (CDCl₃): δ (ppm) = 1.0 (s, 3H, Me-19), 1.1 (s, 3H, Me-18), 1.3–2.2 (m, –CH and –CH₂), 2.0 (s, acetate proton), 2.3 (s, methyl ketone proton), 4.6 (m, 1H, C-3 proton in acetate), 5.3 (m, 1H, C-6 olefinic proton), 6.7 (m, 1H, 16-vinyl hydrogen); ¹³C NMR (CDCl₃): δ (ppm) = 31.5 (C-1), 32.2 (C-2), 76.6 (C-3), 46.0 (C-4), 144.4 (C-5), 121.9 (C-6), 30.1 (C-7), 31.5 (C-8), 50.3.0 (C-9), 38.1 (C-10), 20.6 (C-11), 27.7 (C-12), 36.7 (C-13), 36.8 (C-14), 31.5 (C-15), 140.2 (C-16), 144.4 (C-17), 196.8 (C-18), 19.2 (C-19), 20.6 (C-20), 21.4 (C-21), 15.7 (C–CH₃COO), 170.5 (C–C=O); IR (CHCl₃): 2933, 2851, 1732, 1666, 1435, 1245 cm⁻¹.²³

3.1.4. Chitosan–16-dehydropregnenolone acetate ketimine product. The IR spectrum of the chitosan–dehydropregnenolone acetate ketimine product shows peaks at 3394 (br) cm⁻¹ for –OH stretching, 2929 and 2852 cm⁻¹ for C–H stretching, 1702 cm⁻¹ for C–O stretching of the acetyl group (amide II), and at 1649 cm⁻¹ corresponding to C=N stretching (ketimine); the N–H deformation (NH₂) peak is shifted to 1564 cm⁻¹, 1406 cm⁻¹ is assigned to the asymmetric C–H bending of the CH₂ groups and 1605 cm⁻¹ is attributed to the CO-bridge stretching of the glucosamine residues.

The ¹H NMR spectrum revealed signals at δ = 3.2 ppm for the –NHCO– groups, at 1.1 ppm for the –NH– groups, 4.6 ppm (s-br, for H-1) for the acetal proton of chitosan and 3.2–3.5 ppm (s-sh, br, H-2, H-3, H-4, H-5 & 6) for the CH₂OH groups as well as peaks at 2.4 (s-sh), 1.0 (s, 3H, H-18), 1.3 (s, 3H, H-19), 7.2 (16-vinyl hydrogen), and 4.5 ppm (s, 3H, methyl ketone).

The ¹³C NMR spectrum of the product shows some new peaks at δ 49.8 (C–N–C), 176.5 (–C=N–, ketimine) and 71.4 ppm (CH₂OH) along with the other peaks of the steroid and chitosan parts.

3.1.5. SEM and TEM analysis. The general morphology of the product was investigated using SEM. Fig. 3a and b show representative images of the nanorods with image scale bars of 50 and 30 μm, respectively. Moreover, the latter shows that the particles are aggregates consisting of much smaller growing amorphous particles and the very small (300–500 nm) amorphous particles generate a high surface area. On the other hand, these particles are already amorphous, which makes them sufficiently stable in a system different from the starting one, in which they used to grow.⁷ The presence of both, single isolated amorphous particles and complex aggregates, suggests that the growth process is promoted by two types of nanoparticles: (i) a single isolated bred released from the aggregates and (ii) complex aggregates comprising a large number of single bred nuclei.

Fig. 3 SEM photographs of chitosan–16-DPA (a and b).

The detailed microstructure of the product CHDPA nanorods was further investigated using TEM. The examination of the amorphous products shows that a great part of the material is in the form of random aggregates²⁴ and the structure, self-aggregation behavior, etc., of CHDPA are shown in the TEM images in Fig. 4a and b. The shapes of CHDPA nanoparticles observed were mostly spherical and cubical. The diameters of the nanoparticles were 300–500 nm.

Fig. 4 TEM photographs of chitosan–16-DPA (a and b).

Nevertheless, abundant intergrowth was observed in the products and the number of isolated well-shaped amorphous particles was limited. The most likely origin of the abundant intergrowth is the presence of nanoparticles, which govern the growth in the system. The TEM images in Fig. 4a and b represent the two types of particles. The domination of the aggregates in the product is in good agreement with the above suggestion. The mass of the aggregated nanoparticles growing into amorphous particles is much higher than that of single bred nuclei. The low number of single isolated structures can be explained by either limited breeding or by the close proximity of the released bred nuclei to the parent aggregate, and thus their development into amorphous particles resulting in complex aggregates. Therefore, the formation of nanoparticles with narrow particle size distribution cannot be expected if the growth in the system is promoted by complex aggregates, even being of nanometric size.

3.2. Biology

The synthesized compound showed a good antifungal activity in general. The data and the experimental photographs are given in Table 1 and Fig. 5(E1)–(E5).

Table 1 The inhibitory effects of the samples on mycelia growth of the control and 7 days after inoculation (percent inhibition)

Samples	Concentrations (ppm)
	100	200	300	400	500
	mean ± SD	mean ± SD	mean ± SD	mean ± SD	mean ± SD
CHDPA	74.7 ± 0.98	75.8 ± 0.51	78.4 ± 1.57	81.0 ± 0.65	83.6 ± 0.86
Control	Full growth of the fungus

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Fig. 5 Antifungal activity: C = control, E1–E5 = experimental concentrations of 100, 200, 300, 400 & 500 ppm.

The antifungal activity of the new modified chitosan (CHDPA) was studied against the fungus Colletotrichum gloeosporioides. The inhibitory effects of the samples were tested in vitro on mycelia growth of Colletotrichum gloeosporioides using the poisoned food technique with the five concentrations of 100, 200, 300, 400 and 500 ppm. The data reveal that with increasing the concentration, the inhibition percentage also increases.

3.3. DPA loading capacity (LC), encapsulation efficiency (EE) and DPA release study

The synthesized product showed a good drug loading capacity (LC), i.e. 23.2%, and encapsulation efficiency (EE), 20.9%, which are given in Table 2. The product also showed a good release behavior, as shown in Fig. 6.

Table 2 Effective percentage drug loading capacity (LC) and encapsulation efficiency (EE) of chitosan–16-DPA

Samples	%
	Drug loading capacity (LC)	Encapsulation efficiency (EE)
CHDPA	23.2	20.9

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Fig. 6 In vitro drug release study of chitosan–16-DPA.

4. Conclusion

In this contribution, we successfully utilized chitosan, a biocompatible polymer, and a steroid to synthesize linear chitosan–16-DPA nanoparticle aggregates. The chitosan employed herein not only served as the reducing agent and stabilizer, but also led to the assembly of the chitosan–16-DPA nanoparticles. TEM images and UV spectra confirmed the existence of the linear nanoparticle aggregates in solution. One could obtain highly branched long chains and isolated short chains by adjusting the molar ratio of the chitosan repeat unit to 16-DPA. Moreover, the process of growth and assembly for the chitosan–16-DPA nanoparticles was studied using SEM. This method provides a novel way to fabricate linear chitosan–16-DPA nanoparticle aggregates by virtue of its simple one-pot procedure and chain-length tunability. From the analysis results, the compound was found to show a most promising antifungal activity against the fungus Colletotrichum gloeosporioides. The drug loading capacity (LC) was found to be 23.2% and the encapsulation efficiency (EE) was 20.9%. The drug release was investigated using UV spectrophotometry and the study showed good results.

Acknowledgements

The authors thank the Director CSIR-North East Institute of Science & Technology, Jorhat, Assam, for providing the facilities and valuable advice, and also gratefully acknowledge the financial support by DST, New Delhi and CSIR, New Delhi, India.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra16093b

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