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Plant Extract-loaded Bacterial Cellulose Composite Membrane for Potential Biomedical Applications

  • Bacterial cellulose (BC) has been extensively explored as biomaterial for various biomedical applications owing to its non-toxic nature and unique structural morphology and impressive physico-chemical and mechanical properties. However, its high production cost and lack of antimicrobial activity have restricted its large-scale production and therapeutic applications. Therefore, the current study is aimed to devise a strategy for low-cost BC production and develop its composite with bioactive materials to bless it with antimicrobial activity. Herein, 5 mm thick reticulated fibrous and highly porous BC was produced by utilizing the wasted rotten tomatoes as the production medium. The produced bacterial cellulose waste (BCW) (i.e., produced from wastes) was ex-situ modified with bioactive plant extract (PE) obtained from Euclea schimperi, and the bactericidal activity of the developed BCW/PE was evaluated against Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli through disc diffusion and colony forming unit (CFU) count methods. The BCW/PE composite showed high bactericidal activities against S. aureus and produced clear inhibition zone whereas negligible activity was observed against E. coli, indicating its bactericidal activity mainly against the Gram-positive bacterium. Overall, this study illustrates that there is a huge potential for developing valuable biomaterials from food wastes and utilizing their liquid holding capabilities for value-added applications in medical and pharmaceutical fields.
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Plant Extract-loaded Bacterial Cellulose Composite Membrane for Potential Biomedical Applications

    Corresponding author: Muhammad Wajid Ullah, wajid_kundi@hust.edu.cn
    Corresponding author: Mazhar Ul-Islam, mulislam@du.edu.om
  • a. Department of Chemical Engineering, College of Engineering, Dhofar University, Salalah, Sultanate of Oman
  • b. Department of Biomedical Engineering, Huazhong University of Science and Technology, Wuhan 430074, China

Abstract: Bacterial cellulose (BC) has been extensively explored as biomaterial for various biomedical applications owing to its non-toxic nature and unique structural morphology and impressive physico-chemical and mechanical properties. However, its high production cost and lack of antimicrobial activity have restricted its large-scale production and therapeutic applications. Therefore, the current study is aimed to devise a strategy for low-cost BC production and develop its composite with bioactive materials to bless it with antimicrobial activity. Herein, 5 mm thick reticulated fibrous and highly porous BC was produced by utilizing the wasted rotten tomatoes as the production medium. The produced bacterial cellulose waste (BCW) (i.e., produced from wastes) was ex-situ modified with bioactive plant extract (PE) obtained from Euclea schimperi, and the bactericidal activity of the developed BCW/PE was evaluated against Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli through disc diffusion and colony forming unit (CFU) count methods. The BCW/PE composite showed high bactericidal activities against S. aureus and produced clear inhibition zone whereas negligible activity was observed against E. coli, indicating its bactericidal activity mainly against the Gram-positive bacterium. Overall, this study illustrates that there is a huge potential for developing valuable biomaterials from food wastes and utilizing their liquid holding capabilities for value-added applications in medical and pharmaceutical fields.

1.   Introduction
  • Biopolymers are receiving increasing attention as wound dressing materials owing to their potential therapeutic performance and drug-loading and release capabilities (Liu et al., 2010). To this end, the potential of bacterial cellulose (BC), a biopolymer produced by different microbes and cell-free systems (Ullah et al., 2015, 2017; Kim et al., 2019) has received immense consideration in biomedical and other fields. Despite the structural analogy, the BC possesses superior features and broad-spectrum applications in biomedical field as compared with the plant cellulose owing to its pure nature and outstanding physico-chemical, mechanical, and biological properties (Ul-Islam et al., 2015). The porous and fibrous geometry and availability of hydrogen bonding sites in the BC allow the physical adsorption and chemical interaction of different nanomaterials and polymers with the BC, thus leading to the development of its composites for various applications in environment (Shoukat et al., 2019), energy (Li et al., 2014), food (Shi et al., 2014), optoelectronics (Khan et al., 2015a), and support for wide library of catalysts (Kamal et al., 2019a, 2019b, 2019c). Beside these, both pure BC and its composites are extensively explored for their biomedical applications such as wound dressing (Mogoşanu and Grumezescu, 2014; Di et al., 2017), tissue engineering (Lamboni et al., 2019; Wang et al., 2020), additive manufacturing (Aljohani et al., 2018; McCarthy et al., 2019), cancer diagnosis (Ul-Islam et al., 2019), synthetic organs (Geisel et al., 2016; Yang et al., 2018), artificial blood vessels (Klemm et al., 2001), controlled-release drug carrier (Li et al., 2018), injectable embolization agent (Chen et al., 2014), and several others (Ul-Islam et al., 2019).

    Despite the unique features and broad-spectrum applications, the high production cost, low yield, and lack of certain functional features such as antimicrobial activity, adhesion sites (i.e., limited biocompatibility), and conducting and magnetic properties restrict the large-scale production and commercialization of pristine BC and BC-based composite materials. Extensive efforts have been made to overcome such limitations. For example, the yield of the BC has been improved to some extent by developing various bioreactors and developing genetically engineered strains (Islam et al., 2017). Similarly, the production cost has been greatly minimized by utilizing various wastes and low-cost substrates (Ul-islam et al., 2020). Similarly, the BC has been functionalized with various polymers and nanomaterials by different methods such as in-situ, ex-situ, and solution casting not only to impart antibacterial activity, antioxidant and conducting properties but also to improve its existing features like biocompatibility and optical transparency (Shah et al., 2013).

    The traditional medicines are mostly based on old-age knowledge of different plant extracts and their usage in the treatment of various diseases. Various natural products have been extensively explored for their therapeutic uses and have continued to play an important role in improving human health (Giday et al., 2003). The natural products obtained from different plants (i.e., phytochemicals) such as flavonoids, alkaloids, terpenes, and various phenolic compounds are the key sources of drug molecules in modern and folk medicines, food supplements, and nutraceuticals (Wang et al., 2013; Ibrar et al., 2020). For example, Euclea schimperi (Ebenaceae) is a traditional plant commonly used in the treatment of wounds, eye disorders, and teeth infections (Nwachukwu et al., 2010). The leaves of this plant are used in the treatment of skin infections, snake bites, scabies, and acne (Gebre-Mariam et al., 2006). A study has also reported the antimicrobial activity of the extract of this plant against various bacterial strains such as Staphylococcus aureus, Klebsiella pneumoniae, and several other pathogenic microbes (Mekonnen et al., 2018).

    Although the BC has been functionalized with different antimicrobial polymers and nanomaterials for wound dressing applications, its functionalization with bioactive materials of plant origin is still a scarcely explored area. Therefore, the current study was aimed to functionalize the low-cost produced BC with the extract of E. schimperi in an effort to develop a potential low-cost wound dressing material and drug delivery system. The BC was successfully produced by using wasted rotten tomatoes and functionalized with ethanolic extract of E. schimperi, which effectively inhibited the growth of S. aureus and partially of E. coli, indicating a green and low-cost development of healthcare biomaterials.

2.   Materials and Methods
  • The BC-producing Gluconacetobacter hansenii PJK (KCTC 10505BP) strain was cultured in a basal medium containing glucose 10 g/L, peptone 7 g/L, yeast extract 10 g/L, succinic acid 0.2 g/L, acetic acid 1.5 mL/L, and agar 20 g/L, and incubated under shaking at 150 r/min, 30 ℃, and pH 5 for 16-24 h, as reported previously (Ul-Islam et al., 2014). Other microbial strains including E. coli (ATCC 25922) and S. aureus (ATCC 10390) were cultured both in liquid Mueller-Hinton broth and on Mueller-Hinton agar plates. Both strains were incubated at 37 ℃ for 1-24 h.

  • Wasted rotten tomatoes were collected from the local market and autoclaved, followed by blending to extract their juice. The obtained juice was filtered and sterilized by keeping in oven at 70 ℃ for 6 h. The pH of the juice was adjusted to 5, as the optimum pH for growth of G. hansenii (Khan et al., 2016). The prepared juice, without any additional supplement, was used as BC-production medium.

  • The BC sheets were comparatively produced in sterilized rectangular containers by using the basal medium and the wasted rotten tomatoes production medium. Briefly, 1 L of each media were separately inoculated with 5% freshly prepared pre-culture of G. hansenii. The containers were covered with cotton caps to allow air penetration and incubated statically at 30 ℃. After 3, 5, and 7 days, the BC sheets produced at the air-medium interface were harvested and treated with 0.3 mol/L NaOH for 15 min at 103 kPa and 121 ℃ to disrupt and dissolve the cells and debris, as reported previously (Ul-Islam et al., 2013b). The BC produced by basal and wasted rotten tomatoes media were named as BCB and BCW, respectively, and their production was determined after 3, 5, and 7 days. All washed BC sheets were stored at 4 ℃ until further use.

  • The plant E. schimperi (stem and leaves) was collected from Salalah, Oman, shade dried, and grounded. About 300 g grounded plant material was extracted in 1.5 L ethanol in a soxhlet apparatus for 10 h. The resulting extract was filtered by using Whatman® microfilter (0.45 μm pore diameter; GE Life Sciences, Pittsburgh, PA, USA). The extract was concentrated to 13 g crude residue under reduced pressure at 50 ℃ using the Buchi Rotavapor R-200 and named as the 'plant extract' (PE).

  • The PE was ex-situ loaded into the BCW. Briefly, 5 cm × 5 cm BCW pieces were put into the PE and stirred for 5 h at room temperature. The extract was both adsorbed onto the surface and impregnated into the matrix of BCW. The extract-loaded BCW membranes, i.e., BCW/PE, were stored at 4 ℃ for further use.

  • The morphological features of freeze-dried BCB, BCW, and BCW/PE composite were observed through field emission scanning electron microscopic (FE-SEM, Hitachi S-4800 and EDX-350, Horiba, Tokyo, Japan). Briefly, all samples were fixed onto a brass holder and coated with osmium tetroxide (OsO4) using a VD HPC-ISW osmium coater (Tokyo, Japan) prior to FE-SEM observation. Both surface and cross sectional micrographs were taken for pristine BCB, BCW, and BCW/PE composite.

  • The antibacterial activity of pristine BCW and BCW/PE composite was determined against E. coli and S. aureus via the disc diffusion and the colony forming unit (CFU) methods, as reported previously (Khan et al., 2015b; Ullah et al., 2016a).

    The antibacterial activity test via disc diffusion method was performed on agar plates of respective bacterial strains. Briefly, the antibacterial activity of BCW/PE was determined by cutting it into disc shape with a diameter 6 mm and sterilized at 121 ℃ at 103 kPa for 15 min. Next, 100 µL of fresh pre-cultures of each E. coli and S. aureus were spread on respective agar plates, and the disc impregnated with E. schimperi extract (2 mg or 20 µL) was placed on top and incubated at 37 ℃ for 24 h. Finally, the inhibition zones were measured. Herein, the disc prepared from pristine BCW and 10 µg/mL ampicillin were used as the negative control and positive control, respectively.

    By CFU method, the antibacterial activity of BCW/PE was determined by adding 0.02 g/mL of finely sliced and autoclaved BCW/PE sample into the 9 mL growth media of both E. coli and S. aureus in separate tubes. The tubes were seeded with 1 mL of fresh pre-cultures of each E. coli and S. aureus and incubated under shaking at 150 r/min and 37 ℃ for 18 h. After incubation, 100 µL sample from each tube was taken and spread on the respective agar plates and incubated again at 37 ℃ for 24 h. After incubation, the numbers of colonies formed on the agar plates were counted and antibacterial activity was determined by comparing with the reference (i.e., pristine BCW). The experiment was performed in triplicate and average values were recorded.

3.   Results and Discussion
  • Despite a biomaterial of vital importance especially in biomedical field, the widespread applications of the BC have been limited by its high production cost and lack of certain features as discussed above. In the present study, the potential of wasted rotten tomatoes liquid extract was utilized as an alternative culture medium for low-cost BC production. The investigation of total sugar contents (data not shown) in wasted rotten tomato extract indicated a total of 5% sugars, which was sufficient to be utilized as the carbon sources for the BC production. The BC was produced by G. hansenii PJK as a thin layer at the air-medium interface on the second day of incubation by separately utilizing both the basal and waste rotten tomatoes media as the carbon source. The thickness of the BC sheet increased with the time and reached (4 ± 0.13) mm and (3.91 ± 0.10) mm for the BCB and BCW, respectively, on the fifth day of cultivation. The increased thickness of the BC membrane could be attributed to the continuous production of cellulose fibrils on account of utilizing carbon source and addition to the growing membrane (Tang et al., 2010). The average thickness of the BCB and BCW hydrogels reached (5 ± 0.11) mm and (4.93 ± 0.09) mm, respectively, after seven days of incubation (Fig. 1). After treating with NaOH and washing with distilled water, the BCB and BCW hydrogels produced from basal and wasted rotten tomatoes media appeared whitish and pinkish in color, respectively. The efficacy of both media in terms of produced BC was determined after 3, 5, and 7 days, under static conditions by harvesting, washing, and determining the cellulose content. After three days, a maximum of 0.77 and 0.71 g/L BC was produced from basal and wasted rotten tomatoes media, respectively. The production continued with the same trend, and 1.98 and 1.83 g/L BC was produced after five days, respectively. The BC production reached a maximum of 3.83 and 3.71 g/L after seven days, from basal and wasted rotten tomatoes media, respectively. These results show that although the BC production from wasted rotten tomatoes medium is slightly lower than that from the basal medium, its production by utilizing wasted food sources represents a cost-effective approach as compared with the expensive chemically defined basal or other media.

    Figure 1.  A and C, Inoculation and static incubation of Gluconacetobacter hansenii for bacterial cellulose production by using basal and wasted rotten tomatoes-based media, respectively; and C and D, BC produced and harvested from basal and wasted rotten tomatoes-based media, respectively.

  • The FE-SEM micrographs of surface and cross-sectional views of both BCB and BCW clearly indicate a densely arranged three-dimensional (3D) porous and fibrous web-shaped network (Fig. 2). The density and compactness of the BC fibrils are directly associated with the nature and amount of available carbon source, amount of inoculum, type of strain, and culturing conditions, as well as the post-synthesis processing and drying method (Tang et al., 2010; Ullah et al., 2016b). In the network structure of the BC, the fibers are interconnected through reversible hydrogen bonding which can be converted into irreversible hydrogen bonding upon freeze-drying (Ul-Islam et al., 2012) through direct conversion of ice into vapors, thus retaining the pore geometry of the BC (Ul-Islam et al., 2013a). The pores of different sizes (data not shown) provide an ideal environment for penetration of different materials, including liquids and solids of certain sizes and different shapes (Ul-Islam et al., 2012). In this study, the presence of pores in the web-shaped network of the BCW, as shown from the surface and more clearly from the cross-sections views, ensured the adsorption and penetration of the PE under ex-situ shaking, which could be potentially retained by the fibrous network through physical attachment or chemical interaction (data not shown), thus forming the BCW/PE composite. This is also evident from the FE-SEM micrographs of surface and cross-sectional views of BCW/PE composite which illustrate the impregnation of the PE within the fibrous network as well as variation in fibril diameter (Fig. 2). Additionally, the impregnation of the PE into the BCW matrix was confirmed through dry-weight analysis (data not shown). Overall, the morphological observation of BCW has comparable features to that of the BCB and successful impregnation of the PE into its matrix justifies the production of low-cost BC and development of its composites with plant-derived bioactive materials.

    Figure 2.  Representative FE-SEM micrographs of surface and cross-sectional views of BCB, BCW (BC produced by basal and wasted rotten tomatoes, respectively), and BCW/PE (extract-loaded BCW membranes).

  • The inexpensively produced BCW membranes were further subjected to their drug-carrying capabilities to evaluate their potential for biomedical applications. The bactericidal activity of the BCW composite membrane with extract of the medicinal plant E. schimperi was evaluated against S. aureus and E. coli as the model Gram-positive and Gram-negative bacterial species, respectively, via disc-diffusion and CFU count methods, and the results are shown in Fig. 3. The disc diffusion analysis indicated that the BCW/PE composite disc produced clear inhibition zone of about 2 mm by inhibiting the growth of S. aureus, while it failed to produce a very clear inhibition zone against E. coli. On the other hand, the BCW did not produce any inhibitory zone against both S. aureus and E. coli. The findings of the CFU count method were in accordance with the results of disc diffusion method, where the BCW/PE effectively reduced about 90% growth of S. aureus as compared with the reference (i.e., BCW). Similarly, the BCW/PE did not reduce the growth of E. coli. These results indicate that the extract of E. schimperi plant contains bioactive materials having bactericidal activity only against Gram-positive bacteria. As the BC is known for its non-bactericidal nature (Ullah et al., 2016a), while E. schimperi has known antibacterial activity due to the presence of various secondary metabolites and is traditionally used to treat skin infections and wound infections (Mekonnen et al., 2018), the antibacterial activity of BCW/PE against S. aureus could be attributed to the bactericidal effect of the PE. Although bactericidal in nature, it was quite possible that the active compounds in the PE may lose their activities upon physical or chemical interaction with the cellulose chain in the BCW/PE composite; however, the results of the bactericidal effect of the composite indicated that bioactive compounds in the PE retained their bactericidal activity. These findings open a gateway of developing the composites of the BC with different plant extracts to explore their medicinal use for applications in developing medical, cosmetic, and pharmaceutical products.

    Figure 3.  Representative photographs of comparative antibacterial activities of BCW/PE composite membrane discs with positive (ampicillin) and negative (BCW) controls against S. aureus and E. coli through disc diffusion and CFU count methods.

4.   Conclusions
  • Bacterial cellulose was cost-effectively produced by utilizing the recycled wasted rotten tomatoes medium. Furthermore, a bioactive PE obtained from one of the local plants in Oman was incorporated through ex-situ composite development strategy. The BC exhibited high potential of extract holding capacity which in turn conferred it with bactericidal properties against Gram-positive S. aureus. This illustrates that the improvement of existing features of the BC as well as the introduction of additional features is achievable by supplementing it with suitable materials for specific applications. Overall, this study provides a base both for the low-cost production of the BC by utilizing the food wastes and developing its composites with natural materials to simultaneously address the environmental issues and minimizing the cost for BC-based materials for biomedical use.

Conflict of interest
  • The authors declare that there is no conflict of interest associated with the publication of this work.

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