Kinetics and Mechanisms of Chemical and Biological Agents Release

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Kinetics and mechanisms of chemical and biological agents release from biopolymeric microcapsules Marko Vincekovic, Slaven Juri#, Edyta #ermi#, and Snježana Topolovec-Pintari# J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04075 • Publication Date (Web): 12 Oct 2017 Downloaded from http://pubs.acs.org on October 13, 2017

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Journal of Agricultural and Food Chemistry

Kinetics and mechanisms of chemical and biological agents release from biopolymeric microcapsules

Marko Vinceković1*, Slaven Jurić1, Edyta Đermić2, Snježana Topolovec-Pintarić2 Department of Chemistry1, University of Zagreb Faculty of Agriculture, 10000 Zagreb Croatia Department of Plant Pathology2, University of Zagreb Faculty of Agriculture, 10000 Zagreb Croatia

KEYWORDS: agrochemicals, microcapsules, copper, Trichoderma, sustainability

ACS Paragon Plus Environment


1

ABSTRACT: Kinetics and mechanisms of copper cations and Trichoderma viride spores release

2

from uncoated and chitosan coated alginate microcapsules were investigated. The gelation of a

3

fixed amount of sodium alginate at different concentrations of copper ion solutions resulted in

4

distinct kinetics and release mechanisms. The increase in copper cation concentration promoted,

5

but the presence of the chitosan layer on microcapsule surface and increase in microcapsule size

6

reduced the rate of active agent release. Fitting to simple Korsmeyer–Peppas empirical model

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revealed the underlying release mechanism (Fickian diffusion or a combination of the diffusion

8

and erosion mechanisms) depends on copper cation concentration and presence of T. viride

9

spores. The investigation pointed out that proper selection of formulation variables helps in

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designing microcapsules with the desirable release of copper ions and T. viride for plant

11

protection and nutrition.

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1. INTRODUCTION

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The encapsulation of agrochemicals has been developed as a tool for ecological and sustainable

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plant production’s The benefits conferred by encapsulation include a slow release of a bioactive

15

ingredient, more efficient exploitation of the chemicals used, greater safety for the user, and

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better protection of the environment.2 Biopolymer based capsules with a single active agent have

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extensive applications in agriculture and become one of standard formulation techniques. One of

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the most common materials used for the encapsulation are biopolymers such as alginate

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microcapsules coated with a layer of chitosan.

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Plants need food for their growth and development which are supplied from the atmosphere and

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soil water as well as soil minerals and soil organic matter. Nutrients behave differently in the soil

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and undergo complex changes greatly influenced by the soil pH and microflora and that


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ultimately affects their accessibility to plant roots for absorption. Thus, microbial interactions

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with plant roots are known to profoundly affect plant nutrient status and to affect plant resistance

25

to pathogens. Numerous microorganisms, especially those associated with roots, have the ability

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to increase plant growth and productivity. Trichoderma species are among the almost prevalent

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culturable fungi in soils, based upon the frequency of isolation on suitable media. The dual roles

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of antagonistic activity against plant pathogens and promotion of soil fertility make Trichoderma

29

species a promising alternative to standard plant protection and nutrition technologies.3

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Copper cation is an essential micronutrient for plants and it is used for the management of wide

31

range of fungal and bacterial diseases in various crops. It plays key roles in photosynthetic and

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respiratory electron transport chains, in ethylene sensing, cell wall metabolism, oxidative stress

33

protection and biogenesis of molybdenum cofactor. Thus, a deficiency in the copper supply can

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alter essential functions in plant metabolism. Copper has traditionally been used in agriculture as

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an antifungal agent, and it is also extensively released into the environment by human activities

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that often cause environmental pollution. However, excess copper may be potentially toxic to

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plants, causing phytotoxicity by the formation of reactive oxygen radicals that damage cells, or

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by the interaction with proteins impairing key cellular processes, inactivating enzymes and

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disturbing protein structure. It is worth noting that controlled copper release achieved by

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encapsulation in biopolymer matrices maybe a better alternative to traditional application of

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copper formulation for plants protection.4

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In the previous study, we have demonstrated the possibility of simultaneous encapsulation of

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chemical and biological agents in chitosan/alginate microcapsules.5 Due to benefits for crop

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protection and nutrition as well as high compatibility, T. viride spores and copper ions were taken

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as a suitable combination of chemical and biological agents. Experiments performed on T. viride



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spores survival revealed that during storage at the room temperature the encapsulation of T. viride

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spores in the same compartment with the copper cation does not inhibit their activity.

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Characterization of prepared microcapsules showed high copper cation encapsulation efficiency

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and loading capacity as well as good release behavior from microcapsules.

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Controlled release, that is a successful delivery of active agents at the right place and the right

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time, is an attainable and desirable characteristic for all bioactive agent delivery systems. To

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obtain the well-designed microcapsules efficient for simultaneous encapsulation and controlled

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release, it is important to optimize parameters during microcapsule preparation. The aim of the

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present work was to investigate the effect of copper cation concentration, microcapsule size and

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presence of chitosan layer on the release of both, copper cation and T. viride spores from alginate

56

microcapsules with the intention of delivering active agents to plants at the rate that closely

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approximates plant demands over an extended period.

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2. MATERIALS AND METHODS

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2.1. Materials. Low viscosity sodium alginate (ALG) (CAS Number: 9005-38-3; Brookfield

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viscosity 4 - 12 cps (1% in H2O at 25 oC)) was purchased from Sigma Aldrich (USA). Low

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molecular weight chitosan (CS) (CAS RN: 9012-76-4, molecular weight: 100000–300000) was

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obtained from Acros Organic (USA). A commercially available product copper sulfate

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pentahydrate, CuSO4 · 5H2O was used as a copper cation donating substance (Kemika, Croatia).

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All other chemicals were of analytical grade and used as received without further purification.

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An indigenous isolate of T. viride originated from parasitized sclerotia of Sclerotinia

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sclerotiorum was used in all experiments.3 To obtain spore suspensions, the T. viride was grown

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in Erlenmeyer flasks containing potato dextrose broth.5


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2.2. Microcapsule preparation. Microcapsules without chitosan layer (uncoated microcapsules)

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were prepared by ionic gelation while those with chitosan layer (coated microcapsules) were

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prepared in two stages (ionic gelation followed by polyelectrolyte complexation) as was

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previously described.5 Ionic gelation involves the preparation of alginate core microcapsules

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loaded with copper cations (sodium alginate solution dropped into copper cations solution) or

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with copper and T. viride spores (a mixture of sodium alginate and T. viride spores dropped into

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copper cations solution), and polyelectrolyte complexation includes a coating of alginate core

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microcapsules by chitosan. The concentration of sodium alginate (1.5 %) and chitosan (0.5% CS

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in 1.0% CH3COOH), as well as the size of microcapsule) were kept constant, while initial copper

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cations concentration was 5, 10 and 15 mmol dm-3. The microcapsule diameter was determined

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by using nozzle size of 0.45 or 2 mm, respectively (Encapsulator Büchi-B390, BÜCHI

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Labortechnik AG, Switzerland). It must be mentioned that uncoated alginate microcapsules

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represent a monolithic system (bioactive agents are more or less homogeneously distributed

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through matrix), while coated microcapsules represent core-shell microcapsules (bioactive agents

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are located in the alginate core).

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2.3. Determination electrostatic charge and zeta potential of T. viride spores. The

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electrostatic charge and zeta potential (ζ/mV) of T. viride spores suspension was measured by

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Zetasizer Nano ZS (Malvern, UK) which measures electrophoretic mobility based on laser

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Doppler particle electrophoresis. The zeta potential was estimated from electrophoretic

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measurements using Henry equation:

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Ue = 2ε ζ (fκa/3η)

(1),



89

where ζ is the zeta potential, ε is the dielectric constant, Ue is the electrophoretic mobility and η is

90

the viscosity. fκa is in this case 1.5 and is referred to as the Smoluchowski approximation.7

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Deviations ranged within ±1 mV. All measurements were made at room temperature and in

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triplicate.

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2.4. Encapsulation efficiency, loading capacity and swelling degree. Detailed procedures for

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encapsulation efficiency (EE), bioactive agent loading capacity (LC) and swelling degree (Sw)

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determination are presented in Supporting Information (from S2.4.1. to S2.4.3). The

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concentration of copper cations was determined at λ = 795 nm and concentration of T. viride

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(expressed as the number of spores (NS) per 1 g of dry microcapsule) at 550 nm by a

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spectrophotometer (Shimadzu, UV-1700).

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2.5. In vitro copper and T. viride releasing from microcapsules. The release studies of copper

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ions and T. viride spores from microcapsules were carried out at room temperature. Detailed

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procedures of sample preparation for measurements are presented in Supporting Information

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(S2.5.). Results of release experiments are presented as the fraction of released active agent (Cu2+

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or T. viride spores (Tv)) using equation 2:

104

=

(2),

105

where f represents the fraction of Cu2+ or Tv released, Rt is the amount of Cu2+ or Tv released at

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time t and Rtot is the total amount of Cu2+ or Tv loaded in microcapsules.

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2.6. Statistical analyses. The results were statistically analyzed with Microsoft Excel 2016 and

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XLSTAT statistical software add-in. The data are shown as mean values ± standard deviation.

109

Details are presented in Supporting Information (S2.6.).

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3. RESULTS AND DISCUSSION

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3.1. Effect of copper cation concentration on T. viride spores. T. viride belongs to a group of

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organisms that can survive in high concentrations of different metals and have the potential to

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bind them.8 We have shown in the previous paper that the presence of copper cations did not

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inhibit the growth of encapsulated T. viride.5 None of the applied concentrations caused

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inhibition of mycelial growth. On the contrary, the presence of higher copper cations

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concentration promotes growth.

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Singh et al.9 found an inverse relationship between cell surface electrostatic charge (zeta potential

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ranged from – 11 to 42 mV) and cell surface hydrophobicity (ranged from 12 to 52 %), i.e. less

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hydrophobic cell surfaces were more negatively charged. T. viride spores dispersed in water (Fig.

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1a) had negative charge (average zeta potential -35.1 mV) indicating prevailing spore surface

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hydrophilicity. The negative charge on the cell surface arises from various functional groups such

123

as carboxyl, hydroxyl, amine and phosphate.10,11 The increase of copper cations concentration

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from 0.005 to 2 mol dm-3 resulted in less negatively charged spore surface (Fig. 2), and

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consequently aggregation (Fig. 1b) indicating electrostatic interactions.

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Observations from electron microscopy and cell fractionation studies revealed copper ions

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location on the cell wall of T. viride12 pointing this is the place where the interaction between T.

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viride and copper ions occurred. Chemical analyses of the fungal cell walls revealed a very

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complex chemical composition that may be different in the several taxonomic groups.11 Cell

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walls are composed of polysaccharides (mainly glucan and chitin, up to 90%) and glycoproteins,

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as well as of lipids and other minor components (pigments and inorganic salts). Melanin, which

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in other fungi is associated with chitin, was shown to replace this polymer in the spore wall of



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Trichoderma species. The functional groups detected in the FTIR spectrum5 relate well with the

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chemical structure of the cell wall. FTIR spectrum of T. viride spores and copper cations mixture

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have suggested that at least amine, hydroxyl, carbonyl and amide bonds are the major sites for

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binding of copper cations. It can be assumed that the copper cation binding to T. viride is beside

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electrostatic interactions associated with possible ion exchange and some physico-chemical

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interactions.

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3.2. In vitro release of copper cations and T. viride from microcapsules. Various physico-

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chemical processes such as wetting, swelling (penetration of solution into the matrix) and

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polymer stress relaxation (transition of glassy structure to rubbery state), diffusion through the

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matrix, disintegration, dissolution or erosion of the structure, or their combination, may be

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included in the release of an active agent from a hydrophilic microcapsule.13 Design of controlled

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delivery systems involves optimization of many parameters (microcapsule chemical composition

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and geometry, preparation technique of microcapsule, the environmental conditions during

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release) among which are the most important type and concentrations of both, biopolymer and

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gelling cation.14 Knowledge of bioactive agent kinetics and mechanism of release from

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microcapsules is important for the optimal development of formulations for a particular

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application. Having in mind all of the factors involved in the release and the combinatorial effect

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of different microcapsule composition and the concentration of cross-linking cation15 we

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prepared uncoated and chitosan-coated alginate microcapsules of diameters 0.45 and 2 mm at the

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fixed amount of alginate and T. viride spores, and with various concentrations of copper cations.

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Release profile curves were analyzed by a simple empirical Korsmeyer-Peppas model16-18

154

=

(3),


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where k is a kinetic constant characteristic for particular system considering structural and

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geometrical aspects, n is the release exponent representing the release mechanism, and t is the

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release time. The release exponent n for spherical microcapsules can be characterized by three

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different mechanisms (Fickian diffusion, anomalous (non-Fickian diffusion) or Type II transport.

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Values for n < 0.43 indicate the release is controlled by classical Fickian diffusion that is the rate

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of active agent diffusion is much less than that of polymer relaxation. Values for n > 0.85

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indicate the release is controlled by Type II transport that is the rate of active agent diffusion is

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much larger than that of polymer relaxation. Values of n between 0.43 and 0.85 indicate the

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anomalous transport kinetics determined by a combination of the diffusion mechanism and Type

164

II transport; diffusion and polymer relaxation almost equally contribute to the release.

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3.2.1. Encapsulation efficiency, loading capacity and swelling behavior. Encapsulation efficiency

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(EE) and bioactive agent loading capacity (LC) determination were performed to obtain

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information on the yield and amount of copper cations and T. viride spores encapsulated in

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microcapsules. Results on copper cations encapsulation efficiency and loading capacity are

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presented in Figs. S1a,b and 2a,b,c respectively. The increase of copper cations concentration

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decreased copper ions encapsulation efficiency for all microcapsules indicating even the lowest

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copper cations concentration was enough for gelling the amount of added alginate (S1a,b). No T.

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viride spores presence in filtrate after microcapsules preparation indicated almost 100%

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encapsulation of T. viride spores added. Copper ions loading capacity slightly increased with

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copper concentration for all microcapsules (S2a,b). Small microcapsules loaded with both active

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agents exhibited higher copper ions loading capacity than those loaded only with copper ions

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(S2a). Coating with chitosan layer slightly decreased, while T. viride spores addition enhanced

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the copper ions loading capacity of smaller microcapsules (Fig. S2a). A slightly greater influence



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of chitosan is observed on larger microcapsules (Fig. S2b). Irrespective of whether they are filled

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with one or two active agents, coated microcapsules exhibited higher copper ions loading

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capacity. Results on T. viride spores loading capacity expressed as the number of spores per 1 g

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of dry microcapsules are presented in Fig. S2c. The increase in copper cation concentration

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slightly increased T. viride spores loading capacity, except for those uncoated small

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microcapsules that had almost constant loading capacity.

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The most important rate-controlling release mechanisms from hydrophilic microcapsules are

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swelling and dissolution/erosion at the microcapsule surface.13 When dispersed in deionized

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water, hydrophilic polymer microcapsules swelled thus influencing the release of active agents

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from them. The effect of increasing copper cations concentration on swelling degree is presented

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in Figs. 3a,b. It can be seen that in the range of investigated copper ions concentrations both,

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small (Fig. 3a) and large (Fig. 3b) microcapsules prepared at the lowest copper cations

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concentration exhibited the highest degree of swelling. At higher copper cations concentrations

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the swelling degree exhibited smaller and almost constant values. However, at the highest copper

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ions concentration the swelling degree of chitosan coated microcapsules increased again.5

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The results obtained show that the degree of swelling depends on the concentration of copper

194

ions and chitosan layer. Chitosan-coated microcapsules exhibited somewhat higher swelling

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degree when compared with the uncoated one. This may be ascribed to the high swelling and

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water uptake capabilities of chitosan.19 The concentration of copper cations affects the properties

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of alginate microcapsule, that is, the size of cavities inside alginate bead which accommodate

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water.15 With high concentrations of copper ions available for alginate gelation, the gel beads

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formed faster, becoming smaller and stiffer, because of a higher degree of reticulation and a

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lower retention of water in the gel. A gelation in low concentration of copper ions was slower and


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led to brittle gels. Thus, the obtained differences in the swelling behavior can be primarily

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attributed to the effects of copper cations concentration on the microcapsule structure.

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3.2.2. In vitro release from uncoated microcapsules. The release of copper actions and, both

204

copper cations and T. viride spores from microcapsules prepared without chitosan and with

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increasing concentration of copper cations is presented in Figs. 4a,b and 5a,b, respectively. All

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release profiles are characterized by rapid initial release followed by slower release. Comparison

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between differently sized microcapsules prepared without T. viride spores (Figs. 4a,b) shows

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somewhat slower copper cations releasing from larger microcapsules. In order to obtain better

209

correlation (R2 > 0.98) copper cations release profiles from microcapsules prepared without T.

210

viride spores were analyzed instead Eq. 3 by Eq. 420

211

= +

(4),

212

where a is the y-axis intercept characterizing the burst effect.

213

The values of the release constants k, exponents n and intercept a are given in Table 1. It can be

214

seen that the release constant k increases with initial copper cation concentration. Relatively high

215

n values (n > 0.43) at the lowest copper cation concentration indicates copper cations release

216

follows anomalous kinetics (diffusion and polymer relaxation). The rate and duration of copper

217

cations release are therefore regulated by the swelling and the dimension of swelled layer. Lower

218

n values than 0.43 for microcapsules prepared at higher copper cation concentrations indicates

219

that the release process is controlled by diffusion through microcapsule.

220

The addition of T. viride spores diminished the fraction of released copper cations (Figs. 5a,b).

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Fractions of released T. viride spores were higher than those of copper cations in all cases. This

222

may be explained by interactions between alginate and T. viride spores before dropping into



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copper cations solutions as well as to interactions of copper cations and T. viride released in the

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surrounding solution.5 Previously we have detected interactions with amine, carboxylate and CO

225

groups as well as enhanced intermolecular hydrogen bonds in alginate and T. viride spores

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mixture.5 During gelation copper cations cooperatively interact with a block of L-guluronic acid

227

groups forming ionic crosslinks between different polymer chains. It is likely that T. viride

228

interactions to alginate functional groups and the concomitant gelation differ from those without

229

the T. viride spores associated with alginate, thus causing changes in gel network properties.

230

The increase of copper cation concentration increased both active agents release; the impact is

231

more pronounced with T. viride spores. We have shown previously the high resistance of T.

232

viride spores to the presence of copper cations.5 The presence of copper cations did not inhibit the

233

growth of fungi, i.e. none of the applied concentrations caused inhibition of mycelial growth. On

234

the contrary, the increasing concentrations of copper cations promotes growth, e.g. at the highest

235

copper cation concentration (20 mmol dm-3) the growth of T. viride was the most homogeneous.

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Higher concentration of T. viride spores then loaded (Fig. 5a) measured in samples with small

237

microcapsules prepared at the highest copper cations concentration can be ascribed to the

238

germination of released T. viride spores and formation of germ tube biomass in the surrounding

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medium. Actually, copper cations released from microcapsule promoted T. viride spores

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germination in water.5 The increasing amount of T. viride spores in the dispersing medium is

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closely related to two processes, one is the release from microcapsules and the other is

242

germination. It seems that the amount of released copper cations from microcapsules prepared

243

with lower copper cations concentration was insufficient to stimulate germination.

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The set of curves for both active agents release from microcapsule loaded with copper cations

245

and T. viride exhibit rapid initial release followed by slower release. The values of the release


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constant k and exponent n obtained by Eq. 3 are listed in Table 1. The release of both active

247

agents from smaller microcapsules was faster than from larger microcapsules and increased with

248

initial copper cation concentration. Lower n values than 0.43 indicate that the release mechanism

249

is controlled by a classical Fickian diffusion in all cases.

250

3.2.3. In vitro release from coated microcapsules. A common way to improve the sustained

251

release rate from alginate microcapsule is coating with oppositely charged biopolymers, like

252

chitosan, which represents an additional barrier that slows transport from the microcapsule to the

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surrounding solution.21-23 Electrostatic interactions between positively charged amino groups of

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chitosan and negatively charged carboxylic acid groups of alginate create a polyelectrolyte

255

complex on the microcapsule surface that reduces porosity and increase the stability of alginate

256

microcapsules.24

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In comparison with uncoated microcapsules the chitosan layer on coated microcapsule surface

258

diminished, both copper cation and T. viride spores release (Figs. 6a,b and Figs. 7a,b).

259

Microcapsules prepared without T. viride spores (Figs. 6a,b) exhibited rapid initial release

260

followed by slower release obeying power law equation, while those prepared with T. viride

261

spores (Figs. 7a,b) exhibited an initial lag time. The release profiles of microcapsules prepared

262

without T. viride were analyzed by Eq. 3, while those prepared with T. viride spores by

263

modifying Eq. 3 to initial lag time, l,25

264

= ( − )

(5),

265

The lag time is thought to be equivalent to the time required for the microcapsule to hydrate and

266

reach equilibrium before the beginning of active agents releasing. The processes involved during

267

the lag phase are penetration of water and filling of the microcapsule surface pore with water, as



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well as transport of active agents through the alginate matrix and the water-filled chitosan pores

269

without transport into the surrounding media.

270

The values of the release constants k and exponents n are listed in Table 2. As is expected, the

271

release of active agents from coated was slower than from uncoated microcapsules and increased

272

with increasing copper cation concentration. Lower n values than 0.43 indicate that the release

273

mechanism of copper cations is controlled by a classical Fickian diffusion. The rate-controlling

274

mechanism for T. viride spores release from small microcapsules was Fickian diffusion, whereas

275

the release from large coated microcapsules is controlled by a combination of diffusion and the

276

polymer swelling and relaxation (n > 0.43).

277

3.2.4. Principal component analysis and agglomerative hierarchical clustering. The principal

278

component analysis was used to indicate multivariate dependence between the selected variables.

279

Principal component weighting of the results was made for these attributes: swelling degree (%)

280

– Sw%, loading capacity for copper ions – LC Cu2+, the release constant (k), exponent (n) and

281

release time for copper cations at t0h, t2h, t4h, t24h, t48h, t80h – R-txh. Bartlett's Sphericity test was

282

used to compare the correlation matrix with the matrix of zero correlations. The risk for the

283

rejection of the null hypothesis H0 while it was true was

Kinetics and Mechanisms of Chemical and Biological Agents Release

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