Encapsulation of testosterone by chitosan nanoparticles

Accepted Manuscript Title: Encapsulation of testosterone by chitosan nanoparticles Authors: P. Chanphai, H.A. Tajmir-Riahi PII: DOI: Reference:

S0141-8130(17)30118-6 http://dx.doi.org/doi:10.1016/j.ijbiomac.2017.02.007 BIOMAC 7061

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

9-1-2017 22-1-2017 1-2-2017

Please cite this article as: P.Chanphai, H.A.Tajmir-Riahi, Encapsulation of testosterone by chitosan nanoparticles, International Journal of Biological Macromolecules http://dx.doi.org/10.1016/j.ijbiomac.2017.02.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Chanphai and Tajmir-Riahi

Testosterone-chitosan delivery

Revised manuscript: IJBIOMAC-2017-107 for Int. J. Biol. Macromol. The revisions made are shown in red color throughout the text

Encapsulation of testosterone by chitosan nanoparticles P. Chanphai and H.A. Tajmir-Riahi* Department of Chemistry-Biochemistry and Physics University of Québec at TroisRivières, C. P. 500, TR (Quebec) Canada G9A 5H7

* Corresponding author: Fax: 819-376-5084; Tel: 819-376-5011 (ext. 3326); E-mail: [email protected]

Graphical abstract

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Research Highlights ►Testosterone loading by chitosan nanoparticles is investigated here. ► Major conjugation of testosterone by chitosan was observed. ► As chitosan size increased more stable steroid conjugates were formed. ► The loading efficacy was increased as chitosan size increased. ► Major chitosan morphological changes occurred by testosterone encapsulation.

Abstract The loading of testosterone by chitosan nanoparticles was investigated, using multiple spectroscopic methods, thermodynamic analysis, TEM images and modeling. Thermodynamic parameters showed testosterone-chitosan bindings occur mainly via H-bonding and van der Waals contacts. As polymer size increased more stable steroid-chitosan conjugates formed and hydrophobic contact was also observed. The loading efficacy of testosterone-nanocarrier was 40 to 55% and increased as chitosan size increased. Testosterone encapsulation markedly alters chitosan morphology. Chitosan nanoparticles are capable of transporting testosterone in vitro.

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Abbreviations: ch, chitosan, test, testosterone, TEM, transmission electron microscopy, FTIR, Fourier transform infrared

Keywords: chitosan, testosterone, conjugation, loading efficacy, thermodynamic, TEM images

1. Introduction Polymers are extensively used for the delivery of active pharmaceutical drugs. They can form a matrix or membrane that can control the release of a drug over a prolonged period of time. Among the potential natural cationic polymers used in drug delivery, chitosan has attracted major interest due to its unique chemical properties [1-5]. Chitosan is a nontoxic, biodegradable and biocompatible polysaccharide of b(1-4)-linked D-

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glucosamine and N-acetyl-D-glucosamine [6]. Chitosan and its derivatives have the desired properties for safe use as a pharmaceutical drug delivery tool. This has accelerated research activities worldwide on chitosan micro and nanoparticles as drug delivery vehicles. Chitosan nanoparticles were used for delivery of therapeutic proteins, peptides and small drug molecules [7-9]. Testosterone is the main androgenic hormone which controls many physiological processes such as, sexual functions and secondary sex characteristics, muscle protein metabolism, plasma lipid and bone metabolism [10]. Since a substantial part of steroids is bound to serum proteins in vivo, the potential application of serum proteins in steroid delivery has been reviewed [11]. Synthetic polymers are used as potential nanocarriers to deliver steroids in vitro and in vivo [12,13]. Chitosan and its derivatives were also tested as delivery tools for transporting steroids [14,15]. Therefore, it was of interest to examine the potential application of chitosan nanoparticles for testosterone delivery in vitro using spectroscopic, thermodynamic and microscopic analysis. The loading of testosterone by chitosan-15 and chitosan-100 kDa was determined using multiple spectroscopic analysis, thermodynamic parameters, TEM imaging and molecular modeling. Structural information regarding testosterone-chitosan interactions and the effects of polymer size and hydrophobicity on the steroid loading are presented here. 2. Experimental section 2.1. Materials Purified chitosans 15 and 100 kDa (90% deacetylation) were from Polysciences Inc. (Warrington, USA). Testosterone or 17β-hydroxy-4-androsten-3-one was from Steraloids

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Inc. and used as supplied. Other chemicals were of reagent grades and purified before sample preparation. 2.2. Preparation of stock solutions and testosterone-chitosan- conjugates Solutions of testosterone (in ethanol/H2O 50/50 %) 120 M was prepared and diluted to various concentrations in 10 mM Tris-HCl (pH 7.4). Chitosan was dissolved in acidic solution (0.1 M HCl) (pH 5-6) and diluted to various concentrations using 10 mM Tris-HCl. Testosterone-chitosan conjugates were prepared by the addition of different chitosan concentrations (1 to 60 µM) to a testosterone solution (60 µM). The characterization of each steroid-polymer conjugate by multiple spectroscopic methods and TEM imagings is described below. 2.3. Transmission electron microscopy The TEM images were recorded using a Philips EM 208S microscope operating at 180 kV. The morphology of the conjugates of testosterone with chitosan-15 and 100 kDa in aqueous solution at pH 7.4 were monitored using transmission electron microscopy. One drop (5–10 µL) of the freshly-prepared mixture [chitosan solution (60 µM) + testosterone solution (60 µM)] in Tris–HCl buffer (24 ± 1◦C) was deposited onto a glow-discharged carbon-coated electron microscopy grid. The excess liquid was absorbed by a piece of filter paper and a drop of 2% uranyl acetate negative stain was added before drying at room temperature. 2.4. UV spectroscopy The UV-Vis spectra were recorded on a Perkin-Elmer Lambda spectrophotometer with a slit of 2 nm and scan speed of 400 nm min-1. Quartz cuvettes of 1 cm were used.

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The absorbance measurements were performed at pH 7.4 by keeping the concentration of testosterone constant (60 µM), while altering chitosan concentrations (1 µM to 60 µM). The binding constants of trypsin-protein adducts were obtained according to the method described by Connors [16,17]. 2.5. FTIR spectroscopy Infrared spectra were recorded on a FTIR spectrometer (Impact 420 model), equipped with deuterated triglycine sulphate (DTGS) detector and KBr beam splitter, using AgBr windows. Solution of steroid was added dropwise to the chitosan solution with constant stirring to ensure the formation of homogeneous solution and to have testosterone contents 15, 30 and 60 M with a final chitosan concentration of 60 M. Spectra were collected after 2h incubation of polymer and steroid solutions at room temperature, using hydrated films. Interferograms were accumulated over the spectral range 4000-600 cm-1 with a nominal resolution of 2 cm-1 and 150 scans. The difference spectra [(chitosan solution + testosterone solution) – (chitosan solution)] were generated using chitosan band around 900 cm-1, as standard [18]. 2.6. Docking The docking studies were carried out with ArgusLab 4.0.1 software (Mark A. Thompson, Planaria Software LLC, Seattle, WA, http://www.arguslab.com). The chitosan structure was obtained from the literature [19] and the testosterone three-dimensional structure was generated from PM3 semi-empirical calculations using Chem3D Ultra 11.0. The docking runs were performed on the ArgusDock docking engine using regular precision with a maximum of 150 candidate poses. The conformations were ranked using the Ascore scoring function, which estimates the free binding energy. Upon location of the

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potential binding sites, the docked complex conformations were optimized using a steepest decent algorithm until convergence, with a maximum of 20 iterations. Chitosan donor groups within a distance of 3.5 Å relative to the testosterone were involved in complex formation. 3. Results and discussion 3.1. TEM analysis of testosterone-chitosan conjugates We determined the morphological dynamics of chitosan nanoparticles upon steroid conjugation. The effect of steroid-chitosan interactions on the shape of chitosan nanoparticle was determined by using transmission electron microscopy. The shapes of uncomplexed Ch-15 and Ch-100 kDa alongside with their testosterone conjugates are shown in the TEM micrographs (Fig. 1). TEM micrographs show that uncomplexed chitosan had a markedly different shape depending on its size; Ch-15 has sphericalshaped, while Ch-100 is needle-shaped with smooth surface and narrow size distribution of about 90 nm [21,22 ]. Similar differences were observed for AFM images of Ch-15 and Ch-100 kDa where the result is attributed to the degree of polymer aggregation, as chitosan size increases [23]. However, marked differences were observed in the morphology of the testosterone–chitosan aggregates. TEM images clearly showed the appearance of the aggregates of irregular shapes dispersed in solution when Ch-15 conjugates with testosterone (Fig. 1). In addition, the bound Ch-100 with testosterone showed major changes of the polymer morphological shape (Fig. 1). An increase of the spherical-shaped aggregates can be seen from TEM micrograph, suggesting that the elongated shapes were lost in favor of spherical-shaped in the testosterone–chitosan conjugates (Fig. 1).The loss of the elongated shape of chitosan nanoparticles after

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complex formation with testosterone is likely to be the result of the steroid encapsulation. This is consistent with major particle size increase as encapsulation occurs (Fig. 1). Indeed, testosterone binding to chitosan which is a linear polysaccharide that has multiple sites of interaction and therefore should be regarded as core–shell system with steroid (core) and chitosan (shell) [24-28]. Therefore, if a tightly bound conjugate between polymer and steroid is formed, it can change the initial shape of polymer in favor of the steroid shape. The results suggest that the binding of testosterone to chitosan may play a role in altering the predefined shape of the nanocarrier due to encapsulation. 3.2. Binding features of testosterone-chitosan conjugates by UV-Visible spectroscopy Testosterone-chitosan binding results in the absorption spectra changes of the testosterone and the observed changes can be used to calculate the steroid-polymer binding constant [16,17]. The UV spectra of testosterone-chitosan conjugates are presented in Figure 2. Steroid-chitosan complexation occurred with an increase in the intensity of testosterone absorption band at 250 nm [29]. The testosterone-chitosan binding constants were calculated as described earlier in materials and methods [16], using plots of 1/(A-A0) vs (1/chitosan concentrations) (Fig. 2). The double reciprocal plot is linear and gives the overall binding constant for each conjugate with Ktestosterone-ch-15 =5.0 (±0.5) x 104M-1 and Ktestosterone-ch-100 =5.9 (±0.9) x 105M-1 (Fig. 2 and Table 1). The calculated binding constants show a strong affinity between chitosan and testosterone, which correlates well with steroid hydrophobicity. The increase of polymer size, results in an increase of the stability for testosteronechitosan conjugates (Fig. 2 and Table 1). This is due to an increase of the number of positively charged NH2 groups as polymer size increases, which contributes to more

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hydrophilic interactions. However, evidence regarding hydrophobic, hydrophilic or Hbonding contacts comes from the thermodynamic analysis of steroid-chitosan conjugates discussed below. 3.3. Thermodynamic parameters of testosterone-chitosan interactions The interactions between chitosan nanoparticles and testosterone in aqueous solution can be driven by hydrogen bonding, hydrophobic and electrostatic interactions [29,30]. The thermodynamic parameters of the complexes are determined, in order to characterize the nature of the driving forces between chitosan and testosterone. The thermodynamic parameters (standard enthalpy changes, ΔH; standard entropy changes, ΔS and standard Gibbs free energy changes, ΔG) for the testosterone-chitosan interactions were calculated (Table 2). According to the data of ΔH and ΔS, the nature of the testosterone-chitosan interaction can be characterized [29,30]. The main force of interaction is hydrophobic and H-bonding if ΔH < 0 and ΔS > 0. If ΔH > 0 and ΔS < 0, electrostatic interaction is the predominant force and finally, if ΔH < 0 and ΔS < 0 van der Waals forces and hydrogen bonding are established. The thermodynamic parameters for the interaction of chitosan with testosterone at 298.15, 308.15 and 318.15 K are presented in Figure 3 and Table 2. The negative sign of ΔG means that the binding process between chitosan and testosterone is spontaneous. Furthermore, the testosterone-polymer conjugates studied here have negative ΔH, which means the complex formation between steroid and chitosan is an exothermic reaction. The negative ΔH and negative ΔS for steroid-polymer conjugate indicate that H-bonding and van der Waals contacts are predominant in these testosterone-chitosan conjugates. However, as polymer size increased positive value for ΔS was observed, indicating the presence of hydrophobic contact in testosterone-chitosan

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interactions (Table 2). The presence of hydrophilic and hydrophobic contacts was also observed in several protein-chitosan complexes [31,33]. The loading efficacy (LE) for testosterone-chitosan conjugates was determined as previously reported [34]. % Efficiency = Final absorption intensity- Initial absorption intensity X 100 Initial absorption intensity The loading efficacy was estimated to be 40% for chitosan-15 and increased to 55% with chitosan-100 kDa, in these steroid-chitosan conjugates. Steroid loading efficacy increases as chitosan size increases. This is also consistent with the stability of testosteronechitosan-100 kDa, which is ten times larger than chitosan-15kDa (Fig. 2). 3.4. Structural analysis of testosterone-chitosan conjugates by FTIR spectroscopy Testosterone-chitosan conjugation was characterized by infrared spectroscopy. There are major spectral shifting and intensity variations for the chitosan amide I band at 16281621 cm-1 (mainly C=O stretch) and amide II band at 1530-1521 cm-1 (C-N stretching coupled with N-H bending modes) [18], upon testosterone interaction. The difference spectra [(chitosan + testosterone solution) – (chitosan solution)] were obtained, in order to monitor the intensity variations of these vibrations and the results are shown in Figure 4. Similarly, the infrared spectra of the free chitosan in the region of 3500-2800 cm-1 were compared with those of the steroid-polymer adducts, in order to examine the drug binding to OH and NH2 groups, as well as the presence of hydrophobic and hydrophilic contacts in testosterone-chitosan conjugates. At low testosterone concentration (15 M), a minor increase in the intensity was observed for the chitosan amide I and amide II, in the difference spectra of the steroid-

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polymer complexes (Fig. 4, diff., 15 M). The positive features are located at 1597 cm-1 (steroid-ch-15) and 1595 cm-1 (steroid-ch-100) (Fig. 4, diff., 15 M). These positive features are related to the increase in the intensity of the chitosan vibrational frequencies, upon steroid conjugation. As testosterone concentration increased to 60 M, further increase in the intensity of the polymer amide I and amide II vibrations was observed with positive features at 1597 (steroid-ch-15) and 1596 cm-1 (steroid-ch-100), upon testosterone complexation (Fig. 4, diff., 60 M). The increase in the intensity of the polymer amide I and amide II bands is due to testosterone bindings to chitosan C=O, C-N and N-H groups (hydrophilic interaction). As chitosan size increased, more perturbations of the polymer infrared vibrational frequencies were observed (comparing difference spectra of testosterone-chitosan-15 and testosterone-chitosan-100), which is indicative of a stronger steroid-polymer interaction for chitosan-100 than chitosan-15 kDa (Fig. 4). This is also consistent with our UV results that showed more stable steroid-chitosan-100 conjugation than chitosan-15 kDa (Fig. 2). 3.5. Docking studies The models of the docking for testosterone are shown in Figure 5. The docking results showed that testosterone is surrounded by several donor atoms of chitosan residue on the surface with a free binding energy of −4.44 kcal/mol (Fig. 5). Similar binding patterns were observed for the conjugated doxorubicin with chitosan nanoparticles [9]. It should be noted that, conjugation of steroids by serum proteins [11], synthetic polymers [13], and chitosan nanoparticles [14,15] have been reported and the possible applications of these polymers in steroid delivery are discussed. Our study here shows that chitosan nanoparticles form more stable conjugates with steroids than synthetic

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polymers and carrier proteins [11,13], which shows the advantage of non-toxic chitosan nanoparticle over synthetic polymers in steroid delivery systems. 4. Conclusions Based on spectroscopic and microscopic analysis, testosterone binds chitosan nanoparticles via hydrophilic, H-bonding and van der Waals interactions. Hydrophobic contact was also observed as chitosan size increased. The stability of steroid-chitosan conjugation and the loading efficacy were enhanced as chitosan size increased. Steroid encapsulation induces major chitosan morphological changes. Chitosan nanoparticles are capable of steroid delivery in vitro. Acknowledgments This work is supported by grant from Natural Sciences and Engineering Research Council of Canada (NSERC).

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Captions for Figures

Figure 1. TEM micrographs free chitosans Ch-15 and Ch-100 kDa and their testosterone conjugates. Figure 2. UV-visible spectra of testosterone conjugates with chitosan-15 kDa (A) and chitosan-100 kDa (B) with free testosterone 30 µM (a) and chitosan adducts with chitosan at 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 µM (b-m). Inset: plot of 1/( AA0) vs (1/ chitosan concentration) and binding constant (K) for testosterone-polymer complexes.

Figure 3. logK vs. 1/T for testosterone-chitosan systems. Figure 4. FTIR spectra in the region of 1800-600 cm-1 of hydrated films (pH 7.4) for (A) chitosan-15kDa (60 µM) and (B) chitosan-100 kDa (60 µM) and their testosterone conjugates with difference spectra (diff.) (bottom two curves) obtained at different testosterone concentrations (indicated on the figure).

Figure 5. Docking results of testosterone–chitosan conjugate. View of the nearest donor groups surrounding testosterone with the free binding energy.

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Table 1. Variations of the binding constants for testosterone with chitosan-15 and chitosan-100 kDa, at different temperatures Complexes

Ch-15-Testosterone

Ch-100- Testosterone

Temperature (K)

Binding constants K

298.15

4.98 x 104

308.15

3.00 x 104

318.15

2.02 x 104

298.15

5.89 x 105

308.15

5.15 x 105

318.15

4.71 x 105

Table 2. Thermodynamic parameters for testosterone with chitosan-15 and ch-100 kDa Thermodynamic parameters Complexes

Ch-15-Testosterone

Ch-100- Testosterone

ΔH

ΔS

TΔS

ΔG

(KJ. Mol-1)

(J. Mol-1. K-1)

(KJ. Mol-1)

(KJ. Mol-1)

-15.50

-13.00

-3.87

-11.63 (298.15 K)

-4.00

-11.50 (308.15 K)

-4.13

-11.37 (318.15 K)

10.48

-14.30 (298.15 K)

-3.82

35.15

10.83 11.18

-14.65 (308.15 K) -15.00 (318.15 K)