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Vol. 9. Issue 1.
Pages 890-901 (January - February 2020)
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Vol. 9. Issue 1.
Pages 890-901 (January - February 2020)
Original Article
DOI: 10.1016/j.jmrt.2019.11.029
Open Access
Formation mechanisms of chitosan-silica hybrid materials and its performance as solid support for KR-12 peptide adsorption: Impact on KR-12 antimicrobial activity and proteolytic stability
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Johnatan Diosaa, Fanny Guzmanb, Claudia Bernalc,d, Monica Mesaa,
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mmesacad@gmail.com

Corresponding author.
a Grupo Ciencia de los Materiales, Instituto de Química, FCEN, Universidad de Antioquia UdeA, Calle 70 No. 52-21, Medellín, Colombia
b Laboratorio de Síntesis de Péptidos, Núcleo de Biotecnología Curauma, Pontificia Universidad Católica de Valparaíso, Chile
c Instituto de Investigación Multidisciplinario en Ciencia y Tecnología, Universidad de La Serena, Raul Bitran 1305, La Serena, Chile
d Tecnología Enzimática para Bioprocesos, Departamento de Ingeniería de Alimentos, Universidad de La Serena, Raul Bitran 1305, La Serena, Chile
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Tables (3)
Table 1. Chitosan incorporation, silica production yield, surface area, and pore volume for the chitosan-silica hybrid materials.
Table 2. KR-12 peptide properties.
Table 3. Zeta potential values (ξ) of chitosan-silica materials before/after KR-12 peptide adsorption and adsorption parameters at pH 8.0 (±0.1) after fitting with Langmuir and Freundlich types mechanisms.
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Abstract

Chitosan-silica materials offer a specific environment for the adsorption of biofunctional molecules, such as the antimicrobial peptide KR-12. The objective here is to rationalize the changes in the physicochemical properties of these chitosan-silica materials in function of the synthesis pH, using 0.02w/v % chitosan as catalyst and aggregation agent and, to correlate these characteristics with the loading/delivery and activity/stability of KR-12 antimicrobial peptide. The CS-6 material, prepared at pH 6, exhibits higher surface area (745m2/g) and total pore volume (0.58cm3/g) due to the lower incorporation of chitosan swollen chains (9.36wt %). Higher pHs produced denser materials (CS-7 and CS-8) with higher entangled chitosan incorporated (> 17wt%). Adsorption of KR-12 peptide was found to take two different mechanisms depending on the chitosan-silica support, Langmuir-type for CS-6 material and Freundlich-type for CS-7 and CS-8 materials. According to the KR-12 release profiles, more hydrophobic interactions were observed in the CS-6 material exposing Lys and Arg residues from the peptide towards the material surface. This was correlated with the higher antimicrobial activity of this material against S. aureus strain (MIC=128μg/mL). Additionally, the KR-12 peptide adsorbed in the CS-6 hybrid support is 34 % more protected from the proteolytic action of α-chymotrypsin than the free one. In conclusion, the proposed models of different porous environments in the studied chitosan-silica materials supports the KR-12 peptide loading/delivery mechanisms, leading to biofunctionalized solid antimicrobial materials exhibiting proteolytic stability. This knowledge is useful for designing new antibacterial materials for biomedical applications.

Keywords:
Chitosan-silica particle mechanism formation
KR-12 peptide adsorption/delivery mechanisms
KR-12 controlled release
KR-12/chitosan-silica antimicrobial solid materials
Proteolysis stability
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1Introduction

The biosilicification process in diatoms and sponges [1,2] occurs in nature under mild conditions of pH (near to neutrality) and temperature (∼25°C), mediated by biomolecules such as peptides, proteins, lipids, and polysaccharides [3,4]. It inspires the biomimetic synthesis of silica by sol-gel chemistry, using natural or synthetic silicificating molecules, being less aggressive and eco-friendly than some gaseous or liquid routes [5].

Chitosan, which is a linear polysaccharide rich in amino and hydroxyl groups, can be used as a biomimetic catalyst of silicification process [6], due to the nucleophilic assistance of amino groups [7], resulting in a hybrid material (chitosan–silica) [8]. Due to the versatile role of chitosan in the silicification reaction as a catalyst, aggregation agent, and structural director [2,9], there is still room to study the interactions that direct these processes. Different properties of chitosan-silica hybrid materials like surface area, morphology, and surface chemistry could be tailored during the sol-gel synthesis due to the pH-dependent interactions between the chitosan and the silica precursors. Some examples include the production of hybrid materials with controlled porosity from sodium silicate and modulating the pH between 2 and 6 [10], the variation of the particle morphology from spherical to starfruit-like at fixed pH in the presence of phosphates at different incubation times [11] and the, improvement of their mechanical properties [12]. Another advantage of the chitosan-silica hybrids is their surface reactivity, that allows further functionalization with molecules of different nature [13,14]. These characteristics make these materials attractive as solid supports for biomolecules such as enzymes. For example, taking advantage of the primary amino groups of chitosan for the covalent binding of β-galactosidase [15] and the enzyme–chitosan non-bonding interactions for manganese peroxidase encapsulation during the sol-gel process [16]. In these two cases, the resulting solid biocatalysts retain the activity of the free enzymes with higher stability and easy handling, improving their performance in practical applications.

Nowadays, there is an increased interest of using antimicrobial peptides (AMP), instead of classical antibiotics, since they exhibit broader action spectrum, rapid bactericidal effect, and low bacterial resistance [17]. AMP have common characteristics such as positive net charge and amphipathicity that allow them to interact efficiently with the negatively charged membrane in bacteria to disintegrate it [18–21]. As in the case of the enzymes, it is important to preserve the antimicrobial activity and stability during their application because the AMP can be attacked by proteases in the action site [22] or suffer fast clearance through the binding of serum proteins [23]. One of the strategies for avoid these negative effects is the AMP adsorption in solid carriers that act as delivery systems, as reported for the LL-37 AMP [24]. These systems have additional advantages for AMP because the controlled release rate could reduce their cytotoxicity and improve their bioavailability [25]. The adsorption of the peptide molecules onto a siliceous solid materials can be mediated by electrostatic and hydrophobic interactions and modulated by the surface chemistry, particle size and porosity [24,26–29]. There are few reports about the influence of these interactions on the activity of AMP [30].

The KR-12 AMP is a fraction from the cathelicidin LL-37 found in the immune system of human beings [31]. Its amphipathic structure has cationic amino acids, which are responsible for the antimicrobial activity (KRIVQRIKDFLR) [31]. This shorter peptide avoids the cytotoxicity issue associated with the LL-37, and its synthesis by FMOC should be easier and cheaper because of its size [32]. Since its identification as the smallest antibacterial fraction of theLL-37 peptide in 2008 [31] the research related with its antibacterial action mechanism [33–36], antibiofilm, antifungical [37], osteogenic [38] and wound healing [39] activities has not stopped.

Having in mind that aspects related with the protection from proteases action [40] and advantages of controlled delivering [41] of AMP deserve special attention, it should be very interesting to combine the advantages of the KR-12 AMP with the possibility of use chitosan-silica supports for control its delivery. Up to date there are not related studies that allow to understand the changes on the bioactivity and proteolytic stability behavior of the adsorbed KR-12 peptide in these chitosan-silica materials, which could be very interesting for future applications on the formulation of pharmaceutical products, wound dressings and medical devices.

The objective here is to rationalize the changes in the physicochemical properties of these hybrid materials as a function of synthesis pH, using 0.02w/v % chitosan as catalyst and aggregation agent and, to correlate these characteristics with the loading/delivery and activity/proteolytic stability of KR-12 antimicrobial peptide in these biofunctionalized hybrid supports.

2Materials and methods2.1Materials

Sodium silicate (Na2Si3O7: 27wt % SiO2, 9.0wt % Na2O) was purchased from Merck Co. Chitosan (CS) with 85 % deacetylation and molecular weight 190–375kDa, acetic acid, sodium acetate, and sodium hydroxide were obtained from Sigma-Aldrich Company. KR-12 peptide (KRIVQRIKDFLR) was kindly gifted by Laboratorio de Síntesis de Péptidos (Núcleo de Biotecnología Curauma, Pontificia Universidad Católica de Valparaíso, Chile) All reagents were used without any further modification.

2.2Synthesis of chitosan-silica hybrid materials

Chitosan–silica materials were obtained through a biomimetic sol-gel process by the polycondensation of sodium silicate using chitosan as a catalyst. Each material was synthesized as follow: 1.226g of sodium silicate was added to 100mL of a 50mM sodium acetate solution. The pH of this mixture was adjusted to 6.0, 7.0, or 8.0 with glacial acetic acid. Then, chitosan was added dropwise from a previously prepared solution (0.5g of chitosan was dissolved in 25mL of 3 v/v % acetic acid). The chitosan concentration in the sodium silicate solution was adjusted to 0.02w/v %. The pH of the system was quickly readjusted to the initial condition (pH 6.0, 7.0, or 8.0). After that, the mixture was left reacting at 22°C, under quiescent conditions for 5h. For recovering the solid material, the obtained dispersions were washed three times with deionized water by centrifugation and dried at room temperature overnight. The produced materials were labeled as CS-Y where Y is the pH of synthesis (6.0, 7.0, and 8.0).

2.3Chitosan–silica materials characterization2.3.1FTIR

Functional groups were determined by FTIR with KBr pellets (1:100 sample dilutions) in transmission mode from 4000cm−1 to 500cm−1 using a Spectrum One IR spectrometer (Perkin Elmer). The samples were dried 2h at 100°C before the analysis to reduce the interference from water.

2.3.2Thermogravimetric analysis (TGA)

The TGA analysis was carried out in air at 10°C/min from 30°C to 800°C using a Q500 TGA (TA Instruments). The chitosan incorporation was measured from the weight loss between 200°C and 700°C and expressed as percentage respecting to the net dried weight of the recovered material. The silica production yield was calculated by Eq. 1, where mc is the weight of the recovered hybrid material after washing and drying (as described in 2.2 section), Xsil is the silica fraction in the hybrid material obtained from TGA (Xsil=100 %-Chitosan loss %), and mex is the expected amount of silica according to the silica precursor concentration.

2.3.3Zeta potential

The surface charge of each material at pH 8 (adjusted with 0.1M NaOH), was determined before and after KR-12 adsorption using a Malvern Zetasizer Nano Z instrument, in a disposable capillary cell. The result is the average between 10–15 measurements for each sample at 25°C.

2.3.4Nitrogen adsorption isotherms

The apparent specific surface area was determined from the nitrogen adsorption isotherm at 77K using the Brunauer-Emmett-Teller (BET) method. Pore size distributions were obtained from the adsorption branch using the BJH (Barrett, Joyner, Halenda) model. The total pore volume was obtained at a relative pressure P/P0 of 0.995. Prior to the analysis, the materials were degassed at 100°C for 12h. The characterization was performed in the ASAP 2020 surface area and porosity analyzer (Micromeritics).

2.3.5Scanning Electron Microscopy (SEM)

Morphology of the materials was followed by SEM in the JSM 6490LV microscope (JEOL). The samples dispersed in ethanol were deposited on a graphite tape and coated with gold to enhance the electric conductivity.

2.3.6Dynamic light scattering (DLS)

Particle size and size distributions based on intensity were determined by DLS in Horiba LB 550 equipment. The measurements were carried out at 25°C in aqueous dispersion of the samples. The reported values correspond to the average of five measurements.

2.4KR-12 adsorption on chitosan–silica materials

Adsorption isotherms of KR-12 onto the materials were obtained at 22°C. Solutions of KR-12 with an initial concentration between 25mg/L and 1000mg/L were prepared in a 25mM phosphate buffer at pH 8. To each solution, 5mg of the selected chitosan–silica material was added, and the mixture was stirred for 2h. The adsorbed KR-12 was indirectly determined through quantification of the free peptide in the supernatant after centrifugation. This was done by fluorescence as described in section 2.6. The equilibrium adsorption for each material was determined as follows:

where qe is the equilibrium capacity of the hybrid material (mg of KR-12/g of material), C0 is the initial peptide concentration, Ce is the peptide concentration in solution after adsorption (mg of KR-12/L of solution), V (L) is the volume of the solution, and m (g) is the mass of the material. The obtained isotherms were adjusted to the Langmuir and Freundlich models.

2.5KR-12 release from chitosan-silica materials

In this assay, 5mg of KR-12/CS-Y functionalized materials, prepared by adsorption under conditions described in 2.4, were suspended in 2mL of 25mM phosphate buffer (150mM NaCl) at pH 7.4 and stirred at 22°C. The released KR-12, followed during 24h, was determined by fluorimetric assay as described in section 2.6. The results were reported as percentage of released KR-12 respecting to the KR-12 loaded in each sample.

2.6KR-12 peptide quantification

Briefly, 20μL of fluorescamine (2mg/mL in acetone) was added to a 180μL aliquot from the peptide containing sample. The fluorescence intensity was determined using a Fluoroskan FL Reader (Thermo Scientific, Waltham, MA) with a 360/465nm excitation/emision filters. The KR-12 quantification was made from a specific calibration curve.

2.7Antibacterial assay

The minimum inhibitory concentration (MIC) for the free and KR-12/CS-Y functionalized materials against S. aureus (ATCC 29,213) were determined using a two-step microdilution assay optimized for AMPs [42]. Briefly, i) the bacteria strain (50,000 CFU) was incubated in presence of different KR-12 concentrations (2048μg/mL–1μg/mL) for 2h at 37°C in 10mM Tris buffer (pH 7.4) without tryptic soy broth (TSB), and ii) the peptide and bacteria mixture was added with a small fraction of concentrated TSB and incubated for 20h at 37°C. The MIC was defined as the minimum peptide concentration that inhibited growth by more than 50 %. Growth inhibition was determined measuring changes in absorbance at 590nm and applying the following equation:

where A0h and A20h are the absorbances of a sample well after 0h and 20h of incubation respectively; C0h and C20h are the absorbances of the growth control well (bacteria without antibiotic) after 0h and 20h of incubation.

2.8Stability of free and adsorbed KR-12 peptide against α-chymotrypsin

50μg of KR-12 were solubilized in 25mM phosphate buffer (pH 8) and subjected to enzymatic hydrolysis using α-chymotrypsin. The analysis was performed with a 1:10 (w/w) enzyme to substrate ratio, at 37°C for 30min. After digestion, the remaining KR-12 was followed by reverse phase HPLC using C18 column (Accucore 150, Thermo Scientific) and a linear gradient of acetonitrile (with TFA 0.1 v/v %) from 10 % to 80 % for 10min at a flow rate of 0.5mL/min. The effluent absorbance was followed at 214nm. For the adsorbed peptide the enzymatic digestion proceeds in the same way, but the peptide was previously mixed with 150μg of CS-Y materials for 2h in the same buffer. A protection factor against α-chymotrypsin was defined as the KR-12 area ratio between the free peptide and the adsorbed one after the enzymatic digestion applying Eq. 4:

where Am is the peak area of adsorbed KR-12 peptide after enzymatic digestion and AKR-12 is the peak area of free peptide after enzymatic digestion.

3Results and discussion3.1Synthesis and characterization of chitosan-silica hybrid materials

The silica sol-gel biomimetic synthesis route adopted in this work, at pH 6.0–8.0 and 0.02w/v% chitosan initial concentration, without further treatments for eliminating the organic counterpart, allows obtaining chitosan-silica hybrid materials with different chemical compositions and textural properties (Table 1). The silica sol-gel biomimetic conditions promote the interactions between the siliceous precursor and chitosan moieties during the silica polycondensation for 5h at 22°C. These solid materials can be easily recovered after centrifugation, exhibiting different silica reaction yields (Table 1). As a control experiment, a synthesis without chitosan was followed for 5h at neutral pH, without producing recoverable silica particles. This is related to the low polymerization degree of silica reported around neutral pHs, as consequence of the low amount of ionized orthosilicic molecules (ca. 0.16 %) for contributing to the nucleophilic substitution mechanism (SN2) of silica polycondensation [43]. These results corroborate that chitosan acts as a catalyst during the polycondensation reaction, as reported before under similar silica-biomimetic synthesis conditions [16].

Table 1.

Chitosan incorporation, silica production yield, surface area, and pore volume for the chitosan-silica hybrid materials.

Material  Chitosan incorporation (%)a  Reaction yield (%)b  Apparent specific surface area – BET (m2/g)c  Total pore volume-Vtp (cm3/g)c 
CS-6  9.4±0.6  57.4±8.7  745  0.58 
CS-7  16.7±0.5  26.6±2.1  542  0.33 
CS-8  21.9±1.7  18.7±0.8  374  0.29 
a

Determined from TGA thermogram, in dried base.

b

Calculated with eq. 1.

c

From N2 adsorption branch.

The FTIR analysis confirms the presence of chitosan and silica in the obtained materials, independent of the pH of synthesis (Fig. 1). The band between 2800cm−1 to 3000cm−1 corresponds to C–H stretching vibrations. The band near to 1545cm−1 is due to the –NH2 deformation. Those signals are representative of chitosan [44]. The band at 1090cm−1 is related with the Si–O–Si stretching vibrations and that at 960cm−1 corresponds to free Si−OH groups of silica [44].

Fig. 1.

FTIR spectra for chitosan (left) and hybrid materials (right): CS-6 (black line); CS-7 (red line), and CS-8 (blue line).

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The TGA-DTA thermograms for the hybrid materials are shown in Fig. 2. The weight loss due to adsorbed water is observed between 30°C and 200°C. Two events between 200°C and 700°C coincide with the thermal degradation region of the chitosan, reported by other authors [45–47]. The weight loss related to the condensation of free silanol groups in silica overlapped at temperatures higher than 500°C [48], is not considered because it represents less than 5 % weight loss in a pure silica (Fig. S1) and it is neglected in chitosan-silica hybrid materials [10]. The silica reaction yield and the chitosan incorporation calculated with the TGA weight losses between 200°C and 700°C for all samples are summarized in Table 1. There is a strong correlation between the pH of synthesis and the physicochemical characteristics: The incorporation of chitosan increases and there is a concomitant decrease of the silica reaction yield, surface area and pore volume when the pH of synthesis is raised (Table 1). On the other hand, we hypothesize that the TGA weight loss centered ca. 300°C, which occurs at the same temperature independent of the analyzed sample (Fig. 2a and b), corresponds to the degradation of lightly attached chitosan, which is more prone to a combustion in a similar way to the free chitosan [44]. The degradation step between 520°C and 650°C depending on the sample, could be associated with a fraction of more entrapped chitosan in the siliceous network, which confers thermal protection, as seen in homologous organic-inorganic systems [49]. In this sense, the chitosan incorporated in CS-6 sample (thermal event at 590°C, Fig. 2b) is more protected by the silica network, than in the CS-7 and CS-8 samples (524°C and 550°C, respectively, Fig. 2b).

Fig. 2.

a) TGA and b) DTG curves for CS-6 (black), CS-7 (red), and CS-8 (blue) materials.

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The obtained materials exhibit type-IV N2 sorption isotherms (Fig. 3a), typical for mesoporous solids with low contribution from micropores in the low-pressure region. The wide capillary condensation step in the mesoporous region of the adsorption branch gives a wide pore size distribution (Fig. 3b), which is representative of materials with disordered and heterogeneous textural porosity [50]. This indicates the absence of a chitosan pore-templating effect. This is because the chitosan and silica species are randomly associated during the sol-gel synthesis by this biomimetic process, instead of a silica deposition on a preformed chitosan mold [51]. The pores between 1.5nm–3.5nm in all the samples (Fig. 3b) correspond to the interstices between the primary nanoparticles (ca. 100nm) and their agglomerates (583nm766nm) (SEM and DLS, Fig. 4, left and center sides, respectively). This textural porosity contributes to the specific BET surface area and total pore volume of these materials (Table 1).

Fig. 3.

(a) Nitrogen adsorption/desorption isotherms and (b) pore size distributions from adsorption branch for chitosan-silica materials. CS-6 (black), CS-7 (red) and CS-8 (blue).

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Fig. 4.

SEM micrographs of recovered solid materials (left side, showing primary particles and agglomerates with arrow and circle, respectively), DLS agglomerates size distribution in aqueous dispersion (center) and representation of the porous environment in a portion of agglomerated nanometric particles that form the solid materials (right side) for the CS-6 (a), CS-7 (b), and CS-8 (c) samples. The black dots and blue lines represent the silica network and chitosan chains, respectively. The interchain crowding reduces the available surface area and porous volume, being the CS-8 material the denser one.

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3.2Formation mechanism proposal for chitosan–silica hybrid materials

Here, we attempt to rationalize the changes in silica reaction yield, surface area, and textural porosity of the chitosan-silica hybrid materials in function of synthesis pH. The objective is to discuss about the formation mechanism of these hybrid materials and its consequences on the porous environment, developed during the synthesis by the adopted silica sol-gel biomimetic route at each pH. The different porous environments are schematized, representing a portion of agglomerated nanometric particles that form the solid materials (Fig. 4, left side). It is important to have in mind for the following discussion, that the balance between the fraction of protonated amino groups on chitosan (68 %, 17 % and 2 % at pH 6, 7 and 8, respectively, calculated with amine pKa=6.32) [52] and the deprotonated silicic acid molecules (0.016 %, 0.16 %, and 1.6 % at pH 6, 7 and 8, respectively, calculated with Si(OH)4 pKa=9.8) [43] affects the electrostatic interactions between chitosan chains, and the favored mechanism for the silica polycondensation in the presence of chitosan (proton-transfer or nucleophile-activated) [53].

At pH 6.0, the highest protonation degree of chitosan chains promotes the silica polycondensation through the proton-transfer process [53]. Furthermore, these protonated chains repeal each other creating a swelled chitosan structure (Fig. 4a, left) that allows an effective catalytic interaction with the neutral silica species and in consequence, the measured higher reaction yield (CS-6 sample, Table 1). Moreover, these inter- and intra-chains repulsions favor the highest available surface area and porous volume of this CS-6 material (Table 1). By the contrary, when the synthesis pH is increased to 7.0 and 8.0, the protonation degree of the chitosan chains decreases, leading to the contraction of the chitosan molecules favored by inter- and intra-chains hydrogen bonds. The entangled chitosan chains can be entrapped by the silica network, increasing the incorporated chitosan up to ∼22 % and, denser hybrid particles are formed (Fig. 4b and c, left), which agrees with the lower surface area and porous volume in CS-7 and CS-8 samples (Table 1). On the other hand, the available neutral amine groups, for catalyzing the nucleophilic-assisted silica polycondensation [53], are reduced due to the chitosan coiling, decreasing the silica reaction yield (CS-8, Table 1) despite the highest percentage of SiO species from silicic acid. Probably, the formation mechanism of CS-8 sample can be more related with the aggregation role of the chitosan, avoiding the repulsion between the negative charged silica species [54–56], under the conditions of this work.

3.3KR-12 peptide adsorption/release on chitosan–silica materials

Due to the described physicochemical characteristics for the CS-Y materials, they could offer different environments for loading/delivery of biofunctional molecules, such as the KR-12 antimicrobial peptide, whose intrinsic properties are summarized in Table 2. This hypothesis is studied here from the KR-12 adsorption isotherms and kinetic release profiles.

Table 2.

KR-12 peptide properties.

Peptide sequence  Isoelectric pointa  Net charge at pH 8.0a  Equivalent size (nm)b 
KRIVQRIKDFLR  12.21  +4  1.5 
a

Calculated from https://www.biosyn.com/peptidepropertycalculatorlanding.aspx.

b

Calculated from http://www.calctool.org/CALC/prof/bio/protein_size.

The adsorption isotherms for the KR-12 peptide over the evaluated materials, at pH 8 and keeping constant the adsorption time (2h), are shown in Fig. 5. This adsorption process could be a consequence of several interactions such as hydrogen bonds, hydrophobic, Van der Waals, and electrostatic interactions [28] between the amphiphilic cationic peptide and the chitosan-silica hybrid materials, which exhibit amine, methoxyl [57] and silanol [52] groups (Fig. 1). The importance of each one will depend on the surface chemistry and textural properties of the solid surface, but also on the intrinsic properties of the peptide (Table 2). The electrostatic interactions can be the motor force for KR-12 attraction towards the surface of all materials. That is because at pH 8, the surface of the three materials exhibits negative charge (ξ, zeta potentials, Table 3) from the deprotonated silanol groups [43] and the peptide is positive charged (pI 12.21, Table 2) through the protonated amino groups of Lys and Arg residues. However, the presence of other kind of interactions cannot be neglected, because the neutral amine groups of chitosan can be also exposed to the surface, as shown by TGA weight loss ca. 300°C for less entrapped chitosan chains (Fig. 2a), even if they do not contribute to the ξ value. The screening of the negative charges in the CS-8 material after the peptide adsorption (Table 3) could be associated with the effect of electrostatic interactions with the peptide molecules, which are in the outer surface of this material. By the contrary, the similarity of the ξ values before and after adsorption in CS-6 material suggests that the other interactions, different to the electrostatic ones, are highly involved in the peptide loading into this support. Taking into account the origin of the textural pores in the evaluated materials, it is not straightforward to say that the KR-12 adsorption inside the pores could be responsible of the non-change of the net surface charge (Table 3), as in the case of organized mesoporous silica nanoparticles.

Fig. 5.

Adsorption isotherms of peptide KR-12 on chitosan-silica materials at pH 8, 22°C for 2h. CS-6 (black), CS-7 (red) and CS-8 (blue).

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Table 3.

Zeta potential values (ξ) of chitosan-silica materials before/after KR-12 peptide adsorption and adsorption parameters at pH 8.0 (±0.1) after fitting with Langmuir and Freundlich types mechanisms.

Material  ξ (mV) at pH 8Langmuir mechanismaFreundlich mechanismb
  Before  After  KL  qm  R2  KF  1/nF  R2 
CS-6  −22.2±1.8  −17.1±1.6  6.7×10-2  189.9  0.960  22.38  0.44  0.932 
CS-7  −25.2±2.4  −18.7±2.5  7.0×10-6  126882.9  0.980  1.10  0.95  0.980 
CS-8  −22.5±1.3  −5.5±1.9  8.0×10-7  537875.2  0.820  0.02  1.45  0.996 
a

qe=qmKLCe1+KLCe, KL (Lg−1) is the Langmuir constant and qm (mgg−1) is the maximum adsorption capacity for monolayer formation on the adsorbent.

b

qe=KFCe1/nF, KF (Lmg−1) is the Freundlich constant, 1/nF indicates adsorption intensity.

The evaluation of the form of peptide adsorption isotherms indicates that the mechanism of KR-12 loading in the evaluated solid supports is different (Fig. 5). The higher slope in the CS-6 at low offered peptide concentration (Fig. 5) indicates a higher uptake of the peptide in this region. After that, a progressive saturation of the solid is seen. This can be favored by the higher surface area and Vtp (total pore volume) (745m2/g and 0.58g/cm3, respectively, Table 1), in addition with a higher probability of hydrogen bonding formation per nm2 between the silanol, hydroxyl, and amino groups from the hybrid surface with the hydrophilic residues in the KR-12 peptide. The adsorption is more hindered in the CS-7 and CS-8 materials at low KR-12 concentrations due to their lower surface area and Vtp, but once the first peptide molecules are adsorbed, they promote the uptake of others at high concentration, especially in the CS-8 material (Fig. 5).

To get a better understanding of the adsorption process of the peptide, two isotherm models (Langmuir and Freundlich) are used to describe the adsorption data [60]. The isotherms from Fig. 5 are fitted in the range of 25mg/L–500mg/L of initial peptide concentration and the derived parameters are summarized in Table 3. According to the correlation coefficient, the adsorption process on the CS-6 material is better adjusted to a Langmuir model, which has been commonly used for describing the protein and peptide adsorption on solids [60]. This model is associated with the loading of the offered peptide forming a monolayer, homogeneously distributed in a finite number of equivalent sites in the surface of the material. The higher surface area in the CS-6 sample (Table 1) offers an environment where the KR-12 molecules can be well-distributed, with maximum 2 peptide molecules (equivalent size=1.5nm, Table 2) per textural pore (sizes between 1.5 and 3.5nm, Fig. 2). The comparison of the KL (Langmuir constant) and qm (maximum adsorption capacity) parameters for the three samples is not possible, because the later does not have a physical meaning in the CS-7 and CS-8 materials, which make to discard the model for these two samples (Table 3). The KR-12 adsorption isotherms in CS-7 and CS-8 materials are better adjusted to a Freundlich model, which has been also used for describing the adsorption of small peptides on bare and amino-functionalized silica particles, below a saturation point, indicating the formation of multilayers on the solid surface [28]. The CS-7 and CS-8 materials are less porous (Table 1) and they could be saturated with peptide at early stages, forming peptide multilayers at higher KR-12 concentrations (Fig. 5). The value of 1/nF (adsorption intensity) shows that the formation of multilayers is even stronger on the less porous CS-8 material, which explains the more efficient screening of the surface negative charge by the KR-12 molecules (greater decrease in zeta potential after adsorption, Table 3).

The kinetic release profiles of KR-12 from CSY materials are determined at pH 7.4 in buffer phosphate and 150mM NaCl, for increasing the ionic strength of the medium (Fig. 6). A burst release of the peptide is observed within the first 1h, delivering 74 %, 61 % and 50 % of the total loaded amount from CS-7, CS-8 and CS-6 solid supports, respectively. The higher percentage of peptide released from CS-7 and CS-8 supports corroborates that the electrostatic interactions are more implied in the adsorption of peptide molecules in the surface in these two samples and therefore, the competence with the Na+ ions promotes a higher desorption compared with the CS-6 one. Moreover, the crowding effect due the peptide multilayers in CS-7 and CS-8 samples can have a concentration-like diffusion effect, releasing more peptide to the medium. The 50 % of peptide release from the CS-6 material (Fig. 6) in the presence of 150mM salt corroborates that other interactions, such hydrophobic and hydrogen bond ones are also contributing to its adsorption, as discussed before for other AMP [28]. After the first hour, a sustained peptide release is observed during the 24h of the experiment. There is not 100 % of delivering in the evaluated sample, which can be related with non-electrostatic interactions involved in the peptide adsorption due to its amphiphilic nature.

Fig. 6.

Release profiles of KR-12 from loaded chitosan-silica materials at pH 7.4 and 150mM NaCl. CS-6 (black), CS-7 (red) and CS-8 (blue). Lines are just a guide to the eye.

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3.4Antimicrobial activity and stability of free and adsorbed KR-12 peptide

The antimicrobial activity of free and adsorbed KR-12 peptide is measured by an optimized broth microdilution method for AMP [42]. An exact amount of the CS-Y material with the adsorbed peptide is taken in order to have the same peptide concentration used in the S. aureus inhibition assay with the free KR-12 molecule. This strain is chosen because it is a common Gram-positive microorganism found in wide variety of nosocomials infections, where the KR-12 peptide could be an antibiotic candidate against it [42]. The bare CS-Y materials do not present antibacterial activity against this strain, meanwhile the KR-12 adsorption in the solid supports leads to biofunctionalized solid materials active against S. aureus (Fig. 7).

Fig. 7.

Growth inhibition assays for free KR-12 and KR-12 loaded in CS-Y materials. Free KR-12 (black), CS-6 (red), CS-7 (blue) and CS-8 (green).

(0.13MB).

The MIC is 128μg/mL for the peptide loaded in CS-6 support and 256μg/mL in both CS-7 and CS-8 ones (Fig. 7). These values are higher than the MIC of free KR-12 AMP (32μg/mL, Fig. 7), which means that the contact of the KR-12 peptide with the cell wall of dispersed bacteria is partially hindered due to the presence of the solid support. On the other hand, the peptide adsorption could affect the peptide helicity and therefore, its interaction with the bacterial cell membranes involved in the action mechanism of the antimicrobial peptides [61]. Specially, the Lys residues, which are key aminoacids for the KR-12 antibacterial activity [42], are less available for their action because they are interacting with the negative charged surface of the CS-Y materials and, therefore the activity of the peptide is reduced. However, this fact has a positive effect for reducing the cytotoxicity of the KR-12 peptide against human cells, since Lys and Arg residues are also involved in the cellular lysis [33]. Another positive effect of the steric hindrance effect of the solid support is that it avoids the action of α-chymotrypsine over the antimicrobial peptide, conferring biological stability that it is not exhibited by the free KR-12 molecules (Fig. 8).

Fig. 8.

Protection factor of the peptide KR-12 against proteolytic hydrolysis upon adsorption on CS-Y materials.

(0.06MB).

This protease was used as a model of proteolytic enzymes because it exhibits structural similarities with those released by microorganisms during infection process [22]. The CS-6 material offers the higher protective effect (ca 34 %, Fig. 8) against the action of α-chymotrypsin, which cleaves specifically on peptide bonds where the carboxyl groups are in Phe, Tyr or Trp residues [62]. This corroborates the important contribution of hydrophobic interactions to the KR-12 peptide adsorption in this support, involving the hindering of residues such as Phe. By the contrary, the higher electrostatic nature of the interactions between the CS-7 and CS-8 supports with the KR-12 peptide could led to a higher exposure of the cleavage site for the enzymatic hydrolysis, especially in CS-8 one. This kind of protection would enhance the lifetime of the peptide in future applications where proteases are present. For example, their concentration is increased in infected tissues [63] and they are present in plasmatic serum, which is used for fabrication of wound dressing hydrogels [64].

The quantity of biofunctionalized solid material necessary for achieving a similar activity than the free KR-12 peptide corresponds to 0.30mg; 0.55mg; and 0.51mg of biofunctionalized CS-6, CS-7 and CS-8 solid per mL, respectively. These concentrations are enough for having colloidal stable dispersion in aqueous systems, which is important for formulation of pharmaceutic products and wound dressings [65,66].

The MIC differences among the biofunctionalized solid materials are highly correlated with the KR-12 peptide adsorption mechanism and delivery behavior of each support. The KR-12/CS-6 material is the most active (MIC=128μg/mL, Fig. 7). The higher loaded amount of peptide, forming a monolayer in the higher available surface area (Langmuir-type isotherm, Fig. 5) in addition with a sustained delivery (Fig. 6) and more available Lys and Arg residues (less screening of the surface charge, Table 3), in this biofunctionalized material favor the effective interaction with the bacteria and the cell wall disruption action mechanism [31]. In contrast, the strong binding interaction between KR-12 peptide and CS-8 material (1/nF, Table 3) mainly by electrostatic interactions and the peptide crowding in the multilayers (Freundlich-type isotherm, Fig. 5) are deleterious for its antimicrobial activity. On the other hand, the KR-12 molecules have more freedom degrees when are adsorbed in a monolayer on the CS-6 material, which help to preserve the antimicrobial activity, than those adsorbed in a multilayer way on materials CS-7 and CS-8.

4Conclusions

This work showed that the physicochemical properties of chitosan-silica hybrid materials such as composition, pore size, total pore volume, and specific surface area were highly affected by the pH at a fixed initial chitosan concentration (0.02w/v%). The changes in these properties were associated with the modulation of the protonation state of chitosan chains and their concomitant entanglement degree, which affect the interaction with the siliceous species. These characteristics affected the KR-12 loading/delivering behavior on hybrid chitosan-silica materials and then, the antimicrobial activity and proteolytic stability of the adsorbed KR-12 peptide.

The CS-6 hybrid support prepared at pH 6 exhibited the highest surface area and total pore volume (745m2/g and 0.58cm3/g, respectively), explained by the type of interactions between the low-incorporated chitosan swollen chains (9.36wt %) and silica species during the material formation. These physicochemical characteristics favored the highest KR-12 loading capacity (200mgKR-12/g) through a Langmuir-type adsorption mechanism at pH 8, which in turn favored the sustained release and higher antibacterial activity (MIC S. aureus=128μg/mL). By the contrary, denser CS-7 and CS-8 supports favored a Freundlich-type adsorption mechanism, mediated mainly by electrostatic attractions affecting the Lys and Arg residues and the secondary structure, which are involved in the action mechanism of the antimicrobial peptides. The higher fast-burst peptide release (60 % in 1h) did not favor the KR-12 antimicrobial activity.

Moreover, the presence of chitosan-silica solid support prevented the hydrolysis of this peptide by α-chymotrypsine, being 38 % more protected than the free KR-12 peptide.

The obtained chitosan-silica solid supports, which allow the KR-12 controlled release and proteolytic protection, are very versatile because they can be easily recovered from the reaction medium or used as dispersion/suspension for their subsequent future applications (pharmaceutical, cosmetics, medical devices, etc).

Conflicts of interest

The authors declare no conflicts of interest.

Acknowledgments

Johnatan Diosa acknowledges for the doctoral scholarship from COLCIENCIAS (2015/727). Thanks to Colciencias for the financial support through the project “Preparación de biomateriales antimicrobianos sólidos mediante inmovilización del péptido KR-12 y análogos en materiales silíceos, y evaluación de su comportamiento en gel de fibrina para apósitos. Código111577757023 contrato 765-2017”.

Appendix A
Supplementary data

The following are Supplementary data to this article:

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