Sol–gel composite films with controlled release of biocides

Journal of Controlled Release 60 (1999) 57–65

Sol–gel composite films with controlled release of biocides a, *, C. Jagota a , J. Trepte a , K.-H. Kallies a , H. Haufe b ¨ H. Bottcher b

a ¨ Feinchemie GmbH Sebnitz, Hohenweg 9, D-01855 Sebnitz, Germany ¨ Gesellschaft zur Forderung von Medizin-, Bio- und Umwelttechnologien e.V., Merseburger Strasse 371, D-06132 Halle, Germany

Received 23 October 1998; received in revised form 3 November 1998; accepted 26 January 1999

Abstract The release of biocides (benzoic, sorbic and boric acids) incorporated into modified silica films was investigated with respect to composite structure. The liberation rates of the embedded acids are proportional to the biocide-to-silica ratio and are changed by adding soluble polymers such as hydroxypropylcellulose. The rates of liberation correlate with biocidal activity, i.e., the growth of microorganisms such as Escherichia coli, Lactobacillus plantarum and Penicillium sp. is strongly suppressed by contact with such composite films. In a similar way, strong fungicide and insecticide effects were observed after impregnating wood with composites containing boric acid.  1999 Elsevier Science B.V. All rights reserved. Keywords: Sol–gel; Liberation; Food preservation; Wood protection; Biocide

1. Introduction The possibility of embedding organic compounds into inorganic oxide matrices [1–7] using the recently developed sol–gel technique offers new and interesting perspectives for controlled release matrix systems [8–14]. Such systems could be used for different therapeutic or antibacterial depot systems, in which the metal oxide is just an inert carrier for the bioactive compound (BC). Using sol–gel films in controlled release systems promises many advantages compared to recently used polymeric systems: (i) the film matrix is cheap, inert, transparent and not hazardous to humans or the environment, (ii) the coatings show excellent mechanical properties and are stable to humidity, light and heat, (iii) the coating process is compatible with all common coating technologies and allows control *Corresponding author.

of layer thickness and film quality, and (iv) a wide spectrum of BCs can be incorporated into the sol–gel film. The procedure for preparing such inorganic– organic composites is as follows (Scheme 1) (see also Fig. 1): Although organic components are often added to the metal alkoxide before hydrolysis of the metal oxide sol (method A), the addition of BCs to the completely hydrolysed sol is more convenient (method B). The composite structure and immobilisation behaviour of both methods are virtually identical [14], since encapsulation actually occurs at the condensation step. From an experimental point of view method B offers many advantages over method A, e.g., the ratio of organic-to-inorganic composite can be altered by simple mixing, plus the composition of the sol can be adapted to take into account the solubility of the BC. To date, controlled release systems using thin metal oxide layers with incorporated BCs are still

0168-3659 / 99 / $ – see front matter  1999 Elsevier Science B.V. All rights reserved. PII: S0168-3659( 99 )00053-X

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¨ et al. / Journal of Controlled Release 60 (1999) 57 – 65 H. Bottcher

Fig. 1. Schematic representation of incorporation and liberation of bioactive compounds within a silica sol–gel matrix.

largely unknown. There have been only a small number of investigations into the diffusion of dyes [15,16] and steroids [17] from porous sol–gel glasses and into the release of perfumed essences and oils from hybrid SiO 2 lyogels [18], xerogels and films [19]. Preliminary investigations have been made into the controlled release of drugs such as nifedipin [20] and antimicrobial substances [14,21] from SiO 2 composites. Applications can be envisaged where bioactive substances slowly diffusing out from the composite layer over a long period cause antimicrobial effects, e.g., the conservation of foods or cosmetics, and the protection of plants and wood against biological destruction. The aim of this work was to prove the antimicrobial effects of such controlled release systems based on sol–gel coatings. With regard to the European Union guidelines on food additives [22], coatings incorporating sorbic acid (SA) for use in packaging materials for preserving sliced bread and cheese, and coatings with

combinations of SA and benzoic acid (BA) for preserving meat and fish are of special interest. Therefore the first part of the investigation studied modified silica coatings containing combinations of BA and SA on polymeric foils. These were placed in contact with microorganisms characteristically found on food products (Escherichia coli, Lactobacillus plantarum and Penicillium sp.). The second part investigated how impregnating wood samples with composites containing boric acid (BoA) protected them from attack by fungi and insects. BoA is one of the most effective and least toxic agents for protecting wood against brown and white rot, the larvae of the house longhorn beetle (Hylotrupes bajulus) and the common furniture beetle (Anobium punctatum) [23]. Use of this biocide is mainly limited to wood not exposed to rain or high humidity [24], because of its water solubility and inability to be fixed. For this reason, it was of interest to investigate the leaching behaviour and biocidal effects of BoA incorporated in silica films.

Scheme 1. The BC-containing gel film on the substrate is formed by solidification of the liquid sol film as a result of evaporation of the solvent.

¨ et al. / Journal of Controlled Release 60 (1999) 57 – 65 H. Bottcher

Coatings of modified silica with embedded BoA were prepared on films and wood, then studies made of their releasing behaviour and the fungicidal and insecticidal effects on the wood fungus Coniophora puteana and wood-destroying termite Reticulitermes santonensis.

2. Experimental details

2.1. Sol preparation (A) To embed SA and BA (Fluka, Buchs, Switzerland), sol A was prepared by stirring together 100 ml tetraethoxysilane, 400 ml ethanol and 200 ml 0.01 N HCl for 20 h at room temperature. For coating of cellite foil, this sol was mixed with different amounts of 10% alcoholic solutions of SA and BA, see Table 1. The viscosity h of the sols was measured at 208C using a Rheology International rotation viscometer (Model RI / 2 / L, Shannon, Ireland) with spindle VL6 and a speed of 80 rpm. (B) To embed BoA (Fluka), sol B was prepared by stirring together 100 ml tetraethoxysilane, 420 ml ethanol and 20 ml 0.01 N HCl for 20 h at room temperature. For coating, this sol was mixed with

59

different amounts of solid BoA, see Table 2. The viscosity h was determined as above.

2.2. Film preparation All substrates (cellite or cellulose acetate foil) were coated on a film casting device LBM 70 (MABA Spezialmaschinen Wolfen, Germany), equipped with a 70 mm slide coating unit and air drying. Layer thickness was controlled by the delivery of the coating solution and varying coating speed between 1 and 6.5 m / min. The antimicrobial composite layers were prepared by mixing 10 ml of silica sol A or B with 1 to 5 ml 10% BA, SA (or their mixtures) or BoA in ethanol. In some cases these mixtures were treated with solid soluble hydroxypropylcellulose (HPC) Klucel  / Aqualon, either high viscosity type H (MW 1 150 000) or low viscosity type E (MW 80 000). The mixed solution was continuously casted onto 70 mm cellulose acetate base at 1 m / min. The prepared layers are listed in Tables 1 and 2. Layer thickness was determined by measuring the optical interference of perpendicularly incident reflected white light using the photodiode array spectrometer MCS 400 with the software programme SDICKM (Zeiss, Germany).

Table 1 Composite layers from silica gels with sorbic acid (SA) and benzoic acid (BA) on 140 mm cellite foil Number of layer

SAa

BAa

HPC b

Ratio BC / silica (w / w)

h 208C (mPa?s)

Layer thickness (nm)

Liberation c SA

BA 2

1 2 3 4 5 6 7 8 9 10 11

3 4 5 3 1.5 2.0 2.5 1.5 – – – a

– – – – 1.5 2.0 2.5 1.5 3 3 3

– – – 0.1% H – – – 0.1% H 0.5% E 1.0% E 1.5% E

46 / 54 53 / 47 59 / 41 46 / 54 47 / 53 54 / 46 60 / 40 47 / 53 47 / 53 47 / 53 47 / 53

2.90 2.89 2.84 10.55 3.08 3.04 2.96 10.50 4.60 7.21 10.90

245 236 239 529 280 264 259 481 851 1664 2133

(mg / cm )

(%)

(mg / cm 2 )

(%)

19.80 25.50 33.70 48.60 19.37 23.56 29.95 23.24 – – –

88.0 84.4 99.6 77.1 90.0 92.3 98.3 71.0 – – –

– – – – 18.30 23.09 32.23 25.13 48.39 53.77 73.34

– – – – 94.2 95.9 98.8 78.0 68.1 66.8 71.4

Millilitre of acid (10% w / w in EtOH) mixed with 10 ml sol A. Hydroxypropylcellulose Klucel  /Aqualon, Type H (MW 1 150 000), Klucel E (MW 80 000). c Liberation into water after 24 h at 258C; percentage liberation in relation to the total amount of BC, analysed by extraction of coated foils in methanol (15 min, 508C, ultrasonic bath) and HPLC determination of SA and BA. b

¨ et al. / Journal of Controlled Release 60 (1999) 57 – 65 H. Bottcher

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Table 2 Composite layers from silica gels with boric acid on cellulose acetate foil Number of layer

BoAa

12 13 14 15 16 17 18

125 210 330 500 210 210 210

HPC b

– – – – 0.5% E 1.0% E 2.0% E

Ratio BC / silica (w / w)

h 208C (mPa?s)

Layer thickness (nm)

Liberation c (mg / cm 2 )

(%)

20 / 80 30 / 70 40 / 60 50 / 50 30 / 70 30 / 70 30 / 70

1.94 2.04 2.22 2.52 4.55 7.31 15.71

366 377 386 352 543 684 1285

0.78 1.51 2.08 4.45 1.96 2.33 4.78

62.0 65.9 66.4 83.6 55.2 43.3 39.3

a

Milligram of boric acid mixed with 10 ml of sol B. Hydroxypropylcellulose Klucel  /Aqualon, Type E (MW 80 000). c Liberation into water after 24 h at 258C. b

2.3. Measurement of the amounts of encapsulated agent released from films The controlled release of the biocides SA, BA and BoA from the silica matrix into water was investigated by leaching of the coated cellite and cellulose acetate films using a thermostatic evolution tester PTWS3 (Pharma Test Apparatebau, Hainburg, Germany) equipped with a paddle stirrer (3.5330 cm 2 film sample, 600 ml distilled water, 258C / 458C, paddle speed 250 rpm). The quantities of SA and BA in the wash-out solution were determined directly by HPLC after 30 min, 1 h, 3 h, 6 h and 24 h (with photodiode array detector Waters 996, 43100 mm column Nucleosil 120 C 18 (Knauer, Germany), eluent 1 ml / min of 0.05 M NaH 2 PO 4 with 4 mmol cetyltrimethylammonium hydrogensulphate / CH 3 OH / CH 3 CN (50 / 35 / 15). The deviation after three liberation measurements was less than 10%. The liberated BoA was quantified by spectrochemical analysis of boron in the wash-out solution using an ICP-OES-spectrometer (inductively coupled plasma optical emission spectrometry) Plasmaquant 110 (Zeiss). The sampling from the wash-out solution occurred after 30 min, 1 h, 3 h, 6 h and 24 h.

2.4. Microbiological investigations The antibacterial and fungicidal properties of the coated cellite foils were tested on various microorganisms commonly found in contaminated food:

Enterobacteriacea (E. coli), lactic acid-forming bacteria (L. plantarum) and moulds (Penicillium sp.). For the biological tests five parallel samples and one reference sample with the pure silica sol were used. The initial concentrations of colonies were about 10 3 colony forming units (CFU) / ml for moulds and 10 4 and 10 5 CFU / ml for the bacteria, applied as a hanging drop. The suspensions of bacteria or mould in contact with the preservation foil were incubated at 378C (bacteria) or 228C (moulds) for 4 days in a humidity chamber. Standard microbiological methods were used to determine the number of colonies present [25]. The fungicidal effects of modified silica coatings with embedded BoA on wood were tested using the wood fungus C. puteana. In each case four parallel samples of pine wood (Pinus sylvestris, L.; 0.53 g / cm 3 ) were impregnated by dip coating (15 min) with two modified silica sols and one pure silica sol as a reference. According to the rules of DIN EN 113 (German version of European standard), the treated pine wood samples were conditioned at 208C and 65% relative humidity. The sterilised samples in contact with inoculated culture medium (agarose gel) were incubated at 228C and 70% relative humidity for 16 weeks. Decreases in the mass of the wood samples were analysed to determine the biocidal activity of each composite layer against C. puteana. Using the impregnated and sterilised pine wood samples confirmed biocidal activity against the wood destroying termite R. santonensis, following the rules of DIN EN 117. The insecticidal effects of the sols

¨ et al. / Journal of Controlled Release 60 (1999) 57 – 65 H. Bottcher

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were quantified by determining the mortality of the termites and the decrease in mass of the wood sample.

the smallest additions of high viscosity HPC (type H) causes a strong increase in sol viscosity. 2. Incorporating the strongly swelling HPC within the composite layers promotes water penetration, thus improving diffusion into and out of the layer.

3. Results and discussion

Fig. 2 illustrates the liberation of SA and BA embedded in different composite layers. It shows that there are only small differences between the liberation rates of SA and BA. For both acids, the process of liberation began almost simultaneously, but even after 24 h of vigorous stirring in water, it was not complete. This demonstrates the outstanding long term liberation effect of such composite layers. Further investigations have shown that the liberation rate is increased by raising the temperature, e.g., the rise from 258C to 458C increases the liberation rate by a factor of 1.35 (SA) and 1.54 (BA). Regarding their application in food preservation the relationship between releasing behaviour per unit area and biocide effects for these composite layers was of special interest. The antibacterial and fungicidal properties of the prepared foils were tested in contact with various microorganisms that commonly contaminate food:

In a similar way as for previously investigated modified silica layers with incorporated BA [14], transparent stable films were obtained by embedding SA either alone or combined with BA, see Table 1. It is imperative that gentle drying conditions are used when preparing composite films containing SA and / or BA. Both organic acids are highly volatile and can leave the layer during the drying process, especially if alcohol and water are present. Therefore it is recommended that the composite layers are dried slowly at temperatures below 508C. To evaluate the purely thermal loss of SA and BA during drying, their thermal liberation from dried composite layers was investigated between 25 and 808C by HPLC determination of the SA and BA content before and after the thermal treatment. Even after 2 h annealing at 608C, the loss of SA and BA was very small. However after 2 h at 808C, 21% SA and 22% BA had been removed from the composite layer, indicating that essential losses occur during drying and are connected with the evaporation of water and alcohol. It follows from this that the ratio of silica-to-organic acid in the sol and the layer are different, and it is necessary to check the actual levels of SA and BA within the layer by entire leaching and HPLC analysis. Table 1 shows that these levels depend on sol composition and layer thickness, the latter being controlled by coating parameters and sol viscosity (for dip coating cf. Refs. [26,27], for continuous coating cf. Ref. [28]). For layers 9–11, the layer thickness increases with sol viscosity and is proportional to the liberated BA. Therefore, it is possible to control the releasing behaviour of the composite layers by adding swelling polymers such as HPC, which improve the liberation of the embedded organic acids in two ways: 1. By increasing sol viscosity and thus layer thickness, the absolute quantity of product available to be released is increased, see layers 4, 8–11. Even

1. Enterobacteriacea (E. coli), a typical indicator of faecal contamination that causes rotting, 2. Lactic acid-forming bacteria (L. plantarum), which typically cause spoilage of meat due to overacidification, and 3. Moulds (Penicillium sp.), responsible for the spoilage of bread. For all the microorganisms it was found that the releasing behaviour of BA and SA correlates well with their antimicrobial activity. The inhibition of the growth of L. plantarum is shown in Fig. 3. In a similar way the growth of E. coli and Penicillium sp. were completely inhibited by contact with a layer liberating more than 10 mg SA / cm 2 and 10 mg BA / cm 2 , respectively. These quantities are considerably smaller than normally used in food conservation and much smaller than the maximum permitted amounts of preservatives (2 mg / dm 2 ) in packing foils for food [22]. It could also be shown that packaging a foodstuff such as bread or barbeque sausages in foil coated with the biocidal ingredients

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¨ et al. / Journal of Controlled Release 60 (1999) 57 – 65 H. Bottcher

Fig. 2. Liberation of sorbic acid (solid line) and benzoic acid (dashed line) from different composite layers into water at 258C.

under investigation inhibits the growth of microorganisms clinging to its surface and substantially improves its storage stability. Similarly, embedding BoA within a silica matrix using the procedure described above results in transparent stable composite layers, see Table 2. Again it is essential to keep the drying conditions

gentle on account of the volatility of BoA in steam. As for the food preservative composites, the BoA containing silica films should also preferably be dried slowly and at low temperatures, about 258C. The results of leaching and ICP-OES analysis show that the amounts of released BoA depend on the sol composition, see Table 2 and Fig. 4. On one

Fig. 3. Colony forming units (CFU) of L. plantarum in contact with different silica layers with embedded sorbic acid (see Table 1).

¨ et al. / Journal of Controlled Release 60 (1999) 57 – 65 H. Bottcher

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Fig. 4. Liberation of boric acid from different composite layers into water at 258C.

hand, the liberation of BoA from pure SiO 2 -matrices is controlled by the ratio of BoA-to-silica. For layers 12 (20:80, w / w), 13 (30:70, w / w) and 14 (40:60, w / w) the amounts of liberated BoA (layer 12, 62.0%; layer 13, 65.9%; layer 14, 66.4%) are proportional to the increase of this ratio. The discontinuous rise of liberation at layer 15 (50:50, w / w; liberation: 83.6%) demonstrates that only up to 40% w / w BoA can be effectively encapsulated in a silica matrix. On the other hand, it is possible to control the release of BoA by adding polymers such as HPC, which have a twofold influence on the liberation of the encapsulated BoA: 1. As for the composites containing SA / BA, adding HPC increases sol viscosity and thus layer thickness. Therefore, the absolute quantity of releasable BoA per unit area is raised, see layers 13, 16–18. 2. Macromolecular compounds that contain hydroxyl groups within their chain architecture, e.g., HPC, have a pronounced tendency to complex with BoA. Calculations for liberation into water after 24 h with respect to the absolute content of BoA per unit area (layer 13, 65.9%; layer 16, 55.2%; layer 17, 43.3%; layer 18, 39.3%) illustrate the possibility of control using complexes

formed between BoA and simultaneously incorporated polyhydroxy compounds. The formation of complexes between the cellulose derivatives and BoA was proved by conductimetric measurements. The resistance of impregnated pine wood samples to fungi and insects was tested with organisms relevant to the approval process for wood preservatives: 1. C. puteana, L. — fungus used according to the rules of DIN EN 113, 2. R. santonensis — wood-destroying termites according to DIN EN 117. For all the ligniperdous organisms 30% w / w of BoA within the silica gave sufficient protection to the impregnated wood. Table 3 illustrates the inhibition of growth of C. puteana. Even after 20 weeks of incubation no decrease of mass was observed. The relatively low decrease of mass in the case of samples treated with pure sol B could be explained with the changed hygroscopicity and increased hardness of the wood–silica composite which have an effect on fungoid growth. The test involving termites resulted in 100% mortality and no decrease of mass

¨ et al. / Journal of Controlled Release 60 (1999) 57 – 65 H. Bottcher

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Table 3 Biocidal activity of BoA containing silica sols Layer

Silica sol B 13 15

Coniophora puteana

Reticulitermes santonensis

Decrease of mass (%)

Decrease of mass (%)

Mortality (%)

Treated wood

Untreated wood

Treated wood

Untreated wood

0.5 0 0

42.0 26.6 19.4

13.3 0 0

26.2 26.2 26.2

49.0 100 100

(see Table 3). The results of both leaching and microbiological investigations in conjunction with the low ecotoxicity of the compounds considered suggest that these composite layers have great potential as ecologically beneficial wood preservatives.

Acknowledgements

4. Conclusions

This study is financially supported by the Deutsche Bundesstiftung Umwelt: grant number 08001 / 02.

The results confirm that the release of biocidal compounds from the silica film can be controlled by the mass ratio of silica to organic compound and by modifying the silica matrix, especially by adding soluble or swelling penetration agents such as HPC. Not just composition but also the drying process is important. This governs porosity and the relationship between nanopores and mesopores within the matrix, i.e., the releasing behaviour. One advantage of applying these systems is that the antimicrobial compound is only liberated in the presence of moisture (‘‘liberation on demand’’). The results demonstrate that the sol–gel technique is a versatile new method for embedding and immobilizing biocidal additives within an inorganic matrix and offer an interesting alternative to the present widely-used techniques with organic polymers or supermolecular carriers. The sol–gel matrix is transparent, inert, nontoxic, stable to heat and light, and can be applied to different substrates such as polymer foil, paper, tissue or wood. Oily and high viscosity substances of pharmaceutical interest, e.g., nitroalcohols and panthenol can easily be incorporated into a silica xerogel matrix up to levels of 50% w / w, producing dry bulk products or film coatings [19]. Therefore, sol–gel composites with embedded BCs could be of interest for new biological and medical depot systems with excellent long-term

effects, e.g., transdermal or dermal therapeutic systems.

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