N-Reacetylated Oligochitosan: pH Dependence of Self-Assembly

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N-Reacetylated Oligochitosan: pH dependence of Self-Assembly Properties and Antibacterial Activity Inesa V Blagodatskikh, Sergey N Kulikov, Oxana V Vyshivannaya, Evgeniya A Bezrodnykh, and Vladimir E. Tikhonov Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b00039 • Publication Date (Web): 04 Apr 2017 Downloaded from http://pubs.acs.org on April 6, 2017

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N-Reacetylated Oligochitosan: pH dependence of SelfAssembly Properties and Antibacterial Activity Inesa V. Blagodatskikh1, Sergey N. Kulikov2, 3, Oxana V. Vyshivannaya1, Evgeniya A. Bezrodnykh1, Vladimir E. Tikhonov1* 1

A.N. Nesmeyanov Institute of Organoelement Compounds of Russian Academy of Sciences, Russia, Vavilov st. 28, Moscow, 119991 Russia 2

3

Kazan Federal University, Kremlyovskaya st. 18, Kazan, 420008 Russia

Kazan Scientific Research Institute of Epidemiology and Microbiology, Bolshaya Krasnaya st. 67, Kazan, 420015 Russia

KEYWORDS: chitosan, oligochitosan, acetylation, solution properties, light scattering, antibacterial activity

ABSTRACT: Oligochitosan (short chain chitosan) is more soluble in acidic aqueous media than a high molecular weight (MW) chitosan but its antimicrobial activity decreases with increase in degree of acetylation (DA) and increase in pH above a critical pH threshold point. In the present study, oligochitosans varying in MW were additionally N-acetylated and their self-assembly properties and antibacterial activity towards S. aureus and E. coli were investigated in a wide pH range as a function of MW and DA. Light scattering studies reveals that reacetyleted oligochitosan with Mw ≤ 11 kDa is completely soluble in alkaline media (up to pH 12.5), if its


1

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DA is not less than 16%. Reacetylated chitosans with DA ~ 30% are solubile in the entire pH range up to 12.5, if their Mw are not higher than 25 kDa, but they aggregate and precipitate from the solution at pH ≥ 8 when their Mw was above 25 kDa. Considering the influence of DA and MW, the antibacterial activity of reacetylated oligochitosans is maximal in the short interval of DA 16–28% at pH 7.4. These results are promising for expanding practical application of oligochitosan in pharmaceutical, cosmetic and food compositions.

INTRODUCTION Chitosan (CHI) is industrially produced by a partial or complete deacetylation of chitin [poly(1→ 4)-β-D-N-acetylglucosamine] - a constituent of the cell walls of most fungi and shells of insects and crustaceans, and the term “chitosan” describes a group of unbranched polysaccharides consisting of glucosamine and N-acetylglucosamine (or glucosamine only) and differing in their molecular weight (MW) and degree of acetyl-groups content (DA). As to MW, chitosan might be conditionally categorized depending on MW into a high molecular weight (HMW) chitosan, low molecular weight chitosan, oligochitosan, and chitooligosaccharides (COS), the last two being the products of a deep depolymerization of chitosan. Although the boundaries between these groups are fluid, the term “oligochitosan” (oligoCHI) can be used for molecules with fewer than 100 glucosamine units, i.e. Mw < ~16 kDa.1, 2 Many reviews of the physicochemical properties, applications and antimicrobial activities of chitosan, oligochitosan and COS against bacteria, fungi and viruses have been published so far.1,3–9 In terms of safety, biocompatibility and biodegradability all forms of chitosan are considered

acceptable

and

potential

candidates

for

pharmaceutical

and

biomedical

applications.1,10–16 Also, they show synergistic/additive effect in combination with antibiotics,


2

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which indicates that chitosan and its derivatives can be used to enhance antimicrobial activity of pharmaceuticals.17–20 Consequently, chitosan and its derivatives have found application in biotechnology, medicine, food and cosmetic compositions, and agriculture. Unfortunately, the application of chitosan is restricted in some formulations. The problem is that chitosan dissolves in acidic aqueous media, and its solubility and antimicrobial activity dramatically decreases as pH increases above critical pH (pHc) threshold point at ~6.2–6.621–24 that coincides with pKa value. Low solubility of chitosan and insufficient aggregative stability of its solutions at and above pHc value represents a major limitation of chitosan applicability to some medical formulations.25As it has been shown in some publications, physicochemical properties of chitosan (including its solubility) are mainly determined by three parameters: MW and DA values and acetyl groups (random or block wise) distribution along chitosan chains. Thus, varying these parameters, water solubility of chitosan can be improved.4,24,26,27 As to MW, oligochitosan has several advantages over HMW and COS in common: a) oligochitosan is more soluble in aqueous media and forms solutions of much lower viscosity than HMW chitosan; b) it has better absorption profiles and is more compatible with technologies and components of consumer goods; c) oligochitosan has much higher antimicrobial activity in comparison with lowest COS having Mw < 2 kDa.1,4,7,8,15,17,28–37 Although below pHc threshold point oligochitosan is more soluble than HMW chitosan, it has an insufficient solubility in neutral and alcaline media as well.38 The aggregative stability of its aqueous solution above pHc point is low too, and an increase in solution pH over the point results in a decrease of oligochitosan antimicrobial activity too.35,39–41 There are some strategies to improve the solubility of all forms of chitosan in neutral and alkaline media. The one usually used represents classic chemical derivatization (e.g.


3

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carboxymethylation, sulfatation, quaternization etc) of chitosan. The derivatization makes chitosan soluble in wider pH range but leads to deep altering or losing of its original physicochemical and biochemical properties. Another strategy to increase the solubility includes preparation of a half-N-acetylated (DA ~ 50%) chitosan by either alkaline deacetylation of chitin, the process hard to control, or partial N-acetylation of a highly deacetylated chitosan by acetic acid anhydride in homogeneous conditions, i.e. N-reacetylation of chitosan up to DA value (up to DA ~ 45–55%) at which a half-acetylated chitosan becomes soluble and forms a “clear solution” in wider pH range.38,42–46 Unfortunately, an increase in DA leads to a significant decrease of antimicrobial activity of all forms of chitosan soluble in acidic aqueous media.47–49 An uncertainty in optimal DA values desired to maintain an optimal MW-DA-solubilityactivity relationship of chitosan in alkaline aqueous media requires additional studies of associative properties, solubility and antibacterial activity of N-reacetylated oligochitosan (RAoligoCHI) samples differing in MW in aqueous solution since so far these studies have been carried out for chitosan with Mw >70 kDa only.13,21,43,44,50–58 To make matter worse, selfaggregative properties of reacetylated oligochitosan in aqueous solution have been studied very obscurely.38,44,45 As a result of previous investigations, it has been shown that a random distribution, but not block wise, of acetyl groups along chitosan chains is required for a halfreacetylated chitosan to be completely soluble at neutral and slightly alkaline pH values. Indeed, the development of a water-soluble oligochitosan with an optimal DA value is a prerequisite to its successful application in wider pH range. However, the fundamental information that has been reported until now is scanty. Considering further application of acid-free-water soluble oligochitosan as the additive for food, pharmaceuticals and skin care products, some additional


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investigations are required. Among them, self-assembly properties and antimicrobial activity of reacetylated oligochitosan in neutral and alkaline aqueous media should be considered in details. The main objective of this study was to prepare a series of N-reacetylated oligochitosans varying in MW and DA, investigate their self-assembly properties in a wide pH range and determine the pH range of solubility as a function of MW and DA together with correlation between the solubility and antibacterial activity towards S. aureus and E. coli in acidic and sligtly alkaline aqueous media. MATERIALS AND METHODS Two HMW chitosan samples were the product of BIOPROGRESS (Russia). All other reagents and solvents were the chemical grade products of SIGMA-ALDRICH. Preparation of Parent Chitosan and Oligochitosan Hydrochloride Samples. The parent CHI and oligoCHI hydrochloride samples used for preparation of RA-CHIs and RA-oligoCHIs were obtained by hydrolysis of HMW chitosan (the sample with Mw 334 kDa, DA 4%) in 1 M hydrochloric acid solution at 70°C for 4–24 hr in the accordance with the earlier published protocol.32 In addition, two oligoCHI hydrochloride samples were prepared by hydrolysis of other HMW chitosan (Mw 870 kDa, DA 20%) with hydrogen peroxide following the protocol published in the literature.59 Preparation of RA-oligoCHI and RA-CHI Samples. The samples were prepared by the modified method described in.60 Shortly: CHI or oligoCHI hydrochloride (1 g) was dissolved in deionized water (25 mL), and the solution pH was ajusted to pH 6.0 with 0.1 M sodium hydroxide solution at intensive stirring. The resultent solution was diluted with equial volume of methanol and cooled to 20°C. A required quantity of acetic acid anhydride was added to the solution at intensive stirring. In 1 hr, concentrated aqueous ammonia (5 mL) was added to the


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solution. The mixture (solution or suspension) was completely dialyzed against deionized MilliQ water using 1 kDa (D2272) or 12 kDa (D9777) SIGMA dialyzing tubes for some days and then lyophilised after the adding of 1 mL of concentreted hydrochloric acid. All sample hydrochlorides were additionally dried twice under vacuum: over solid NaOH and then over solid P2O5. The product yield was 0.90–0.95 g; the residual water content determined by Fischer’s method was found to be 9.0–9.3%; molar relation Cl/N = 0.98 (microanalytical data). The samples used were practically completely (≥ 99.9%) soluble in deionized Milli-Q water (solubility test for “Chitosan hydrochloride”, European Pharmacopeia 5.0, pp. 1248–1249). Degree of Acetylation (DA, mol. %). DA values were determined by 1H-NMR method.61 High Performance Size Exclusion Chromatography (HP SEC). Agilent 1200 Series Chromatography system equipped with an isocratic pump, refractive index detector, and PL-OH mixed column was applied for HP-SEC. Buffer solution (0.3 M CH3COOH/0.225 M CH3COONH4, pH 4.5) was filtered through a 0.22 µm hydrophilic polyvinylidene fluoride Durapore membrane, degassed and afterwards used as the eluent at flow rate of 0.8 mL min−1. The column and detector temperatures were kept at 25°C, injection volume was 50 µL, and sample concentration was 0.001 g mL−1. A series of monodispersed pullulans (Fluka) with Mp from 1.08 to 710 kg mol−1 were used as calibration standards. Light Scattering. SLS and DLS measurements were performed using a PhotoCor Complex spectrometer (PhotoCor Instruments, Russia) equipped with a He–Ne laser (λ = 633 nm, 10 mW) as the light source and a pseudo cross-correlation system of photon counting. The real-time correlator was employed in the logarithmic configuration. Measurements were performed in dilute solutions at 25°С and scattering angle 90°. Distributions over decay time and hydrodynamic radius were obtained by means of a nonlinear regularized inverse Laplace


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transform method (CONTIN). Apparent self-diffusion coefficients D were determined for each diffusive mode in accordance with the relation D = 1/τq2, where q = (4πn/λ)sin(θ/2) is the wave vector magnitude, and τ is the relaxation time. The corresponding hydrodynamic radii Rh were calculated from Stokes-Einstein equation: Rh = kT/6πηD, where k is Boltzmann’s constant, η is the solvent viscosity. The pH dependencies of LS intensities and Rh values were studied as follows: a sample hydrochloride was dissolved in either 0.1 M acetic acid or in TRIS/HCl buffer (pH 6.0) containing 0.15 M NaCl at concentration of 3–5 mg mL−1 solution. The solution was filtered throw a membrane filter Durapore 0.45 µm into a glass cuvette, and it was titrated with 1 M TRIS solution as a titrant under pH control by a pH-meter (Mettler-Toledo FE20 with microelectrode InLab Micro Pro) up to pH 8.0. At pH > 8, 1 M NaOH was used as a titrant. LS intensity and autocorrelation function were measured at angle 90° after addition of each portion of the titrant. The inflection points of LS intensity vs pH were considered as pHc values. Minimal Inhibitory Concentration (MIC) Assay. The E. coli ATCC 25922 and S. aureus ATCC 35591 bacterial strains were obtained from the American Type Culture Collection. A stock RA-oligoCHI hydrochloride solution in distilled water was filtered through a 0.22-µm PVDF filter (Millipore) and stored at 4°C until needed. The MICs of the samples were determined by a microdilution assay in accordance with the modified method described in the literature.62 All experiments were carried out in triplicate. The MIC (mean value ± 10%) was defined as the lowest concentration required suppressing cells multiplication. RESULTS AND DISCUSSION Samples Preparation and Characterization. Industrial deacetylation of chitin is carried out in heterogeneous condition (solid state chitin in 30–50% aqueous sodium hydroxide solution)


7

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and leads to an irregular (blockwise) distribution of residual acetyl groups along chitosan chains due to semi-crystalline character of the initial chitin so that the industrially produced chitosan usually has DA 1–30% and MW varying in a wide range. Generally, solubility is the property of a substance to dissolve in a solvent to form a stable homogeneous solution and is usually expressed in molar units or mass units per volume. From this point of view, basic form of chitosan is insoluble in water. Chitosan solution is usually prepared in water in the presence of acetic, hydrochloric, lactic, or glycolic acid. Organic or inorganic acid converts chitosan in aqueous media to the corresponding chitosan salt and makes it water soluble. It has been shown that the molar amount of acid required for the dissolution is as much as needed for protonation of ≥ 60% chitosan amino groups. In fact, solubility and chitosan salt solution viscosity and stability in aqueous media is the manageable parameters dependent on MW, DA, solution pH, and ionic force mainly4. In order to improve the solubility and reduce chitosan solution viscosity, a variety of depolymerization technologies are usually used to reduce molecular weight of chitosan, but some of them lead to changing its chemical structure. In this work, three series of oligoCHI samples differing in MW and DA were prepared and used in light scattering and microbiological studies. In order to prepare the first series of parent oligoCHI samples differing in MW, we used the most suitable method for decomposition of HMW chitosan, namely the depolymerization of HMW chitosan (Mw 334 kDa, DA 4%) by hydrochloric acid. This method was chosen since a) its impact on the chemical structure of oligochitosan is minimal in comparison with the other methods; b) it requires the usage of neither an expensive purified form of chitinase/chitosanase enzyme or a cheaper complex of these enzymes, whose usage, in turn, requires separation of oligochitosan from the enzymatic dirt and leads to enzyme-type dependent antimicrobial activity of oligochitosan.33 The method of


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depolymerization of chitosan in hydrochloric acid solution is more specific to break up the glycosidic bonds between block-conjunct N-acetylglucosamine units rather than between Nacetylglucosamine-glucosamine or glucosamine-glucosamine units, and it doesn’t modify chitosan chemically.63 Partial N-deacetylation was also observed during the hydrolysis and as a result the degrees of acetylation of oligochitosans obtained after the acidic hydrolysis were lower than that of the parent chitosan in accordance with the previously published data.64 At this stage, a parent series of oligoCHI and CHI hydrochlorides with the content of residual acetyl groups of 1–2% but differing in MW values were prepared and afterwards used for the preparation of RAoligoCHI and RA-CHI samples. The reaction of reacetylation was performed by means of acetic anhydride as an acetylation agent at ambient temperature in homogeneous water/methanol medium in order to induce a random distribution of acetyl groups along the polymer chains.61 Consequently, this series included the samples with Mw 7–121 kDa and DA 10–35% (Table 1). In the effort to evaluate the influence of DA distribution on oligoCHI self-assembly properties, two oligoCHI samples were prepared by hydrolysis of another HMW chitosan (MW 870 kDa, DA 20%) with hydrogen peroxide. This method has been shown to be a site-specific towards a preferable

cleavage

of

glycosidic

bonds

between

glucosamine-glucosamine

units.59

Consequently, the oligoCHIs with Mw 10 or 12 kDa and blockwise distributed acetyl-groups (DA 6 or 9%) were prepared and used as such, i.e. without additional N-reacetylation, for comparative SLS-DLS studies. After the preparations, all samples were analyzed by HP-SEC and 1H-NMR methods to determine their molecular characteristics: weight average (Mw) and number average (Mn) molecular weights, polydispersity index (Pi = Mw/Mn) and DA shown in Table 1.


9

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Table 1. Dependence of pHc points of RA-oligoCHIs, oligoCHIs and RA-CHIs on DA and MW. pHc b) point Sample name

Mw, kDa

Pi

DA, %

Rhaggr±1, a) nm

Tris/HCl+ 0.1 M AcOH 0.15 M NaCl

RA-oligoCHIs RA-oligoCHIs-1

9±1

2.1

10

65

7.4

7.3

RA-oligoCHIs-2

10 ± 1

1.6

16

79

8.0

8.2

RA-oligoCHIs-3

10 ± 1

1.9

23

93

absent c)

absent c)

RA-oligoCHIs-4

7±1

2.1

28

61

absent c)

absent c)

RA-oligoCHIs-5

9±1

2.1

35

75

absent c)

absent c)

RA-oligoCHIs-6

11 ± 1

2.1

35

80

absent c)

absent c)

OligoCHIs OligoCHIs-1

10 ± 1

1.9

1

68

6.4

6.4

OligoCHIs-2 d)

10 ± 1

2.1

6

35

6.5

6.4

OligoCHIs-3 d)

12 ± 1

2.3

9

40

6.7

6.6

RA-CHIs RA-CHIs-1

121 ± 5

3.2

32

130

7.4

7.5

RA-CHIs-2

57 ± 2

3.5

27

130

7.9

8.0 e)

RA-CHIs-3

25 ± 2

2.6

28

125

absent

absent

a)

Rh of aggregates after dissolution in 0.1M AcOH

b)

Determined at concentration of 5 mg mL−1 (RA-oligoCHIs) or 3 mg mL−1 (RA-CHIs and oligoCHIs); c)

Titration with 3 M TRIS up to pH 8.5 followed by titration with 1 M NaOH up to pH 12.5;

e)

Stable colloidal solution (particles with Rh 275 nm) was observed at pH > pHc in a week;

d)

The sample was prepared by HMW hydrolysis with hydrogen peroxide.


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Solution Properties of RA-oligoCHIs. Oligochitosan chains include neutral (hydroxyl groups), cationic (charged amino groups), and hydrophobic (acetyl groups) sites. Inter- and intrachain interactions of these groups in the presence of counterions lead to formation of reversible junctions so that a diversity of aggregates and clusters can be found in oligochitosan solution. The aggregation phenomenon occurring in solutions of oligochitosan was demonstrated by dynamic and static light scattering (DLS and SLS) techniques,32,40,41 where it was shown that hydrodynamic radii of aggregates Rhagg were virtually independent of solution pH below pHc value despite a continuous variation in degrees of ionization. At pHc value, a macro-phase separation of oligochitosan/water system to a gel phase and supernatant was started so that a coexistence of two aggregation modes (primary aggregates and their clusters) was observed by DLS studies. These studies of self-assembling effects occurring in oligoCHI and RA-oligoCHI solutions at neutral and basic pH values might be of fundamental interest not only for accurate samples characterization, but also for practical application of oligochitosan in medicine and biotechnology since this phenomenon can affect not only its solution properties but its biological properties as well. Indeed, in order to thoroughly understand practical applicability of RAoligoCHIs their molecular heterogeneity and self-assembling properties should be considered too. In this research, self-assembly properties of a series of RA-oligoCHIs and oligoCHIs (Mw from 7 to 12 kDa) varying in DA values from 1 to 35% were studied in solution as a function of pH by SLS-DLS method. The pHc values were estimated by titration of sample solutions of various ionic strength (0.1 M AcOH and 0.15 M NaCl). The inflection points, where LS intensity and Rh started to grow dramatically were considered as pHc (Figures 1a, 1b). The corresponding pHc points of this series of oligochitosans with various DA are listed in Table 1. The results obtained


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clearly show that RA-oligoCHIs with Mw 7–11 kDa and DA ≥ 23% have no phase separation threshold point and do not demonstrate essential changes of scattered intensity and Rh in the entire tested pH range up to 12.5, and the solvent salinity does not virtually influence the pHc values.

50 40

1 2 3 4

a

30 20

I/I0

10 4 3 2 1 2

3

4

5

6

7

8

9

10

11

12

13

14

pH

5000 4000

1 2 3 4

b 3000 2000

Rh, nm

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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300 250 200 150 100 50 2

3

4

5

6

7

8

9

10

11

12

13

14

pH

Figure 1. pH-dependence of scattered light intensity (a) and hydrodynamic radius (b) of oligoCHI-1 (1) and RA-oligoCHIs varying in DA: RA-oligoCHI-1 (2), RA-oligoCHI-2 (3), RAoligoCHI-5 (4) solutions in 0.1 M AcOH/NaOH solutions.


12

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As it follows from the DLS data, RA-oligoCHIs and RA-CHIs in solutions are partially aggregated: the main peak (the intensity impact 92–97%) belongs to the aggregates, whose sizes (Rh 61–130 nm) are virtually independent of DA and pH in the single-phase regions (Table 1, Figure 1). The examples of size distributions in the sample solutions are shown in Figure 2.

1.2

1 2

0.8

I/Imax

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0.4

0.0

10

-1

10

0

1

10

10

2

3

10

10

4

Rh, nm

Figure 2. Distributions of scattered light intensity (angle 90°) over hydrodynamic radius of RAoligoCHI-5 (1) and RA-oligoCHI-2 (2) solutions in 0.15 M NaCl at pH 7.5. Taking into account that the intensity of scattered light can be written as I ~ cM ~ cRx [where: c is mass concentration, M is molecular mass (or mass of aggregate), and the index x varies from 1 to 3 depending on macromolecule conformation (or particle structure)], it is clear that the large impact of aggregates, whose radii are about 30 times as large as the radius of the macromolecule, on the scattering intensity corresponds to their mass fraction of about 1% in the sample solution. The trend of chitosan of various MW to aggregate in aqueous solutions has been well documented and explained by interchain hydrophobic interactions and hydrogen bonding.41,54,65


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It can be concluded from this study that the aggregation ability of RA-CHIs in solutions with pH below pHc point is their inherent property, and, in this sense, RA-oligoCHIs are similar to the parent oligoCHIs of low DA.40,41 For comparison, the pHc values of oligoCHI-3 (Mw 12 kDa, DA 9%) and oligoCHI-2 (Mw 10 kDa, DA 6%) alternatively prepared by degradation of HMW chitosan (Mw 870 kDa, DA 20%) with hydrogen peroxide were determined as well. The corresponding critical points pHc 6.4–6.7 (Table 1 and Figure 3) of these samples were found to be much lower, than pHc of RA-oligoCHI-1 having close Mw and DA values, and almost as low as that of the sample oligoCHI-1 with Mw 10 kDa and DA 1%. This fact may be related to the difference in the type of N-acetylated units distribution along the chains: the products of HMW chitosan degradation by hydrogen peroxide may preserve some block-wise distributed acetyl groups originated from the parent HMW chitosan,59,66,67 whereas the homogeneous Nreacetylation leads to a statistical distribution of N-acetylglucosamine units in chitosan molecules.27,38,43–46,55

70 60

a

50 40

I/I0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

30

1 2

20 10 0 4.5

5.0

5.5

6.0

6.5

7.0

7.5

pH


14

Biomacromolecules

2000

b

1750 1500 1250

Rh, nm

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1000

1 2

750 500 250 0 4.5

5.0

5.5

6.0

6.5

7.0

7.5

pH

Figure 3. pH-dependence of scattered light intensity (a) and hydrodynamic radius (b) of oligoCHI-2 (1) and OligoCHI-3 (2) in 0.1 M AcOH/NaOH solutions. The effect of chain length of RA-CHI and RA-oligoCHI on the pH range of solubility was studied using a series of samples with DA about 30% varying in Mw from 7 to 121 kDa (Table 1). As it was found, the content of acetyl groups in the range of 28–35% in the samples with Mw up to 25 kDa made them soluble in entire pH range (up to pH 12.5). As to other RA-CHIs with higher MW values, the study showed that their pHc points reduced with the increase in their Mw (Table 1). In addition, the aggregative ability of RA-oligoCHIs and RA-CHIs in solutions with pH 7.4 (0.15 M NaCl in 0.1 M TRIS/HCl) was examined by SLS-DLS method throughout a month. In contrast to the prepared by chitin deacetylation partially acetylated HMW chitosans, capable of fast ageing in solution and precipitation from the solution,51,53,54,67 RA-oligoCHI-5 and RAoligoCHI-6 solutions (DA 35%) were shown to be invariable during the observation period. The other RA-oligoCHIs of lower DA (16 and 23%), or RA-CHIs of higher Mw (25–57 kDa), on the


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other hand, demonstrated the phase separation in 1–2 weeks of the storage (data not shown). The sample RA-CHI-1 (Mw 121 kDa, DA 32%) showed a very short storage time partially precipitating in a day of the storage even in a more diluted (c = 1 mg mL−1) solution. To conclude, the random (statistical) N-reacetylation of about 30% of monomer units make the samples with Mw ≤ 50 kDa soluble in alkaline media. Earlier,44,45 pH range of water solubility of half-reacetylated oligochitosans was studied by turbidimetry technique that referred to the measurement of the transmitted light at the same wavelength and direction as the incident beam. In this report, we have used LS technique which is more precise and informative in comparison with turbidimetry, to detect the range of oligochitosan solubility. In this aspect, the results of our investigation are superior to the earlier results that showed that the DA range of water-solubility of N-reacetylated chitosans (Mw 8.8–60 kDa) prepared by the oxidative degradation of HMW chitosan (Mw 600 kDa, DA ~ 25%) with perboric acid was observed in the vicinity of DA ~ 50%. Other samples with DA below 40% and above 60% failed to give clear aqueous solutions in the entire pH range, and the solutions were not completely transparent at pH 6–12 indicating that the samples were not completely soluble under neutral and alkaline conditions. Besides, it should be mentioned here that our results were only in a partial agreement with the results published in the work,38 where the solubility of four partially acetylated HMW (Mw 164– 310 kDa) chitosans (DA 1, 17, 37, and 60%) was investigated as a function of pH. It was shown that all chitosans precipitate between pH 6.0 and 7.5. A peculiarity of this investigation was that after the reacetylation three chitosan samples with lowest DAs were depolymerized by the treatment with nitrous acid. This reaction was very selective and nitrous acid attacked exclusively glucosamine units of chitosan thus reducing the chitosan molecular weight via a


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cascade of reactions.68,69 After the treatment, solubility of the obtained oligochitosans in water at pH 7.5 was determined. In agreement with our results, the most deacetylated oligochitosan (DA 1%) was insoluble at pH 7.5, while the most acetylated oligochitosan (DA 60%) was completely soluble at all pH values. Surprisingly, it was found that all the other oligochitosan samples could be separated into soluble and insoluble fractions at pH 7.5 depending on their DA values. The oligochitosans with Mn 2.5–5.1 kDa were insoluble at DA 12–14%, while being soluble at DA 25–30%. On the other hand, the samples with Mn 9–48 kDa were found insoluble even at DA 34–37% though becoming soluble at DA 44–46%.38 It should be mentiond in addition that pH behavior of a series of reacetylated HMW chitosans (Mw 128–188 kDa) varying in DA was studied by SLS method as well. As it was shown, critical pH values increased from 6.15 to 7.5 for chitosans having a DA equal to 1 and 51%, respectively, and higher acetyl content led to the formation of a dispersion of small aggregates whose size was not pH dependent.52 As to our experimental results, they demonstrate that the reacetylation of oligochitosan in homogeneous conditions seem to be a more acceptable method for preparation of partially acetylated oligochitosan soluble in a wide pH range (including not only physiological values but alkaline pH ones as well) in comparison with the other methods. Moreover, in the present study, we show that RA-oligoCHIs (Mw ≤ 11 kDa) are completely soluble in alkaline media (up to pH 12.5) if their degree of reacetylation is not less than 16%. In addition, it is demonstrated that the samples with DA ~ 30% do not lose their solubility in the entire pH range up to 12.5, if their Mw values are not higher than 25 kDa, but they start to aggregate and precipitate from the solution at pH ≥ 8 when their Mw > 25 kDa.

These results are promising for expanding practical

oligochitosan application since they allow increasing its pH range of solubility up to alkaline pH values.


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Antimicrobial Activity of RA-OligoChis. As a result of many investigations, it is commonly accepted that antimicrobial activity of various types of chitosan depends on its MW and DA, target microorganism, and experimental conditions (solution pH, concentration, incubation medium, method of chitosan sample preparation, etc). In this paper we will not touch the mode of antimicrobial action of chitosan/oligochitosan. Nevertheless, it should be mentioned that the variations in MW and DA leads to two different mechanisms of chitosan/oligochitosan and target microorganism interaction: the first one includes adsorption of oligochitosan onto cell wall leading to cell wall covering, membrane weakening, disruption, and cell leakage, and the second one consists in penetration of chitosan/oligochitosan into a living cell leading to the inhibition of various enzymes and interfacing with the synthesis of mRNA and proteins. These modes of action were determined for living microbial cells, confirmed for artificial model using a Langmuir monolayer technique and discussed in some papers70–75 and reviews.1,6,9 Here, one should take into account that chitosan is usually produced by deacetylation of chitin. The deacetylation is carried out under heterogeneous conditions and results in block-wise distribution of residual N-acetyl-D-glucosamine units. Oppositely, N-reacetylation of chitosan with a very low content of residual acetyl groups leads to a random distribution of acetyl groups along polymeric chains. Consequently, these two forms of chitosan differ in their pHc thresholds and solubility possessing a variable antimicrobial activity, in spite of the chitosan samples being able to have close MW and DA values. For the ﬁrst time,45 the influence of DA and MW parameters of N-reacetylated chitosans on their antimicrobial activity were studied independently. As it was shown, all homogeneously reacetylated chitosan samples having a wide range of DA (2, 12, 24, 41, 56, and 61%) and Mw (from 42.5 to 135 kDa) were soluble in acidic conditions in the range of pH 4.4–6.1, and regardless of the pH, the samples with lower DA (2–


18

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12%) had higher activity in this pH range than more acetylated ones. These results showed that antibacterial activity of chitosan increased with decreasing DA, MW and pH, and the activity was stronger against Gram-negative E.coli than that towards Gram-positive S. aureus cells. Unfortunately, the activity was not determined at higher pH or lower MW values.45

pH 6.0 pH 7.4 1024

a

512 256

MIC, µg/ml

128 64 32 16 8 4 2 1

9 10

16

23

28

35

DA, mol.%

pH 6.0 pH 7.4 1024

b

512 256 128

MIC, µg/ml

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64 32 16 8 4 2 1

9 10

16

23

28

35

DA, mol%

Figure 4. Dependence of bacteriostatic activity of oligoCHIs-1, 3

and RA-oligoCHIs-1–5

towards E.coli (a) and S. aureus (b) on DA at pH 6.0 or pH 7.4.


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In this work, the susceptibility of E. coli and S. aureus cells toward RA-oligoCHIs having Mw 7–11 kDa in comparison with oligoCHI samples with closely related MW values but differening in aggregative properties was tested in vitro as well. The MIC values of the samples were determined both in acidic (pH 6.0) and slightly alkaline (pH 7.4) solutions (Figures 4a, 4b). The study found that the antibacterial effect of oligoCHIs and RA-oligoCHIs was dependent on DA values and acetyl groups distribution (more precisely, on the method of a partially acetylated oligochitosan preparation that, in turn, determined acetyl groups distribution). As to the influence of DA, our studies revealed the 2–8-fold reduction of bacteriostatic activity of oligoCHI and RAoligoCHIs at pH 6.0 with the increase in DA and confirmed, basically, the former results that antibacterial effect of chitosan towards Gram-negative and Gram-positive bacteria reduced with the increase in DA.47–49 On the other hand, the variable impact of DA was observed on antibacterial activity of RA-oligoCHIs at pH 7.4. Thus, the difference between the MIC values at pH 6.0 and pH 7.4 was found to be much less for RA-oligoCHIs with the DA of 16% or bigger (i.e. for those which had no phase separation in alkaline media). This effect is common for both types of the microorganisms. Considering the MIC values of antibacterial activity achieved in a weakly acidic or slightly alkaline media, the range of optimal acetyl groups level was found to be from 16 to 28%. Actually, among main experimental conditions, solution pH plays the most important role determining positive charge of oligochitosan molecules, solubility and, as a consequence, antimicrobial activity of oligochitosan. An increase in solution pH leads to decrease in the oligochitosan amino groups protonation and simultaneous decrease in antimicrobial activity of chitosan.6,32,33,35,41 As it is mentioned above, an increase in solution pH over a pHc value leads to a decrease in solubility and antimicrobial activity of low acetylated oligochitosans, especially at alkaline media. As a result of reacetylation, the increase in DA from


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10 to 23% causes a simultaneous increase in pHc (Table 1) and solubility that, in turn, led to 8– 16-fold increase in activity at pH 7.4. But further increase in DA from 23% to 28–35% led gradually to decreased activity (Figures 4a, 4b). Thus, an optimal DA value required for a better DA/solubility/activity relationship seems to be in the interval from 10 to 28%. Summing up, N-reacetylation of oligochitosan provides an increase in both solubility and antimicrobial activity in aqueous media only in the short DA interval of 16–28%. CONCLUSIONS As a result of all previous investigations and our resent data, we have to state that aggregation of reacetylated oligochitosan molecules in solution is their inherent property regardless of their molecular weight but the tendenc for aggregation depends on the degree of acetylation and mode of acetyl groups distribution. In this study we use a reproducible method for preparation of a partially acetylated oligochitosan by N-reacetylation of a highly deacetylated oligochitosan with acetic acid anhydride at homogeneous conditions. DLS-SLS studies and antibacterial assay demonstrate that the solubility and bacteriostatic activity of oligochitosan in neutral and alkaline aqueous media, where a parent oligochitosan is less soluble, can be increased after its partial Nreacetylation so the that the reacetyleted oligochitosan is completely soluble in alkaline media, if its DA is not less than 16%. Antimicrobial tests demonstrate that the antibacterial activity of reacetylated oligochitosans is maximal in the short interval of DA 16–28% at pH 7.4. These effects are more pronounced for oligochitosans with Mw ~10 kDa and less. Unfortunately, comparison with earlier literary experimental data is difficult because of the extreme diversity in the reported antimicrobial activity/MW/DA dependences obtained for a wide variety of oligochitosans prepared by different chemical or enzymatic methods and determined in different experimental conditions. Moreover, the influence of acetyl group distribution on antimicrobial


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activity was not examined in details yet. However, such a study would provide a unique insight into the dependence of these two factors (degree of acetylation and distribution of acetyl groups) on antimicrobial activity of different oligochitosans. Given that a partially N-acetylated chitosan can be splitted in human body by some specific (lysozyme, chitotriosidase) and unspecific (lipase, α-amylase etc) enzymes,1,11,13 which exist in various human body fluids and tissues and convert chitosan to lower chitooligosaccharides (glucosamine and N-acetylglucosamine) and afterwords digested, the results of the present investigation are of fundamental interest for practical applications of oligochitosan in medical, cosmetic and food compositions. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]; Phone: +7 (499)1359375; Fax: +7 (499)1355085; Fax: +7-4991355085; Tel.: +7-4991359375 Postal address: 119991, V-334, Moscow, Vavilova st. 28, INEOS RAS ACKNOWLEDGMENTS The authors thank the Russian Science Foundation for supporting of the research of synthesis and antimicrobial activity by the project RSF 16-14-00046. REFERENCES (1) Aam, B.B.; Heggset, E.B.; Norberg, A.L.; Sørlie, M.; Vårum, K.M.; Eijsink, V.G.H. Mar. Drugs 2010, 8, 1482–1517. (2) Tikhonov, V. cross-citation: https://www.researchgate.net/publication/286865133.


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N-Reacetylated Oligochitosan: pH Dependence of Self-Assembly

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