Potentially Immunogenic Contaminants in Wood-Based and Bacterial

Nov 28, 2017 - Knowledge gaps in the biosafety data of the nanocellulose (NC) for biomedical use through various routes of administration call for clo...
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Potentially Immunogenic Contaminants in Wood-based and Bacterial Nanocellulose: Assessment of Endotoxin and (1,3)-#-D-glucan Levels Jun Liu, Markus Bacher, Thomas Rosenau, Stefan M. Willför, and Albert Mihranyan Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01334 • Publication Date (Web): 28 Nov 2017 Downloaded from http://pubs.acs.org on November 29, 2017

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Potentially Immunogenic Contaminants in Woodbased and Bacterial Nanocellulose: Assessment of Endotoxin and (1,3)-β-D-glucan Levels Jun Liu,a, b* Markus Bacher,c Thomas Rosenau,c, d Stefan Willför,d Albert Mihranyan a* a

Nanotechnology and Functional Materials, Department of Engineering Sciences, Box 534, Uppsala University, 75121 Uppsala, Sweden.

b

Biofuels Institute, School of the Environment and Safety Engineering, Jiangsu University, 212013 Zhenjiang, China. c

Department of Chemistry, University of Natural Resources and Applied Life Science (BOKU), Muthgasse 18, 1190 Wien, Austria.

d

Johan Gadolin Process Chemistry Centre, c/o Laboratory of Wood and Paper Chemistry, Åbo Akademi University, Porthansgatan 3-5, FI-20500, Turku/Åbo, Finland. *Corresponding author: [email protected]; [email protected]

ABSTRACT:

Knowledge gaps in the biosafety data of the nanocellulose (NC) for biomedical use through various routes of administration call for closer look at health and exposure evaluation. This work evaluated the potentially immunogenic contaminants levels, e.g. endotoxin and (1,3)-β-

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D-glucan, in four representative NCs, i.e. wood-based NCs and bacterial cellulose (BC). The hot-water extracts were analysed with ELISA assays, HPSEC-MALLS, GC, and NMR analysis. Varying levels of endotoxin and (1,3)-β-D-glucan contaminats were found in these widely used NCs. Although the β-(1,3)-D-glucan was not detected from the NMR spectra due to the small extract samples amount (2-7 mg), the anomerics and highly diastereotopic 6-CH2 signals may suggest the presence of β-(1,4)-linkages with β-(1,6) branching in the polysaccharides of NCs’ hot-water extracts, which were otherwise not detectable in the enzymatic assay. In all, the article highlights the importance of monitoring various watersoluble potentially immunogenic contaminants in NC for biomedical use.

Keywords: Nanocellulose; Biosafety assessment; Immunogenic contaminants; (1,3)-β-Dglucan; Endotoxin; Biomedical application.

INTRODUCTION Nanocellulose is a new family of cellulose-based material with unique nano-scaled structure which mainly include cellulose nanocrystals (CNCs, also terms as nanocrystalline cellulose, i.e. NCC), cellulose nanofibrils (CNFs, also terms as nanofibrillated cellulose, i.e. NFC), and bacterial cellulose (BC) 1. Generally, nanocelluloses are prepared by mineral acid hydrolysis and mechanical defibrillation approach. Chemical or enzymatic pretrements can be applied to assist the mechanical treatment and/or to introduce desired functional groups onto the cellulose fibril surface 2. As an abundant and bio-based material, nanocellulose which is derived from plant biomass, bacteria, algae, and tunicates, holds potential to be widely used in various biomedical and/or pharmaceutical areas, such as drug delivery 3-6, tissue engineering 7, wound healing

8-10

, contact lenses

11-14

, blood vessels

manufacturing of protein-based pharmaceuticals

7, 19, 20

15, 16

, hemodialysis

17, 18

, and

due to their inherent properties, such

as nontoxicity, biodegradability, excellent structure strength and stiffness, and thermal 2

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stability 1. However, to fulfill the requirements for biomedical and pharmaceutical application, more biosafety assessment and evaluation is needed. The ISO-10993 standard for Biological evaluation of medical devices

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requires that

biological and chemical risk of extractables and leachables of a material should be considered as part of the biocompatibility evaluation before it can be approved for biomedical or pharmaceutical application. Extractables are organic or inorganic chemicals that are released from the material into a predefined solvent system under controlled conditions, including extreme conditions that are normally not encountered in a process. Leachables, on the other hand, are chemicals that migrate from the material under operational conditions intended for the final use. Identification and quantification of extractables and leachables that might migrate out of the material and their potential toxicological risk is a critical part of biosafety assessments of biomaterials. By the evidence of supporting various cell lines survival, proliferation, and differentiation, it is normally conceived that nanocellulose is biocompatible and nontoxic

22, 23

. However, the source, preparation method, and chemical modifications of

nanocellulose directly determine the physical-chemical properties of the final product, thereby also affecting their toxicological behavior 24, 25. Currently, little information is available on the leachables and extractables in the nanocellulose-based material and their potential detrimental biological impact on biomedical and/or pharmaceutical applications 26. Several leachable compounds can have a strong immunogenic effect. The endotoxin contamination is a common problem due to the potential risk of stimulating a systemic inflammatory response up to endotoxin shock, sepsis, tissue damage, and death

27, 28

.

Leachable (1,3)-β-D-glucans are another important group of impurities in cellulose-based materials

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. Leachable (1,3)-β-D-glucans, which were identified in various cellulose-based

membranes for hemodialysis, as well as parenteral drug products that came in contact with cellulose-based filters, were shown to cause a strong immune response

30-33

. Furthermore,

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leachable (1,3)- β-D-glucans need to be monitored as they can cause a false-positive diagnosis of fungi infection. Also, interference of leachable (1,3)-β-D-glucans from cellulose-based products has been reported to cause falsely high endotoxin levels due to the activation of an enzyme clotting cascade in the LAL assay 34, 35. The potential of nanocellulose to elicit (pro-)inflammatory immune response has been reported earlier by Lopes et al.

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, Yanamala et al.

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, Shvedova et al.

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, and Clift et al.

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.

However, none of these authors considered the levels of immunogenic leachable compounds and their cross-interference. In our recent study, potential leachables and extractables, as well as other impurities like heavy metals or microorganisms, were monitored in the nanocellulose from Cladophora sp. algae

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. In particular, the (1,3)-β-D-glucan and endotoxin

contaminations have been assessed and minimized to clinically insignificant levels. Because of the significant knowledge gaps in the safety information of nanocellulose, a closer look on environmental and health safety of nanocellulose is needed 22, 40. The present work is aimed at identification and evaluation of the endotoxin and (1,3)-β-Dglucan levels in four common nanocelluloses (Figure 1). In particular, wood-based nanocelluloses (native CNF, carboxymethylated CNF, and CMC-grafted CNF)

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, as well as

bacterial nanocellose (from nata de coco), are investigated. Native CNF with a charge density of 44.2µeq/g is prepared by enzymatic pretreatment of dissolving pulp followed by a highpressure homogenization process (three passes through Z-shaped chamber pair with diameter of 400 µm and 200µm under 105 MPa pressure and five passes through chamber pair with diameter of 200 µm and 100µm under 170 MPa pressure)

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, representing the native

nanocellulose without surface modification. Carboxymethylated CNF is prepared by carboxymethylation of cellulose pulp with charge density of 590 µeq/g followed by highpressure homogenization process (four passes through chamber pair with diameter of 200 µm and 100µm under 40-170 MPa pressure)

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, representing surface modification of the native

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nanocellulose. CMC-grafted CNF is prepared by grafting carboxymethyl cellulose onto the cellulose fibers with a charge density of 160 µeq/g followed by high-pressure homogenisation (one pass through chamber pair with diameter of 200 µm and 100µm under 170 MPa pressure) 44

, representing the nanocellulose with incorporation of other cellulose-based material.

Bacterial nanocellulose (BC) is produced by fermentation of coconut water with celluloseproducing Acetobacter xylinum bacteria 45. Thus, BC represents unmodified nanocellulose.

Figure 1. Schematic illustration of four different types of nanocellulose

MATERIALS AND METHODS Materials Wood-based nanocelluloses (native CNF, carboxymethylated CNF, and CMC-grafted CNF) were purchased from Innventia AB, Sweden. The products were produced from softwood sulphite dissolving pulp consisting of Norway Spruce (60 %) and Scots Pine (40 %) (Domsjö mill, Sweden). Carboxymethylated CNF had a degree of carboxymethyl substitution (D.S.) of

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0.098. CMC-grafted CNF was carboxymethyl (Mw 1000 kg/mol) grafted nanocellulose with D.S. of 0.4. Bacterial nanocellulose (BC) was obtained from nata de coco (Vietst, Vietnam).

Methods Analysis of hot-water extracts Isolation of extractabls of all the nanocellulose materials (native CNF, carboxymethylated CNF, CMC-grafted CNF, and BC) was conducted with hot-water for 24 h in a Soxhlet apparatus following the ISO standard methods (ISO 10993-12). A thimble was used as blank or negative control extraction. The total amount of the extracts was determined gravimetrically after freeze-drying. All glassware used in the experiments was subjected to dry heat sterilization at 250 ºC for 30 min for depyrogenation.

Molar mass measurement with HPSEC-MALLS The weight- and number-average molar masses (Mw and Mn) of the hot-water extracts were determined by HPSEC

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. A multi-angle laser light scattering (MALLS) detector

(miniDAWN, Wyatt Technology, Santa Barbara, USA) and a refractive index (RI) detector (Shimadzu Corporation, Japan) were used. A two-column system, 2 × UltrahydrogelTM linear 300 mm x 7.8 mm column (Waters, Milford, USA), in series was used. 0.1 M NaNO3 was used as the elution solvent at a flow rate of 0.5 mL/min and dn/dc value of 0.150 mL/g 47. The samples were filtered through a 0.22 µm nylon syringe filter before injection. The injection volume was 100 µL.

Carbohydrate analysis with GC The non-cellulosic carbohydrate composition in the nanocellulose of native CNF, carboxymethylated CNF, CMC-grafted CNF, and BC, and their hot-water extracts was determined by gas chromatography (GC) after methanolysis and silylation 48. GC analysis was done on a PerkinElmer AutoSystemXL instrument (Norwalk, USA) equipped with HP-1 and HP-5 column, (25m x 0.2 mm, 0.11 µm film). The temperature program used was: 100 °C -

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175 °C, 4 °C/min, 175 °C - 290 °C, 12 °C/min. The temperatures of injector and detector were 260 °C and 290 °C, respectively.

(1,3)-β-D-glucan and endotoxin detection The maximum leachable (1,3)-β-D-glucan and the endotoxin contaminants (in the hot-water extracts) were measured using the (1,3)-β-D-glucan / endotoxin-specific Limulus Amebocyte Lysate (LAL) endpoint test kits, Glucantell® and Pyrochrome®, respectively (Associates of Cape Cod Inc., UK) according to the manufacturer’s instructions. In brief, the (1,3)-β-Dglucan or the endotoxin (E. coli O113:H10) control standard and the hot-water extracts of the cellulose materials were mixed with the LAL reagents in 96-well plates, and were incubated at 37 °C for the recommended time period. The diazo coupling reagents were added to the reaction mixtures for color development. The optical density of the mixtures in the microplate was measured at 540-550 nm using a microplate reader (Tecon Infinite M200, Switzerland). The Glucantell® assay is specific to the (1,3)-β-D-glucan detection. The possibility of false positive endotoxin detection was avoided by using a Glucashield® buffer which selectively blocks the interference of (1,3)-β-D-glucan during the LAL endotoxin test. NMR All NMR spectra were recorded on a Bruker Avance II 400 (resonance frequencies 400.13 MHz for 1H and 100.63 MHz for 13C) equipped with a 5 mm observe broadband probe head (BBFO) with z–gradients at room temperature with standard Bruker pulse programs. The samples (2-7 mg) were dissolved in 0.6 mL of D2O (99.8 % D). Chemical shifts are given in ppm, for calibration TMSP-d4 (Trimethylsilyl-3-propionic-2,2,3,3-d4 acid sodium salt) was added. All two-dimensional experiments were performed with 1k × 256 data points, while the number of transients and the sweep widths were optimized individually. HSQC experiments were acquired in the edited mode using adiabatic pulse for inversion of

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C and GARP-

sequence for broadband 13C-decoupling, optimized for 1J(CH) = 145 Hz.

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RESULTS AND DISCUSSION The appearance of different nanocelluloses and their hot-water extracts is shown in Figure 2. All nanocellulose samples were provided in the form of high water content hydrogels. As it is seen in Figure 2, native CNF, and CMC-grafted CNF had a paste-like, relatively fluid texture, while carboxymethylated CNF was a translucent semisolid. BC was in the form of mechanically resilient sheets. Extended hot-water extraction was applied to all studied samples to reveal information about potential leachables. The extracted products were freezedried to yield a brownish, flake-like powder as shown in Figure 2.

Figure 2. Pictures of four different types of nanocellulose and their hot-water extracts.

Table 1. Non-cellulosic polysaccharide composition of nanocelluloses (wt.-% of NCs).

Monosaccharide

Native CNF

Rhamnose Arabinose

0.02 0.06

CMCCarboxymethylated grafted CNF CNF