Differences in levan nanoparticles depending on their synthesis route: Microbial vs cell-free systems

doi:10.1016/j.ijbiomac.2019.06.128

International Journal of Biological Macromolecules

Volume 137, 15 September 2019, Pages 62-68

https://doi.org/10.1016/j.ijbiomac.2019.06.128 Get rights and content

Highlights

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Polyfructoses CAC is depending on the synthesis route.
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Differences in MWs were observed at concentrations above the CAC.
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Polymer thermal analysis is not dependent on the synthesis route.
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Both synthesis routes provided non-porous polymers.
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Stable and spherical nanoparticles were obtained for both systems.
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Nanoparticles showed a low non-specific adsorption.

Abstract

Differences between the levan obtained from bacteria and from cell-free systems were studied in this work. Results showed that both polymers are non-porous solids (type II isotherm with 20 m²/g) with a main thermal decomposition at 200 °C and a negligible value of protein adsorption. Microbial levan produced nanoparticles of 90 nm in diameter whereas nanoparticles of 110 nm were obtained with the polymer obtained from a cell-free system. Both polymers behave as aggregates depending on the critical aggregation concentration. At the same time, that concentration depends on the technique used for the polymer synthesis. Cell-free system aggregation concentration is 0.24 mg/mL whereas a concentration of 0.05 mg/mL was found for the microbial system. In both cases, the average molecular weight of the aggregate is higher than 2000 kDa. These results highlight the existence of aggregation equilibrium for both polymers that has to be taken into account for future applications.

Introduction

Biopolymers are biodegradable and biocompatible materials that usually have functional groups on their side chains that can be modified to perform chemical functionalization (or reaction). Due to their particular and promising properties, their use is very interesting for several biomedical applications. Among these polymers, levan is a biopolymer constituted by fructose units bonded by β(2–6) linkages, with some ramifications β (2–1), having different applications in biomedicine, food or energy [1].

One of the most promising characteristics of levan is its ability to form nanostructures in water by self-assembly, forming a polymeric micelle, which makes it a good carrier in drug delivery systems [2,3]. However, there is reduced information in the literature concerning the mechanism of nanoparticle formation and the key parameters that control that process (i.e. critical aggregation concentration).

Levan has been traditionally produced from different bacteria species as a component of the extracellular polysaccharide matrix. These bacteria (Gram-positive or Gram-negative), under certain growing conditions (excess of sucrose and a slightly acid pH), can secrete an enzyme (levansucrase) that is able to hydrolyze the sucrose in the culture medium to subsequently polymerize the derived fructose units (using water as acceptor of fructose units). On the other hand, several authors have isolated the enzyme from bacteria [4,5] and a new approach for the synthesis by directly using the enzyme in a solution of sucrose is being explored. The use of cell-free systems introduces several advantages in comparison with the traditional microorganism-based synthesis, such as avoiding culture contamination, easier downstream processing and a faster polymerization reaction [6]. As an example, Szwengiel et al. [5] optimized the synthesis conditions using levansucrase from Bacillus subtilis, including different substrates, temperature, pH, and the effects of some cofactors such as Mg or Mn, which control the hydrolytic and transferase activity of the enzyme.

However, the differences between both syntheses on the resulting polymer properties are still unknown. In particular, for biomedical applications, properties such as molecular weight or particle size are key features regarding the stability in water, aggregation and even a possible antitumoral effect [7]. In this context, although levan nanoparticles obtained from microorganisms and their properties have been previously studied, there is still a lack of knowledge regarding nanoparticles derived from cell-free systems.

On the other hand, the main drawback of polymeric micelles (as levan or polyfructoses) for systemic administration is the disaggregation process that happens when the particles (micelles) are introduced in the blood stream. The high volume of blood reduces the concentration of nanoparticles; and if this is lower than CAC, the chains constituting the particles will disaggregate and the drug will be released without control. Therefore, a deep study of levan physicochemical properties may help to clarify this phenomenon. In this context, our main hypothesis is that levan and polyfructoses can have a similar behavior in terms of aggregation phenomenon. Moreover, polymer properties can be modified depending on the synthesis route.

Based on the previous facts, the main objective of this work is to perform a comparison between polymers that are produced from bacteria and from a cell-free system. Both systems will be compared in addition with commercial levan from Erwinia herbicola and will be characterized by studying different properties regarding nanoparticle sizes and molecular weight. Moreover, studies concerning CAC and protein adsorption have been performed in order to further characterize the polymer as well as to provide information regarding the phenomenon of nanoparticle formation.

Section snippets

Microbial growth and polyfructose isolation

Acinetobacter nectaris (CECT 8127) was selected to produce polyfructoses as was described by González-Garcinuño et al. [8] This bacterium was purchased from the Spanish type culture collection of microorganisms (CECT). The strain was cultured in Erlenmeyer flasks (volume 500 mL) (Duran, Germany), with the following culture media: 7 g L⁻¹ yeast extract (Sigma Aldrich), 2.5 g L⁻¹ KHPO₄, 1.6 g L⁻¹ NH₄SO₄, 0.4 g L⁻¹ MgCl₂, and a variable amount of sucrose (from 60 to 150 g L⁻¹). The pH of culture

Polymer produced by cell-free system

The polymer was obtained by following the methods described in Section 2.2. and the yield was calculated as the percentage of the polymer compared to the amount of sucrose that was used as substrate. The yield ranged from 18% to 22% (mg polyfructose/mg initial sucrose). This value was higher than the value reported by Bersaneti et al. [14], who obtained a yield of about 12% or Santos-Moriano et al. [15] with a yield of around 15% in FOS polymerization with levansucrase. This increase can be

Nanoparticle formation by self-assembly

Previous results show information about polyfructose nanoparticle formation by self-assembly (Fig. 7). In both systems (bacterial and cell-free), sucrose is incorporated to the active site of the enzyme levansucrase (glucose is established as acceptor for polymerization) [step 1]; and after that, another molecule of sucrose is hydrolyzed and its fructose is transferred to form 6-kestose (trisaccharide) [step 2]. Based on our molecular weight results and CAC determination, further steps involve

Conclusions

There were not significant chemical differences between the polymers obtained from levansucrase in a cell-free system and from secreted levansucrase in a culture medium. Cell-free systems give rise to smaller nanoparticles than those obtained from a microorganism although with similar zeta-potential values (−4 mV). Despite the low zeta-potential values, nanoparticles are still stable mainly due to interactions with the solvent, which assures colloidal stability due to the presence of van der

Acknowledgments

Authors acknowledge Junta de Castilla y León for project SA004U26. A. González-Garcinuño acknowledges Spanish ministry of Education for his PhD fellowship (FPU14/04914). Authors thank the Department of Inorganic Chemistry at University of Salamanca for the support with FTIR experiments and thank Prof. María Gracia García Martín (Department of Organic Chemistry and Pharmaceutics of the University of Sevilla) for her help with the GPC experiments.

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Citation Excerpt :
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Differences in levan nanoparticles depending on their synthesis route: Microbial vs cell-free systems

Highlights

Abstract

Introduction

Section snippets

Microbial growth and polyfructose isolation

Polymer produced by cell-free system

Nanoparticle formation by self-assembly

Conclusions

Acknowledgments

Biotechnol. Adv.

Carbohydr. Polym.

J. Mol. Catal. B Enzym.

Curr. Opin. Biotechnol.

Int. J. Biol. Macromol.

Process Biochem.

Int. J. Biol. Macromol.

J. Mol. Catal. B Enzym.

Int. J. Biol. Macromol.

Int. J. Biol. Macromol.

Process Biochem.

Int. J. Biol. Macromol.

J. Control. Release