Transforming biomass into bioactive coatings by atmospheric plasma

Abstract

A method for forming a coating from biomass using plasma technology operated either at atmospheric or at low pressure is disclosed.

Description

TRANSFORMING BIOMASS INTO BIOACTIVE COATINGS BY ATMOSPHERIC PLASMA PRIORITY DOCUMENT

[0001] The present application claims priority from Australian Provisional Patent Application No.2023902971 titled “TRANSFORMING BIOMASS INTO BIOACTIVE COATINGS BY ATMOSPHERIC PLASMA” and filed on 14 September 2023, the content of which is hereby incorporated by reference in its entirety. TECHNICAL FIELD

[0002] The present disclosure relates to bioactive coatings. In a particular form the present disclosure relates to processes for forming bioactive coatings using plasma. BACKGROUND

[0003] Wound infection and subsequent healing impairment is a complex process with a significant global impact, affecting millions of individuals worldwide.[1,2] In particular, chronic wounds result in substantial morbidity due to extended healing times and often the inability to heal.[3] This ubiquitous problem highlights the need for innovative materials and methods capable of enhancing wound healing and the rate of recovery. Beyond alleviating symptoms, such as pain, effective wound treatment can help reduce hygiene-related complications and the risk of infection, which pose serious threats to successful healing. The clinical need, combined with the growing wound care product market, projected to reach between $15 and $22 billion annually by 2024,[4] underscores the opportunities for the introduction of novel wound treatments.

[0004] Wound infections arise from contamination by pathogenic bacteria, leading to tissue colonization and the expression of virulence factors.[1,2,5] Factors influencing wound infections highly depend on the type of wound, the patient’s overall health status, and the healthcare environment. With an estimated 1%–3% of all surgeries in the United States resulting in surgical site wound infections, and up to 50% of diabetic foot ulcers becoming infected,[1] the management of wound infections is of paramount importance. Antibiotic treatment is the principal intervention used to curtail infections.[1,2] The overuse and misuse of antibiotics are known to lead to the development of antibiotic resistance and are rapidly becoming the most urgent problem in healthcare.[6] Silver-based dressings have been widely used in the treatment of infected wounds.[7–9] Nevertheless, the wound-healing effectiveness of these dressings is compromised by the cumulative toxicity of silver.[10,11] Furthermore, a concerning development is the emergence of bacterial silver resistance in wound care, posing a serious challenge.[8,12,13]

[0005] Microalgae, such as Spirulina maxima, have emerged as a promising alternative in the quest for effectively treating wound infections.[14,15] It should be noted that S. maxima has the most recent scientific name Limnospira maxima;

[0016] however, due to the common use, S. maxima name is used throughout this specification. These unicellular organisms possess a simple reproductive system that generates a biomass containing bioactive compounds, including phycocyanin, anthocyanin, and active oligosaccharides.[16–18] Notably, these microalgae exhibit potent antioxidant activity that can counter the damaging effects of inflammation at the site of the wound, as well as strong antibacterial properties crucial for preventing infections.[14,15] Nevertheless, harnessing the full potential of these microalgae for wound care has traditionally been limited by several key challenges.[19,20] An important challenge arises when attempting to extract, refine, and encapsulate the valuable compounds derived from microalgae. The thick cell walls of microalgae, which serve as a protective barrier in their natural aquatic habitats, pose a significant obstacle, rendering the extraction processes inefficient.[19,20] Further complexity to the refinement and encapsulation procedures can lead to the degradation or loss of the bioactive compounds, consequently diminishing their potential benefits for wound healing.

[0006] There is thus a need to provide processes for accessing the biomass containing bioactive compounds in microalgae. Alternatively, or in addition, there is a need to provide new processes for producing wound healing compositions from microalgae. SUMMARY

[0007] According to a first aspect, there is provided a method for forming a coating directly from biomass using plasma operated either at atmospheric or at low pressure.

[0008] In certain embodiments of the first aspect, the method comprises providing a solution comprising biomass material, coating a substrate with the solution comprising biomass material, drying the coating on the substrate to form a biomass coated substrate, and exposing the biomass on the biomass coated substrate to a plasma formed from a gas at atmospheric pressure or sub-atmospheric pressure to produce a bioactive coating on the substrate.

[0009] In certain embodiments of the first aspect, the method comprises coating the substrate with the solution comprising biomass material to achieve a dry weight density of at least 0.05 mg cm−2. In certain of these embodiments, the method comprises coating the substrate with the solution comprising biomass material to achieve a dry weight density of from about 0.1 mg cm−2to about 0.1 kg cm-2. In certain specific embodiments, the method comprises coating the substrate with the solution comprising biomass material to achieve a dry weight density of about 0.1 mg cm−2.

[0010] In certain embodiments of the first aspect, the coating formed substantially preserves the bioactive components of the biomass.

[0011] In certain embodiments of the first aspect, the coating has antibacterial properties.

[0012] In certain embodiments of the first aspect, the coating has antifungal properties.

[0013] In certain embodiments of the first aspect, the coating has tissue healing properties.

[0014] In certain embodiments of the first aspect, the coating has anti-inflammatory properties.

[0015] In certain embodiments of the first aspect, the substrate material is selected from one or more of the group consisting of metals, ceramics, polymers, and composites.

[0016] In certain embodiments of the first aspect, the coating is deposited on material of complex shapes.

[0017] In certain embodiments of the first aspect, the coating has a tuneable degree of crosslinking.

[0018] According to a second aspect, there is provided a method for extracting compounds of interest from biomass comprising exposing biomass to a plasma operated either at atmospheric or at low pressure.

[0019] In certain embodiments of the first and second aspects, the method comprises exposing the biomass to atmospheric plasma for from about 1 sec cm−2to about 1000 min cm−2, such as about 100 sec cm−2or about 200 sec cm−2.

[0020] In certain embodiments of the first and second aspects, the gas is selected from one or more of the group consisting of argon, helium, nitrogen, oxygen and air. In certain specific embodiments of these aspects, the gas is an inert gas. In certain of these specific embodiments, the inert gas is argon.

[0021] In certain embodiments of the first and second aspects, the plasma is formed at a gas flow rate of from about 0.01 L / min to about 1000 L / min, such as about 10 L / min.

[0022] In certain embodiments of the first and second aspects, the plasma is located about 30 mm above the surface of the biomass.

[0023] In certain embodiments of the first and second aspects, the biomass is selected from one or more of the group consisting of algae, seaweed, and other biologically derived material. In certain of these embodiments, the biomass comprises Spirulina maxima. In certain other of these embodiments, thebiomass comprises Spirulina platensis.

[0024] According to a third aspect, there is provided a bioactive coating formed by the method of the first aspect.

[0025] According to a fourth aspect, there is provided a plasma deposited bioactive coating formed directly from a biomass material. BRIEF DESCRIPTION OF THE FIGURES

[0026] Embodiments of the present disclosure will be discussed with reference to the accompanying figures wherein:

[0027] Figure 1 shows a schematic representation of the setup of Ar-AAJ: (A) Schematic representation of the integrated plasma jet system with the optical emission spectroscopy characterization systems; and (B) outlet of the plasma jet reactor;

[0028] Figure 2 shows plots of the colour intensity of untreated and Ar-APJ treated microalgal samples at concentrations of (A) 0.1 mg cm-2and (B) 0.6 mg cm-2after submerging in water at 37 °C for various time points: 1, 12, and 24 hours, n=3;

[0029] Figure 3 shows (A) standard curve of total polysaccharide concentration; (B) standard curve of total protein concentration; and (C) standard curve of antioxidant ability;

[0030] Figure 4 shows schematic representations of the response mechanism of DAPI and phalloidin. DAPI (4′,6-diamidino-2-phenylindole) is a DNA-specific probe that binds to A-T rich DNA sequences in the minor grove to generate a fluorescent complex.

[0025] Phalloidin, a poisonous, quickly acting bicyclic peptide derived from the toadstool Amanita phalloides, causes G-actin to polymerize into filamentous forms

[0026] ;

[0031] Figure 5 shows an overview of argon atmospheric plasma jet (Ar-APJ) system. (A) Photograph showing the 5 setup argon atmospheric plasma treatment (Ar-APJ) used to transform Spirulina maxima biomass into 6 a robust ultrathin coating. (B) Photograph showing the argon atmospheric plasma jet (Ar- APJ). (C) OES 7 spectra of Ar-APJ at the argon flowrate of 10 LPM and voltage at 10 kV. (D) Illustration of how Ar-APJ 8 can turn S. maxima into a contiguous ultrathin coating;

[0032] Figure 6 shows OES spectra of argon atmospheric plasma jet produced at (A) 20 PLM, 10 kV; (B) 20 PLM, 7.6 kV; (C) 20 PLM, 5.2 kV; (D) 10 PLM, 10 kV; (E) 10 PLM, 7.6 kV; (F) 10 PLM, 5.2 kV; (G) 5 PLM, 10 kV; (H) 5 PLM, 7.6 kV; (I) 5 PLM, 5.2 kV;

[0033] Figure 7 shows morphological analysis of S. maxima following Ar-APJ treatment, observed using epifluorescence (left) and scanning electron microscopy (SEM) (right) imaging techniques. Morphology of A) untreated S. maxima. S. maxima treated with Ar-APJ for a duration of B) 100 seconds and C) 200 seconds over a surface area of 1 cm2, resulting in significant changes in the cell wall structure. D) Photographs showing the durability of coatings of untreated (Sm) and treated S. maxima (PSm) with the conditions of 200 sec m−2. Original coatings of Sm and PSm were then washed with water to examine their durability;

[0034] Figure 8 shows SEM images of morphology of S. maxima. (A) and (B) Morphology of S. maxima before plasma treatment. (C) and (D) Morphology of S. maxima before treating with plasma treatment with 200s over 1 cm2;

[0035] Figure 9 shows the results of water contact angle were measured on Sm and PSm surfaces with two concentrations including 0.1 and 0.6 mg / cm2. Data are presented as mean ± standard deviation, n = 3. ** p<0.01, ****p<0.0001;

[0036] Figure 10 shows the results of mapping the biochemical changes of untreated (Sm) and Ar- APJ-treated (PSm) coatings using synchrotron macro attenuated total reflection Fourier-transform infrared (ATR-FTIR) microspectroscopy. (A) Heat map images of different chemical components in S. maxima, including lipid, amide I / II (protein), and polysaccharide regions across Sm and PSm coatings. Scale bar = 20 µm. (B) PCA score plots of Sm and PSm. (C) PCA-PC1 loading spectra. (D) Second derivative FTIR spectra;

[0037] Figure 11 shows XPS spectra of Sm and PSm in (A) C 1s, (B) O 1s, and (C) N 1s regions;

[0038] Figure 12 shows XPS characterization of untreated and plasma-treated samples of S. maxima: (A) XPS survey. (B) P 2p spectra. (C) Mg 1s spectra. (D) K 2p spectra. (E) Ca 2p spectra. (F) Na 1s spectra;

[0039] Figure 13 shows the effects of treatment on bioactive components of S. maxima. (A) The diagrams showing the process to evaluating bioactive compounds of treated (PSm) and untreated (Sm) S. maxima. (B) Total polysaccharide concentrations, and (C) total protein concentrations of Sm and PSm. (D) The concentrations (µg mL−1) of released C-phycocyanin in PBS (10 x 10−3M, pH 7.4) from the Sm and PSm coatings. (E) DPPH assay used to quantify antioxidant activity of these coatings. n = 3, all data represented as mean ± SD, *p < 0.05,**p < 0.01, ***p < 0.001, ****p < 0.0001;

[0040] Figure 14 shows evaluation of antibacterial properties of untreated (Sm) and Ar-APJ-treated S. maxima (PSm) against Gram-negative Pseudomonas aeruginosa and Gram-positive Staphylococcusaureus. (A) Confocal laser scanning microscopy (CLSM) images and corresponding plots quantifying the antibacterial efficacy of Sm and PSm against (B) P. aeruginosa and (C) S. aureus. Scale bar = 10 µm. n = 3, all data represented as mean ± SD. ****p < 0.0001. (C) Illustrated schematic depicting the antibacterial mechanism of Sm and PSm involving reactive oxygen species (ROS) and membrane potential;

[0041] Figure 15 shows SEM images showing the morphology of (A) P. aeruginosa and (B) S. aureus on Sm and PSm. Scale bar (top row) = 10 μm, scale bar (inset - bottom row) = 2 µm;

[0042] Figure 16 shows the antifouling capacity of Sm and PSm against S. aureus and P. aeruginosa. (A) 3D model representations of Z-stack micrographs captured by CLSM. (B) Biovolume measurements. (C) Biofilm thickness. (D) Safranin absorbance study. n = 3, all data represented as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001;

[0043] Figure 17 shows membrane potential and intracellular ROS generation. (A) CLSM micrographs and ratio of polarized to depolarized membrane and fluorescence intensity of ROS created when incubated P. aeruginosa with Sm and PSm. (B) CLSM images and ratio of polarized and depolarized membrane and fluorescence intensity of ROS created when incubated S. aureus with Sm and PSm. Scale bar = 10 µm. n = 3, all data represented as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001;

[0044] Figure 18 shows evaluation of biocompatibility of Sm and PSm against THP-1 macrophage. (A) CLSM images of THP-1 macrophage cell in 1 day after incubating on Sm and PSm substrates. (B) Viability percentage normalized to tissue culturing plates (TCP) of THP-1 cell after incubating with Sm and Psm substrates for 1 and 3 days. (C) IL-6 concentration of THP-1 (pg mL−1) after incubating with Sm and PSm for 1 and 3 days. n = 3, all data represented as mean ± SD, *p < 0.01; and

[0045] Figure 19 shows evaluation of the wound healing ability of Sm and PSm. (A) CLSM images of HaCaT cell (blue shows nucleus stained with DAPI, and green shows F-actin stained with phalloidin) over the periods of 1 and 3 d, scale bar = 10 µm. (B) Viability normalized to tissue culture plates (TCP) of HaCaT cell after incubating with Sm and PSm in 1 and 3 days. (C) Wound scratch assay using HaCaT cells after incubation with Sm, PSm, TCP, and negative control (NC) from day 0 to day 2. (D) Scratch width of HaCaT cell after incubating with Sm and PSm (X means complete healing or wound closure). Scale bar = 100 µm. n = 3, all data represented as mean ± SD, *p < 0.001; and

[0046] Figure 20 shows (a) Schematic illustrating the operation process and study timelines; (b) Representative images of the healing process of infected wounds in the rats in the six different groups. (c-d) Wound closure rates at different time points; (e) Masson Trichrome staining of the different groups at 3rd, 7thand 21stday after surgery. (Scale bar, 2mm in Panoramic pictures and 200 μm in high field of view); (f) Statistical analysis of collagen deposition based on Masson Trichrome staining; (g-h) Immunofluorescence of COX-2 in wound area of different groups at day 3. The white arrow indicates the inflammatory infiltration area. (Scale bar, 50 μm). (i-j) Immunofluorescence of CD31 showed effective induction of blood vessels in the plasma treatment Darwin groups at 21st day. The white arrow indicates microvasculature. (Scale bar, 50 μm). Error bar represents mean ± s.d.; n = 5, ***p <0.001, **p < 0.01, *p < 0.05. DESCRIPTION OF EMBODIMENTS

[0047] The present disclosure arises from an innovative approach taken by the inventors to develop ultrathin coatings derived from S. maxima, integrating biotechnology and plasma physics. With the increasing demand and interest in antibacterial coatings, plasma techniques have gained prominence.[21– 23] The present inventors have harnessed the capability of an atmospheric plasma jet operated in an inert argon gas, to convert single cells of S. maxima biomass into a durable and functional bioactive ultrathin coating. Using this technology, all bioactive compounds within the biomass were preserved but also to enable the breakdown of polysaccharides present in the microalgal cell wall. The resulting formation of bioactive linkages significantly enhances the bioavailability of the wound-healing compounds present in the microalgal cells. The bioactive coatings produced from this process exhibit an ensemble of healing properties including promotion of skin cell proliferation and expediting wound closure. Moreover, the coatings possess anti-inflammatory properties, which are crucial in the management of wound-related inflammation and pain, and ensuring effective wound healing.[24,25] Importantly, the coatings display potent antibacterial characteristics, offering a protection from wound infections and reducing the need for antibiotics, and the associated problems with the development of resistance. To the best of the present inventors’ knowledge, this technology is the first of its kind capable of transforming single microalgal cells into a functional bioactive coating. This approach provides a comprehensive solution for wound healing via harnessing the full therapeutic potential of S. maxima using a plasma-assisted transformation of algal biomass into efficient and cost-effective coatings.

[0048] Disclosed herein is a method for forming a coating directly from biomass using plasma operated either at atmospheric or at low pressure. Also disclosed herein is a method for extracting compounds of interest from biomass comprising exposing biomass to a plasma operated either at atmospheric or at low pressure.

[0049] The method for forming a coating directly from biomass using plasma operated either at atmospheric or at low pressure comprises providing a solution comprising biomass material. A substrate is coated with the solution comprising biomass material and the coating is dried on the substrate to form abiomass coated substrate. The biomass on the biomass coated substrate is then exposed to a plasma formed from an inert gas at atmospheric pressure or sub-atmospheric pressure to produce a bioactive coating on the substrate.

[0050] As disclosed herein, an atmospheric plasma system has been designed to transform S. maxima biomass into an ultrathin coating. Based on the mechanism of action of the plasma described elsewhere herein, it is expected that other biomass materials could also be used to form a coating. When treating biomass with plasma, the success of the treatment depends on several variables, including the plasma power, gas flow rate, length of the treatment, and the distance between the plasma source and the sample. These variables may have an impact on the plasma density, temperature, and generation of reactive species, which may have an impact on the effectiveness of the therapy.

[0059] In Figure 1, the use of alternating current voltage (AC) for plasma formation in the treatment of S. maxima offers numerous advantages. First, using AC voltage makes it easier to create more homogeneous plasma, which is necessary for distributing plasma treatment evenly throughout the S. maxima culture.

[0060] This encourages the treatment of the microalga in a more predictable and effective manner. Additionally, the plasma density and temperature can be better controlled by AC voltage, which can be important for optimizing the procedure.

[0061] Manipulating the temperature can prevent thermal damage to the S. maxima culture, whilst manipulating the plasma density can result in the generation of more reactive species. Through an electric discharge, a plasma is produced in several steps. A gas first responds as an insulator when a voltage is applied to it

[0062] . The electric field in the gas, however, becomes powerful enough as the voltage rises to ionize the gas molecules and produce a conductive channel. The breakdown voltage is the name given to this voltage. Free electrons and ions are present in the gas after it has been ionized. These charged particles have the ability to ionize other gas molecules through collisions, releasing additional free electrons and ions

[0063] . Ionization is the process that creates self-sustaining plasma. As the plasma expands, the density of charged particles decreases, and some electrons and ions recombine to form neutral atoms or molecules. This process can also produce photons, which can contribute to the optical emission of the plasma. The ionization process and subsequent collisions between charged particles can also lead to heating of the gas, which increases the temperature of the plasma

[0064] .

[0051] In the methods disclosed herein, argon gas is used as an exemplary gas to generate plasma for the treatment of S. maxima due to several benefits associated with its usage. Argon is a popular choice for working gas in plasma treatment due to its inert nature, as it does not react with the material being treated and remains chemically stable in most plasma conditions

[0065] . This makes it an ideal gas for treating sensitive materials such as microalgae like S. maxima. Additionally, argon possesses a low ionization energy, which implies that it requires less energy to ionize when compared to other gases, such as nitrogen or oxygen

[0066] . This characteristic allows for the easier initiation of plasma and efficient plasma generation at lower voltages. The low ionization energy also minimizes the likelihood of damage to the S. maxima culture during the plasma treatment process

[0066] . Moreover, argon plasma can generate reactivespecies such as reactive oxygen species (ROS) and reactive nitrogen species

[0035] , which can be useful in treating S. maxima. These species help in breaking down the cell walls of S. maxima and enhance its digestibility, making it more accessible for consumption or extraction of valuable compounds.

[0067]

[0052] Whilst the use of argon has particular advantages as described, it is contemplated that other gases such as helium, nitrogen, air or oxygen could also be used to form the plasma.

[0053] The methods disclosed herein allow for the direct formation of a coating from biomass using plasma and without any additional chemical or physical steps required to form a coating and / or access bioactive compounds present in the biomass.

[0054] As used herein, the term ‘biomass’ means a quantity of living organisms. The methods described herein are particularly suitable for processing of biomass material in which the living organism(s) contain one or more bioactive compound or other compound of interest. The biomass may be an algae biomass. The algae biomass may include members of green algae, brown algae and red algae including, but not limited to, members of the blue-green algae Spirulina species including Spirulina maxima, and Spirulina platensis, members of the green algae Chlorella species including Chlorella ellipsoidea, and Chlorella vulgaris, members of the green algae Haematococcus species including Haematococcus pluvialis, members of the green algae Dunaliella species including Dunaliella salina, and members of the red algae Porphyridium species including Porphyridium cruentum. A particularly suitable algae biomass is Spirulina maxima. Another particularly suitable algae biomass is Spirulina platensis. Alternatively, or in addition, the biomass may be a seaweed biomass. The seaweed biomass may include members of the red, brown and green seaweeds including, but not limited to, Ecklonia cava, Ascophyllum nodosum, Cladosiphon okamuranus, Undaria pinnatifida, Saccharina longicruris, Saccharina latissima, Sargassum polycystum, Laminaria japonica, Fucus vesiculosus, Asparagopsis taxiformis, and Fucus serratus. Alternatively still, the biomass may be other biologically derived material. A solution comprising the biomass material is coated onto a substrate. The solution can be any aqueous, non-aqueous of mixed solvent solution of the biomass material. The concentration of biomass material in the solution may be from about 0.1 mg mL−1, such as 0.1 mg mL−1, 0.2 mg mL−1, 0.3 mg mL−1, 0.4 mg mL−1, 0.5 mg mL−1, 0.6 mg mL−1, 0.7 mg mL−1, 0.8 mg mL−1, 0.9 mg mL−1or 1.0 mg mL−1. In exemplary embodiments, the concentration of biomass material in the solution is about 0.5 mg mL−1.

[0055] The solution comprising the biomass material can be coated onto a substrate using any suitable coating technique including spray coating, dip coating, roller coating, etc.

[0056] The solution comprising the biomass material can be coated onto a substrate in order to achieve a desired density of biomass material on the surface. The desired density may be at least 0.05 mg cm-2, such as from about 0.1 mg cm−2to about 0.1 kg cm-2or from about 0.1 mg cm-2to about 0.6 mg cm-2,including 0.1 mg cm-2, 0.2 mg cm-2, 0.3 mg cm-2, 0.4 mg cm-2, 0.5 mg cm-2or 0.6 mg cm-2. For example, when the biomass material comprises S. maxima the desired density may be about 0.1 mg cm-2.

[0057] The substrate can be formed from any suitable material such as metals, ceramics, polymers, and composites. The substrate may be planar, curved, rigid, flexible or of complex shape. For example, the substrate may be a gauze, such as a wound dressing. Alternatively, the substrate may be a medical device such as a vascular graft, stent, bandage, glove, etc.

[0058] Once the solution comprising the biomass material has been coated onto a substrate at the desired density, the coating is dried on the substrate. Any suitable drying method can be used including air drying, room temperature drying, etc.

[0059] The dried coating is then exposed to a plasma formed from an inert gas at atmospheric pressure or sub-atmospheric pressure. The biomass in the dried coating may be exposed to atmospheric plasma for from about 1 sec cm−2to about 1000 min cm−2or about 50 sec cm−2to about 500 sec cm−2, such as about 50 sec cm−2, 100 sec cm−2, 150 sec cm−2, 200 sec cm−2, 250 sec cm−2, 300 sec cm−2, 350 sec cm−2, 400 sec cm−2, 450 sec cm−2or 500 sec cm−2. In some exemplary embodiments the biomass in the dried coating is exposed to atmospheric plasma for 100 sec cm−2or 200 sec cm−2.

[0060] The gas flow rate of inert gas used to form the plasma may be from about 0.01 L / min to about 1000 L / min, such as about 5 L / min to about 50 L / min. For example, the gas flow rate of inert gas used to form the plasma may be 5 L / min, 10 L / min, 15 L / min, 20 L / min, 25 L / min, 30 L / min, 35 L / min, 40 L / min, 45 L / min or 50 L / min. In some exemplary embodiments the gas flow rate of inert gas used to form the plasma is 10L / min.

[0061] The plasma may be positioned from about 1 mm to about 5000 mm above the surface of the biomass, such as from about 1 mm to about 500 mm above the surface of the biomass such as 5 mm, 10 mm, 15 mm, 20 mm, 25 mm, 30 mm, 35 mm, 40 mm, 45 mm or 50 mm above the biomass. In some exemplary embodiments the plasma is positioned about 30 mm above the biomass.

[0062] The plasma treatment results in the formation of a bioactive coating on the substrate. The coating may have antibacterial properties, antifungal properties, tissue healing properties and / or anti- inflammatory properties. It will be appreciated that the properties of the coating will depend on the composition of bioactive compounds within the biomass material.

[0063] Advantageously, the coating formed substantially preserves the bioactive components of the biomass.

[0064] In certain embodiments of the first aspect, the coating has a tuneable degree of crosslinking.

[0065] Also disclosed herein is a bioactive coating formed by the method disclosed herein.

[0066] Also disclosed herein is a plasma deposited bioactive coating formed directly from a biomass material. EXAMPLES

[0067] Example 1 - Plasma System for the Treatment of Microalgae Biomass

[0068] The configuration of the integrated atmospheric plasma jet system for the treatment of microalgae biomass was set up as shown in Figure 1A. An AC power supply (Variac SRV-5, POWERTECH, AU) was employed in conjunction with an electronic transformer (NP-10000-30, NeonPro Co, USA) to power the jet system. The power supply maintained a fixed frequency of 24 kHz, while the voltage was set to 10 kV. A tungsten-steel rod (150.0mm × 2.5 mm) inserted into a dielectric ceramic tube (150 mm length, inner diameter 12.0 mm, outer diameter 14.0 mm) was connected to the high voltage power supply, while a cone-shaped grounded electrode was positioned at a distance of 15 mm from the nozzle. To prevent the plasma from turning into an arc discharge, a 10 kΩ resistor was installed. The plasma jet was ignited directly from the nozzle, which was located 30 mm above the samples (Figure 1B), with argon gas supplied at a flow rate of 10.0 standard LPM. A PLASUS EMICON MC spectroscopic plasma monitor and process control system (PLASUS, DE) with EMICON MC version 4.6 software was used to record the optical emission spectroscopy

[0011] of argon plasmas. The optical sensor head was positioned at a distance of 250 mm from the plasma jet (Figure 1).

[0069] S. maxima (OxyMin Spirulina Organic) was obtained as the dry biomass from OxyMins (QLD, Australia). Most of these cells were found to be nonviable. The compositions of S. maxima are shown in Table 1.

[0070] Table 1 – Composition of S. maxima

[0071] Nutrient information of commercial product and spraying of S. maxima solution

[0072] The powder of S. maxima was bought from OXYMIN®SPIRULINA with nutrient information presented in Table 1. The microalgae with concentration 0.5 mg mL−1Milli-Q water to create a S. maxima solution, which was coated on surfaces by spraying about 5 sprays per well of 48-well plate and 1 spray on 1 cm2silicon wafer.

[0073] Effects of Ar-APJ treatment on morphology of S. maxima cells

[0074] Prior to plasma treatment, S. maxima was coated on various surfaces (silicon wafers, coverslips, and glass slides) to achieve a dry weight density of at least 0.1 mg cm−2. Firstly, clear round glass coverslips were coated by applying microalgal solution to achieve densities of 0.1 and 0.6 mg cm-2. These coverslips were then submerged in 1.0 ml of Milli-Q water for 1 hour, 12 hours, and 24 hours. At each time interval, the coated coverslips were removed, dried, and subsequently photographed under identical conditions. Finally, ImageJ software was used to quantify the green colour intensity originating from microalgae.

[0075] The plasma treatment conditions are shown in Table 2.

[0076] Table 2 – Summary of atmospheric plasma treatment conditions

[0077] As seen in Figure 2A, microalgae coatings that underwent plasma processing exhibit enhanced durability on round coverslip surfaces. This enhancement is in stark contrast to the non-plasma treated coatings, which tend to fully detach after 24 hours of water immersion. As shown in Figure 2B, coatings with the density of 0.6 mg cm-2are less durable. Therefore, 0.1 mg cm-2was found to be the optimaldensity for the plasma treatment. Then, it was treated with atmospheric plasma in 100 and 200 s cm−2. The glass slides were stained for 5 minutes with a 0.01% calcofluor white solution (Sigma-Aldrich, Inc., Saint Louis, MO), and potassium hydroxide was added to improve sensitivity in detecting microalgae. The image of S. maxima cell wall with and without treating plasma was observed by Olympus IX83 inverted epifluorescence microscope.

[0051] Prior to scanning electron microscopy, the microalgae were coated with 2 nm platinum. Scanning electron microscopy was performed at Flinders Microscopy and Microanalysis using an FEI Inspect F50 SEM (https: / / doi.org / 10.25957 / flinders.sem). Electron micrographs were obtained with an electron beam voltage of 5 kV.

[0078] Sessile drop water contact angle measurements

[0079] Sample wettability was studied using a RD-SDM02 contact angle measuring equipment. Sm and PSm samples with concentration 0.6 and 0.1 mg cm−2deposited on round glass coverslips were used as substrates for the wettability measurements. A small droplet of 3 µL Milli-Q water was carefully placed on the surface to determine the advancing contact angle. Subsequently, the droplet’s images were captured using a microscope camera and were used to measure using ImageJ.

[0080] Synchrotron ATR-FTIR microspectroscopy

[0081] Synchrotron ATR-FTIR microspectroscopy was carried out as previously described.

[0038] A single FTIR image covering a 350 x 350 mm2sampling area was captured using an Agilent FTIR microscope equipped with a liquid-N2 cooled Stingray FPA detector and a 15x objective lens. Each FTIR image was made up of a 32 x 32 array of spectra that represented chemical data that was collected from a region on the sample plane that was around 10.9 x 10.9 mm2. For each biological replication, Resolution Pro was used to get at least five excellent FTIR spectral map. Prior to each sample spectral image measurement, IR imaging software with an 8 cm−1resolution, 128 co-added scans, and background measurements were carried out.

[0082] XPS analysis

[0083] A Thermo Scientific K-alpha XPS spectrometer equipped with Al K ^^ X-ray source (1486.7 eV) and concentric hemispherical electron analyzer was used for XPS experiments. All S. maxima samples were prepared on silicon substrates. The XPS data were then analyzed with CasaXPS software. C 1s peak at 285 eV was used as a charge reference.[40,52] To minimise possible damage to the samples caused by prolonged X-ray exposure, XPS was carried out in the multiplex mode with seven scans for each element and two scans for XPS survey.

[0084] Assessing the durability of Sm and PSm coatings on wound dressing

[0085] To assess the durability of microalgae coatings on wound dressing following Ar-APJ, the Sm and PSm samples were firmly held in place within beakers using clamps. Subsequently, a continuous stream of water was introduced into the beakers for a duration of 15 minutes. Following this step, the Sm and PSm samples underwent drying at a controlled temperature of 50 °C.

[0086] Phytochemical compounds

[0087] To investigate the effect of plasma treatment on the total polysaccharide of S. maxima, an ultrasonic extraction method using NaOH solvent was employed. S. maxima is a blue-green algae with a rigid cell wall consisting of polysaccharides. The ultrasonic method was utilized to effectively break down the cell wall and extract the total polysaccharide content.[41,68] Additionally, NaOH, a strong base capable of breaking down complex organic molecules such as polysaccharides into simpler components, was used to selectively dissolve the polysaccharides and facilitate their extraction. Sm and PSm coatings were incubated with 50 mL of 0.5 m NaOH / urea solvent. The mixture was placed into an ultrasonication apparatus (Sonics VibraCell VXC 500) at frequency of 40 kHz and power of 80 W. The ultrasonication treatment was carried out continuously at room temperature for 30 minutes. After that, the solution was centrifuged at 10,000 rpm in 10 minutes to collect supernatant. This supernatant was used for examining total polysaccharide and protein concentration. The total polysaccharide content was assessed using the phenol / acid sulfuric acid method.

[0053] During this process, concentrated sulfuric acid is used to break down any polysaccharides, oligosaccharides, or disaccharides into monosaccharides. The pentoses (5- carbon compounds) and hexoses (6-carbon compounds) that are produced undergo dehydration to yield furfural and hydroxymethyl furfural, respectively. The result of the reaction between furfural and phenol is a yellow-gold color.

[0069] The total protein content was determined using the Lowry method.

[0054] The standard curve of total polysaccharide, protein, and antioxidant ability are shown in Figure 3.

[0088] Release level of C-phycocyanin

[0089] S. maxima solution was sprayed onto a 12-well plate to achieve a density of 0.1 mg cm−2. Atmospheric plasma jet treatment was then applied to the plate surface as above. Next, 1 mL of PBS (pH = 7.4) was added to each well and incubated at 37 °C in the dark. The release level of C-phycocyanin was evaluated at various time points, including 15 minutes, 60 minutes, 24 hours, and 48 hours. Cell debris was removed from the PBS solution through centrifugation at 13,000 rpm for 10 minutes, and the blue- coloured supernatant was collected. The supernatant was then analysed at wavelengths of 615 nm and 652 nm using a SYNERGY-HTX multiwell plate reader (BioTek, Vermont, USA). C-phycocyanin concentration was determined by applying the equation below.

[0055] C − phycocyanin

[0090] Cell viability of S. maxima biomass

[0091] S. maxima with a concentration of 0.1 mg / mL was coated on silicon wafer and dried for 4 hours at room temperature. Then, they were treated with atmospheric pressure plasma. After that, SYT 9 and PI stock solutions (Thermo Fisher Scientific) were added into samples and incubated in the dark for 30 minutes at 22 °C.

[0070] Images were taken at 63X magnification with SYTO9 and PI-stained cells being detected by confocal laser scanning microscope (Zeiss_LSM880 (Zeiss, Oberkochen, Germany). Based on CLSM images, both non-treated and plasma-treated S. maxima were stained with PI and observed under a fluorescence microscope.

[0092] Antioxidant ability

[0093] A 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay was conducted following the previous method.

[0056] A stock solution was prepared by dissolving 24 mg of DPPH in 100 mL of methanol, which was then stored at −20 °C until further use. The working solution was prepared by mixing 10 mL of the stock solution with 45 mL of methanol to achieve an absorbance at 515 nm using a SYNERGY-HTX multiwell plate reader (BioTek, Vermont, USA). The sample (150 mL) was then allowed to react with 2850 mL of the DPPH solution in the dark for 24 hours. Subsequently, the absorbance was measured at 515 nm. The standard curve was found to be linear in the concentration range of 25nM to 800 mM Trolox. The results were expressed in mM Trolox equivalent / g fresh mass (TE / g). Additional dilution was performed if the measured DPPH value exceeded the linear range of the standard curve. Standard curve of antioxidant ability was presented in Figure 3.

[0094] Evaluating antibacterial capacity

[0095] The Sm and PSm coatings were placed in a 12-well tissue culture plate and then seeded with 200 µL of S. aureus ATCC 25923 and P. aeruginosa ATCC 15692 at a concentration of 105CFU mL−1. After the silicon wafer was incubated for 18 hours, it was gently washed once with PBS. Next, the bacteria were stained for 10 minutes in SYTO9 (Sigma) (excitation and emission maxima, 480 nm and 500 nm) and propidium iodide (PI) (Sigma, excitation, and emission, 490 nm and 635 nm) in the ratio of 1:1. Confocal laser scanning microscope (Zeiss_LSM880, Oberkochen, Germany) was used to observe the percentage of dead bacteria. Dead bacterial Antibacterial percentage = Total bacterial × 100% (8)

[0096] The bacterial cells were fixed in 4% glutaraldehyde for 1 hour at room temperature. Thesamples were dehydrated in a sequential graded ethanol (30%, 50%, 70%, 90%, and 100%). Finally, all samples were coated with 2 nm platinum. Samples were then observed in a FEI Inspect F50 (FEI Company, Oregon, USA) at 5 kV with a working distance of 5 mm.

[0097] ROS quantification

[0098] 100 µL of diluted 2’,7’- dichlorofluorescin diacetate (DCF-DA) solution (Abcam, Australia) was added to each well of a multiwell plate. The plate was then incubated for 45 minutes at 37 °C in a dark environment. Subsequently, the DCF-DA solution was carefully removed from the wells. As a positive control, tert-butyl hydroperoxide (TBHP) was applied to the bacterial samples without S. maxima. The fluorescence measurements were conducted using CLSM at an excitation / emission wavelength of 485 nm / 535 nm. The fluorescence intensity was used to determine the density of ROS. Fluorescence intensity = FItest − FIblank (9) where FItest represented fluorescence of green color of Sm and PSm and FIblank represented fluorescence intensity of background.

[0099] Membrane integrity quantification

[0100] Bacteria on Sm and PSm were stained with 1 µM DiO (Biotium Corp., Hayward, CA) and incubated in the dark for 5 minutes. Then, 2 µM DPA was added to the bacterial solution and incubated for 45 minutes in the dark. Finally, fluorescence images were captured using CLSM.

[0057] ^^ ^^ Ratio of polarized and depolarized membrane =^^ ^^ ^^^^ ^^^^ ^^ ^^ ^^ ^^(10) where FIred represented fluorescence intensity of polarized membrane and FIgreen represented green fluorescence intensity of depolarized membrane.

[0101] Biofilm culture

[0102] A single colony of S. aureus ATCC 25923 and P. aeruginosa PAO1 was transferred to 5 mL of tryptone soy broth (TSB) and incubated overnight at 37 °C until stationary phase. The cell density was inferred from optical density at 600 nm and the overnight culture was diluted to a final cell density of 106 CFU mL−1. The treated and untreated substrates were placed in 24-well plates and sterilized by UV exposure for 20 minutes per side. The sterile substrates were immersed in 1 mL of bacterial cell suspension and then stored in a sealed box with a damp paper towel to maintain a humid environment. The box was placed atop an orbital shaker at 60 RPM in a warm room (37 °C) for 48 hours. After the first24 hours, the cell suspension in each well was replaced with fresh media to remove exhausted nutrients and stationary phase cells.

[0103] 3D biovolume analysis

[0104] The inoculated substrates were gently removed from their cell suspensions and dipped in sterile PBS to rinse off nonadherent cells. The washed substrates were placed in fresh 24-well plates and immersed in BacLight Live / Dead reagents, with an equal proportion of SYTO9 (excitation and emission maxima, 480 nm and 500 nm) and propidium iodide (excitation and emission maxima, 490 nm and 635 nm) in PBS at a concentration of 1.5 µL / mL. The samples were incubated at room temperature for 15 minutes and then imaged on an Olympus FV3000 confocal laser scanning microscope. At 3 random locations per sample, 3D Z-stack images were assembled by taking scans at 0.5 µm intervals along the Z- axis. The Z-stacks were imported into Imaris 3D analysis software and converted to 3D models using the “surfaces” function. Total biovolume was measured by combining the red and green channels.

[0105] Safranin absorbance

[0106] Biofilms were cultured on the substrates as described above. Following incubation, the substrates were gently rinsed in PBS to remove nonadherent cells. Each sample was individually placed in the wells of a clean 24-well plate and immersed in 0.1% safranin for 10 minutes. The safranin solution was removed and gently rinsed off with sterile PBS. The rinse was repeated two times. PBS was removed and the samples were then immersed in a decolorizing solution of ethanol and acetone at a 1:1 ratio. The samples were left to soak in the decolorizing solution for 10 minutes on an orbital shaker at 60 RPM. To measure the intensity of absorbance, 100 µL aliquots were taken from the samples and transferred to a 96- well plate and optical density was measured at 530 nm. The S. maxima surface coating was expected to contribute to the absorption of safranin, so a Sm and PSm control was used without the presence of bacteria. The absorbance values of the Sm and PSm experimental samples were corrected by subtracting out the value derived from the no-bacteria control.

[0107] Biocompatibility and Pro-inflammatory assay

[0108] Macrophages-like THP-1 cell at a density of 105 cells / well were seeded on Sm and PSm coatings. After 24 hours and 72 hours, cytotoxicity assays were performed using MTT assay.

[0109] Specifically, THP-1 cells (TIB-202, ATCC, Virginia, USA) were maintained in RPMI 1640 (Gibco) containing 10% heat-inactivated fetal bovine serum (Gibco-BRL), 1% streptomycin / penicillin (Gibco-BRL) at 37 °C, 5% carbon dioxide (CO2). Briefly, THP-1 cells were cultured in 75 cm2culture flasks (Corning) with the culture medium added 0.1 μg / mL phorbol 12-myristate-13-acetate

[0021] (Gibco-BRL). They were incubated for 2 days at 37 °C in a humidified atmosphere of 5% CO2 to allow them to differentiate into macrophages-like THP-1 cells. Once reaching about 80%–85% confluence, they were harvested using 0.25% Trypsin-EDTA (1X) (Gibco-BRL).

[0022] Cell viability was determined using the MTT assay (Sigma) as per the manufacturer instructions. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; Sigma) was sterilised and filtered in PBS at 5 mg / mL to remove a small amount of insoluble residue present in some batches of MTT. Stock MTT solution (10 µL per 100 µL medium) was added to all wells of an assay at the times and plates were incubated at 37 °C for 4 hours. To dissolve the dark blue crystals, DMSO (ChemSupply) was added to each well and thoroughly mixed. The plates were read on a SYNERGY-HTX multi-well plate reader (Bio-Tek) using a test wavelength of 570 nm after a few minutes at room temperature to ensure that all crystals were dissolved. The percentage cell viability was obtained after normalizing the data by culture medium only control cells. Wells with culture medium only as 100% cell viability.

[0023] CLSM study was processed by fixing the silicon wafer incubated with macrophages-like THP-1 cell and plasma-treated S. maxima by 4% Paraformaldehyde in 20 minutes after incubating with macrophages-like THP-1 cell. It was then washed with PBS 1-2 times. After that, the silicon wafer was incubated with 0.1% Triton-X (Sigma) for 10 minutes and then washed with PBS 3 times. It was then incubated with phalloidin (Thermo Scientific) for 90 minutes (5 µL phalloidin in 200 µL PBS). The sample was washed with PBS 3 times and incubated with DAPI (Thermo Scientific) (2 µL DAPI in 200 µL PBS) for 10 minutes (Figure 4). After, it was washed with PBS. Confocal wavelengths were used 365 nm and 189 nm. CLSM was used to image both phalloidin and DAPI stained samples. The media from each well was collected, followed by stimulation with IL-6. The IL-6 produced in the culture medium was quantified using the IL-6 specific ELISA kit, following the manufacturer’s instructions (Invitrogen, Thermo Fisher Scientific, AU). Media without cells was used as a control.

[0058]

[0110] HaCaT cell proliferation

[0111] HaCaT cells (human epidermal keratinocyte line, cell line services, Eppelheim, Germany) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco, AU) and 1% streptomycin / penicillin (Gibco-BRL, AU). When the cells reached ~80%–85% confluence, they were harvested using 0.25% Trypsin-EDTA (1X) (Gibco- BRL, AU). A volume of 200 µL of HaCaT cells at a density of 105cells mL−1was seeded onto Sm and PSm substrates and incubated for 1 and 3 days. MTT and CLSM assays were carried out on HaCaT cells.

[0112] Wound scratch assay

[0113] A wound scratch experiment was conducted using Incucyte SX5 live-cell analysis instrument (Sartorius). Plasma treated and untreated 24-well plates were seeded with 1 x 106HaCaT cells in triplicates and incubated for 24 hours at 37 °C and 5% CO2until a uniform cell monolayer was formed. These monolayers were consistently scratched using a sterile 100 µL pipette, washed with 1x PBS andreplaced with fresh DMEM. The well plates were placed in Incucyte SX5 live-cell analysis instrument and images were recorded for 2 days. Incucyte 2022A Rev1 software was used to measure the scratch width. The controls involved untreated tissue culture plates (TCP) seeded with cells and the negative control involved cells whose migration were retarded by treating with 1% dimethyl sulfoxide, Sigma (NC).

[0114] Statistical Analysis

[0115] All experiments were conducted in triplicate to ensure accuracy. The cell viability percentage and all other quantitative data are reported as the mean ± standard deviations. Statistical comparisons were made using ANOVA analysis, with p-values less than 0.05 deemed to be statistically significant. The statistical analysis was facilitated using GraphPad Prism version 5.00 for Windows, provided by GraphPad Software, San Diego, CA (www.graphpad.com).

[0116] Results and Discussion

[0117] Designing and Characterizing Argon Atmospheric Plasma Jet Process to Transform S. maxima Biomass into an Ultrathin Coating

[0118] The robust cell wall of S. maxima presents a considerable barrier to the utilization of its bioactive compounds.[19,20] In response to this challenge, we have developed a novel argon atmospheric plasma jet (Ar-APJ) system, capable of selectively disrupting the polysaccharide structures within the algal cell wall. The inertness of argon plasma jet is ideal for transforming the sensitive biological material of S. maxima. This technique not only allows for the reforming of the polysaccharide fragments within the cell wall, but also preserves the essential bioactive compounds. The successful manipulation of polysaccharide linkages paves the way for producing contiguous coatings directly from microalgae biomass. A detailed schematic of the argon plasma jet system can be found in Figure 5 A,B.

[0119] Optical emission spectroscopy was utilized to provide the comprehensive characterizations for the Ar-APJ system. To optimize the utilization of argon plasma, a setup of optical emission spectroscopy was conducted. Three different voltages (5.2 kV, 7.6 kV, and 10 kV) and three gas flow rates (5, 10, and 20 LPM) were selected to generate argon plasma. The results of optical emission spectroscopy

[0011] are presented in Figure 6. It was observed that the emission intensity increased proportionally with higher voltages, indicating an increase in the presence of reactive oxygen and nitrogen radicals in the plasma phase. It is noteworthy that the argon plasma generated at a gas flow rate of 10 LPM exhibited higher emission intensity than the plasma generated at 20 LPM. This difference is probably due to incomplete ionization caused by excessively fast gas flow rates. Therefore, the optimal condition for generating the maximum plasma intensity, namely 10 LPM at 10 kV, was selected for the treatment of S. maxima. Atypical optical emission spectrum and the corresponding emission bands covering a range of 200 nm–950 nm is shown in Figure 5C. The peaks in the wavelength range of 690 nm–912 nm are attributed to the 4p →4s transition of the excited argon species.

[0026] The peaks observed at 316 nm, 337 nm, 357 nm, 380 nm, and 405 nm are associated with the second positive system (SPS) of molecular nitrogen N2 (C3πu→B3πg, Δv = 0).

[0027] The generation of the N2 (C3πu) emission is attributed to the excitation of ground-state N2 (X1Σ+g) molecules by electron collisions, as shown in Equation (1). On the other hand, the low-lying N2 (B3Σ+g) emission is associated with the de-excitation transitions of N2 (C3πu), as described by Equation (2).

[0028] e−+ N2 ( X1Σ+g) υ = 0 → N2 (C3πu) υ’= 0 + e−(1) N2 (C3πu) → N2 ( B3Σ+g) + hv (2)

[0120] The detection of an emission peak of・OH at 309 nm corresponding to A2Σ+→X2π (Δυ = 0) indicates the dissociation of water molecules by high-energy electron collisions [Equation (3)] and the Penning process with excited argon and water vapor [Equation (4)].

[0028] e−+ H2O → e−+ H + ⋅OH (3) Ar∗+ H2O → Ar + H + ⋅OH (4)

[0121] The atomic oxygen lines observed at 777 nm and 844 nm are the result of the 2s22p3(4S0)3s → 2s22p3(4S0)3p transition induced by electron impact collisions with oxygen molecules, as given in Equation (5).

[0028] The generated O can potentially recombine with H radicals, ultimately leading to the formation of・OH, as described in Equation (6).

[0029] O2+ e−→ 2O + e−(5) O + H +M → ⋅OH +M (6)

[0122] It should be noted that despite the use of argon carrier gas, the OES shows the presence of nitrogen and oxygen species that are unavoidable in processes carried out in ambient air.

[0029] The OES spectrum also shows peaks corresponding to the generation of reactive oxygen and nitrogen species (RONS) during treatment, which can further break down S. maxima cell walls, turning them into a robustcoating with further treatments (Figure 5D). Three different voltages (5.2, 7.6, and 10 kV) and three gas flow rates (5, 10, and 20 litres per minute [LPM]) were selected to generate argon plasma. The results of optical emission spectroscopy

[0011] reflecting the Ar-APJ conditions are presented in Figure 6. The optimal condition for generating the maximum plasma intensity, namely, 10 LPM at 10 kV, was selected for the treatment of S. maxima. A summary of the selected conditions is shown in Table 2.

[0123] Furthermore, the Ar-APJ treatment is a substrate-independent process, meaning that the coatings can be prepared on almost any type of biomaterial. For example, the plasma-treated S. maxima coatings were formed directly on a commercial wound dressing.

[0124] Effects of Ar-APJ on S. maxima Biomass

[0125] Before the Ar-APJ treatment, the surfaces of silicon wafers or coverslips were precoated with S. maxima with a density of 0.1 mg cm−2. The cell viability of S. maxima biomass was found to be completely dead. Viability staining with SYTO9 and propidium iodide (PI) is a popular technique in microbiological research.

[0071] Like ethidium bromide, the red-fluorescent nucleic acid stain PI intercalates to DNA with no sequence preference, with one dye molecule per four to five base pairs. When PI is bound to DNA, its fluorescence is increased by 20 to 30 times. Because it penetrates only cells with disrupted membranes and is generally excluded from viable cells, PI is widely employed to identify dead cells in a population and as a counterstain in fluorescent techniques that utilize multiple colours. Conversely, the nucleic acid stain SYTO9, which fluoresces green, can penetrate both living and dead bacterial cells. The fluorescent signal of SYTO9 increases significantly when it attaches to a nucleic acid, but it has a low inherent fluorescence signal when unattached. When both dyes are present, PI has a higher affinity for nucleic acids than SYTO9. Therefore, PI replaces SYTO9, which is displaced from nucleic acids.

[0071] In this study, both samples emitted uniform red fluorescence, which corresponds to the emission colour of propidium iodide (PI). Therefore, S. maxima microalgae used for this study was non- viable.

[0126] The cell wall of S. maxima can be stained with a 0.01% (w / v) calcofluor white solution, so cell walls can be visualized under an epifluorescence microscope (Figure 7). Scanning electron microscopy (SEM) was employed to examine the microalgae morphology post Ar-APJ treatment. The initial condition of untreated S. maxima revealed an intact cell wall and morphology, with a length of ~40 ± 10 µm (Figure 7A). Upon subjecting a 100-s Ar-APJ treatment to a 1 cm2area, the cell wall underwent fragmentation into smaller segments, each ~20–25 µm in length (Figure 7B). Although a majority of the cell wall remained intact, the treatment caused noticeable cracks on the microalgal surfaces. Further extending the treatment time to 200 seconds across 1 cm2area resulted in a significant transformation. S. maxima lost its native structure, completely disintegrated, and subsequently reformed into ultrathin films (Figure 7C).

[0127] Based on epifluorescence and scanning electron microscopy (SEM) analysis, the morphological changes of S. maxima before and after atmospheric plasma treatment were observed, compared, and yielded valuable results. As shown in Figure 8 A, B, the original morphology of S. maxima was approximately 100 µm in length, serving as a reference for comparison with the morphology following plasma treatment. The microalga exhibited multicellular cylindrical trichomes arranged in an open left- hand helix along its entire structure. These trichomes were covered by a thin sheath that showed slight constrictions at the cross-walls. The width of the trichomes varied between 6 µm and 12 µm. Notably, the surface of the microalgae remained intact, as observed in the SEM image.

[0128] Following a 200-second atmospheric plasma treatment applied to a surface area of 2 cm2, the overall structure of S. maxima underwent significant degradation and lost its original form. Figure 8 C, D illustrated this breakdown, as the microalgae fragmented into tiny pieces.

[0129] These observations align with previous studies demonstrating the capacity of atmospheric plasma treatment to disrupt only the peptidoglycan layer of bacteria.[30,31] The degree of cell disruption depends on the treatment duration.

[0032] The high-energy and reactive species generated during the plasma treatment procedure induce chemical reactions within the cell wall, causing the breakage of intermolecular bonds and the rearrangement of molecular structures.[33,34] Moreover, reactive species generated from the plasma treatment, such as reactive oxygen species (ROS) and reactive nitrogen species,

[0035] have been shown to interact with and modify the cell wall components of microalgae, affecting the physicochemical properties of the cell wall, including surface charge, hydrophobicity, and permeability.

[0036]

[0130] Based on these observations, the Ar-APJ treatments of 200 seconds over 1 cm2were selected. Herein, untreated S. maxima coatings are denoted as Sm, and plasma-treated coatings are denoted as PSm. Notably, we found that APJ treated coatings (PSm) exhibit resilience and durability against multiple washings with water. These plasma-treated coatings remain intact even after being submerged in water for a duration of 24 hours and even under the flow of water. The outcomes of the durability tests revealed that the microalgae-layer on the PSm sample displayed remarkable resistance to the washout compared to the Sm sample. These results indicate that the wound dressing, coated with microalgae and treated with atmospheric plasma, exhibits an impressive and robust durability even after undergoing rinsing with water. Notably, the durability of PSm demonstrates that the thin coating will remain intact and will not delaminate when it is applied to its intended wound site. This property enables the coating to remain in place and provide a sustained release of bioactive compounds, such as phycocyanin. The sustained release of these bioactive compounds can inhibit bacterial growth and support the wound healing process. The data are shown in Figure 2.

[0131] The water contact angle results indicate that all PSm samples exhibit greater wettabilitycompared to the corresponding Sm samples at the respective concentrations (Figure 9). At an algae concentration of 0.1 mg cm−2on the surface, the water contact angle of PSm and Sm was 27.7° ± 4.6 and 43.1° ± 3.1, respectively. This trend was also observed at a coating concentration of 0.6 mg cm−2, where the corresponding contact angle of PSm and Sm was 23.5° ± 2.9 and 59.6° ± 2.8, respectively. Consequently, the surface treatment using Ar-APJ appears, involving algae coating at both concentration ranges, significantly enhanced the wettability of the PSm samples.

[0132] Analysis of Changes of Functional Groups of S. maxima

[0133] To characterise the biomolecular changes to the structural components of S. maxima following Ar-APJ treatment, we used synchrotron-sourced macro attenuated total reflection Fourier-transform infrared (ATR-FTIR) microspectroscopy.[37,38] Figure 10A shows representative spectral maps of the regions of interest as a function of FTIR absorption intensity. This was generated by integrating the spectral region for lipids in the methylene region (3000–2800 cm−1), for proteins in the amide region (1705–1600 cm−1), and for polysaccharides and nucleic acid changes in the fingerprint region (1200–950 cm−1). The heat maps indicate variations in the intensity of these regions. Although the maps are normalized, a decrease in intensity in the C─H and fingerprint spectral regions can be observed after plasma treatment, along with an increased intensity of amide I and II peaks. These observations may indicate cellular breakdown, leading to the leakage of intracellular components. We conducted principal component analysis (PCA) to identify distinct changes in the ATR-FTIR spectra between treated and untreated substrates, as shown in Figure 10B. With PC-1 describing 90% of the spectral variation between samples, the two sets were found to be quite variable. Peaks for PC-1, used to identify the wavenumbers and corresponding chemical components that differed the most between Sm and PSm coatings, were obtained from the divergence of PCA loading. The intensity of the loading peaks indicates a degree of difference, with higher intensity showing the most variation. The key peaks are highlighted in Figure 10C, and the corresponding biochemical compounds are summarized in Table 3.

[0134] Table 3 - FTIR band assignments for functional groups found in the 2nd derivative spectra of PSm and Sm.

[0072]

[0135] Within the lipid region, two groups of weaker peaks associated with changes to the methyl / methylene groups (2960 cm−1and 2925 cm−1) of the phospholipids within the cell membrane were observed. These changes likely result from the rupture of the cell membrane due to plasma treatment. Most of the peaks within the PC-1 loadings are in the amide I and amide II bands, as indicated by the bands at 1638 cm−1, 1514 cm−1, and 1452 cm−1. Variations within the polysaccharide regions could be due to alterations in the various structural polysaccharides during plasma treatment, as demonstrated by changes in the 1155 cm−1and 1045 cm−1bands. Thus, these results contribute to our understanding of the effects of Ar-APJ on the functional groups of microalgae.

[0136] Likewise, the changes observed in the PCA loading spectra are readily apparent when examining individual spectra extracted from second derivatives (Figure 10D). These alterations are characterized by heightened amide I and II bands in PSm compared to the Sm coating. Moreover, the amide I band exhibits a subtle broadening and shift, while the amide II band undergoes deformation and a slight shift toward a lower wavenumber after plasma treatment. In the lipid region, the peaks experience a slight shift toward a lower wavenumber, indicating lipid oxidation resulting from the Ar-APJ treatment. However, the most significant variation is observed in the polysaccharide region. Notably, the peak intensity at 1045 cm−1diminishes and exhibits slight broadening, while simultaneously shifting toward a higher wavenumber post-treatment.

[0137] Analysis the Elemental Composition and Chemical State of S. maxima after Ar-APJ

[0138] The effect of Ar-APJ treatment on the chemical composition of S. maxima was investigated using X-ray photoelectron spectroscopy (XPS) (Figure 11; Figure 12). The C 1s, O 1s, and N 1s spectra of S. maxima before and after plasma treatment are shown in Figure 11. The C 1s spectrum in Figure 11A can be deconvoluted into various carbon species, in which the highest peak at 285 eV corresponds to C- H / C-C bonds while the remainder located at ~286.4 eV, 287.9 eV, and 288.6 eV are assigned to C-O / C- N, C=O, and O-C=O functional groups, respectively.[39,40] Interestingly, the PSm samples exhibit a remarkable decrease in C-H / C-C and C-O / C-N groups but increase in O-C=O groups, indicating a strong interaction between the S. maxima samples and ROS generated by the atmospheric plasma. The same trend can also be observed in the O 1s spectra with the O=C / O-C ratio increasing from 0.6 for Sm to 1.4 for PSm coatings (Figure 11B). These findings are consistent with the ATR-FTIR analysis discussed above. In addition, the XPS data revealed a higher nitrogen content in the plasma-treated samples (Figure 11C), likely related to the cytoplasmic proteins released by the cell membrane disruption. This is also evident by the significant increase in the amounts of trace elements (e.g., phosphorus, magnesium, potassium, calcium, and sodium) detected in S. maxima samples after Ar-APJ treatment (Figure 12).

[0139] Effects of Ar-APJ Treatment on Bioactive Compounds of S. maxima

[0140] The effects Ar-APJ treatment on the bioactive components of S. maxima were assessed (Figure 13A). The total protein and total polysaccharide of Sm and PSm coatings were extracted and quantified as previously described.

[0041] As expected, PSm had lower total polysaccharide contents in comparison to untreated Sm, shown in Figure 13B. These findings suggested that Ar-APJ treatment on S. maxima caused a breakdown of some polysaccharides into smaller fragments.

[0030] The decrease in polysaccharide content in PSm can be attributed to the structural changes caused by the treatment, resulting in near- complete destruction of the cell wall. This interpretation is also supported by the ATR-FTIR and XPS analysis of the polysaccharide spectral region which consistently indicated a reduction in polysaccharide abundance. As a consequence, a larger quantity of bioactive compounds was released. As shown in Figure 13C, the percentage of total protein in the Sm and PSm was measured to be 43.7% and 39.9%, respectively. However, there was no significant difference between these samples (p > 0.05), i.e., Ar-APJ treatment did not significantly affect the protein components.

[0141] C-phycocyanin is an important bioactive pigment protein complex of interest in S. maxima and is responsible for antibacterial and antioxidant properties.[42,43] To evaluate the release of C- phycocyanin from S. maxima, Sm and PSm coatings were soaked in a phosphate-buffered saline (PBS, 10 x 10−3M, pH 7.4) solution at 37 °C for 0.25 hours, 1 hour, 24 hours, and 48 hours (Figure 13A). The study revealed a consistent increase in C-phycocyanin release for both Sm and PSm. More specially, Sm released 10.3 µg mL−1, while PSm released ~9.6 µg mL−1of C-phycocyanin at the 15 minute mark(Figure 13D). After 48 hours, Sm released cumulatively 21.1 µg mL−1of C-phycocyanin, compared to 19.4 µg mL−1for PSm. Notably, PSm consistently exhibited lower levels of C-phycocyanin release compared to Sm at all time points. The formation of a new matrix of polysaccharides after Ar-APJ treatment may contribute to the better retention and slower release of C-phycocyanin. In the context of wound healing applications, the slower release of C-phycocyanin from PSm may be advantageous. A sustained and slower release may allow for prolonged delivery of C-phycocyanin over an extended period, thereby maintaining its anti-inflammatory, antioxidant, and wound-healing properties.[42,43] The availability of C-phycocyanin in PSm for an extended duration could lead to persistent therapeutic effects.

[0142] Antioxidants contribute to wound healing by shielding tissues and cells from harmful oxidative species that can impair the healing process.[42,43] As shown in Figure 13E, the antioxidant properties of Sm and PSm coatings were sustained over a 48 hour period. A strong antioxidant activity for both Sm and PSm after 15 minutes (0.25 hours) was observed. The antioxidant performance remained stable for both coatings after 1 hour. At durations greater than 1 hour, there was no significant difference in antioxidant performance between the Sm and PSm samples. This indicates that the Ar-APJ process does not interfere with the antioxidant functionality of the bioactive components.

[0143] Antibacterial and Antifouling Capacity of Ar-APJ-Treated S. maxima

[0144] The antibacterial activity of Sm and PSm was assessed using confocal laser scanning microscopy (CLSM, Figure 14). For Pseudomonas aeruginosa, both Sm and PSm had a high rate of antibacterial activity, with 82% and 93% dead cells, respectively. Furthermore, qualitative SEM analysis (Figure 15A) revealed a reduction in cell density and disruption to typical morphology of P. aeruginosa. For Staphylococcus aureus, the Sm coating had an antibacterial efficacy of 35%, which was increased to 73% on the PSm substrate. This observation was also accompanied by an observation of reduced cell density.

[0145] To elucidate the capacity of Sm and PSm to inhibit biofilm formation, we cultured S. aureus and P. aeruginosa on the substrates for 48 hour and measured biofilm thickness, biovolume, and absorbance of safranin (Figure 16). Significant reductions in all metrics for Sm and PSm coated surfaces, compared to the untreated control substrate were observed. The biovolume of S. aureus and P. aeruginosa was reduced by 70%–80% on the Sm and PSm substrates compared to the untreated control substrates. The biofilm thickness of S. aureus was reduced by 55%–60% on the Sm and PSm substrates, while for P. aeruginosa, the reduction was 75% in the case of PSm. These results were corroborated by a safranin absorbance assay, which measured a decreased absorbance by ~50% for S. aureus and ~78%- 80% for P. aeruginosa on both substrates. These results confirm that the bactericidal activity of Sm and PSm surfaces inhibits the formation of biofilm.

[0146] Examining Antibacterial Mechanisms of Both Sm and PSm Coatings

[0147] The fluorescence intensity analyzed via CLSM was utilized to assess the membrane potential and intracellular ROS of both P. aeruginosa and S. aureus on Sm and PSm. The results, shown in Figure 17 demonstrate that more ROS were formed for both P. aeruginosa and S. aureus on PSm compared to Sm. This aligns with previous research which has shown that antimicrobial compounds can lead to the intracellular generation of ROS, imposing secondary bactericidal effects beyond the primary target mechanism of the antimicrobial compound.

[0044] This process is thought to occur via the overstimulation of electrons via the tricarboxylic acid cycle, the activation of Fenton chemistry. Therefore, the higher ROS generation on PSm compared to Sm could be attributed to this mechanism. Indeed, the intracellular generation of ROS has been proposed as a common end-point in the bactericidal mechanism of multiple antimicrobials with differing primary targets,

[0045] making it plausible that a similar occurrence is observed with Sm and PSm. Figure 17 further reinforces this observation, showing a lower ratio of polarized to depolarized membrane for both P. aeruginosa and S. aureus on PSm compared to Sm. This indicates that there is more membrane damage on PSm and points to the major antibacterial mechanism being the production of intracellular ROS, leading to cell death.

[0148] Biocompatibility Assay

[0149] We further evaluated the biocompatibility of untreated (Sm) and treated (PSm) S. maxima coatings. Macrophages grown from differentiated THP-1 monocytes were selected as the in vitro cellular model.

[0046] A standard MTT assay which measures the metabolic activity of cells was used to determine cell viability. No significant reduction in cell viability was noted for either Sm or PSm for 24 hours (Figure 18A). The THP-1 cells proliferated to full confluency after 72 hours on PSm. The CLSMmicrographs of THP-1 cells (stained with DAPI and phalloidin) on both Sm and PSm in Figure 18A demonstrate that the cells adopted their normal metabolically active morphology. The cells appear to be more proliferated on PSm compared to Sm. Based on these results, Sm and PSm were safe and do not possess any cytotoxicity, according to ISO 109993-5 standards.

[0047] These results were consistent with previous literature, which indicated that S. maxima is safe for use with human cells.

[0048]

[0150] We also evaluated the pro-inflammatory response of THP-1 macrophage to Sm and PSm by measuring the secretion of IL-6. IL-6 is a pro-inflammatory cytokine crucial in chronic inflammatory diseases. It is produced by cells such as macrophages, Tcells, and B-cells, participating in the immune response against infections and tissue damage.

[0049] The concentrations of IL-6 were measured at two different time points as shown in Figure 18C. After 1 day of incubation, IL-6 levels on Sm and PSm were detected to be 172.3 pg mL−1and 112.6 pg mL−1of IL-6, respectively. And after 3 days of incubation, the concentration of IL-6 secreted by THP-1 cells on PSm was significantly lower than that of Sm. This indicates that PSm coatings exhibit superior anti-inflammatory properties, which may be beneficial forwound healing.

[0151] Keratinocyte Proliferation and Wound Scratch Assay

[0152] The impact of Sm and PSm coatings on mammalian cell proliferation was investigated using HaCaT cells as a model skin cell line. CLSM micrographs (Figure 19A) showed qualitatively that HaCaT cell density was comparable on all tested surfaces after 3 days of incubation. Furthermore, the MTT assay revealed that there was no significant difference in HaCaT viability between the Sm and PSm surfaces (Figure 19B).

[0153] Keratinocyte migration plays a pivotal role in wound closure and re-epithelialization. The ability of Sm and PSm to promote the migration of human epidermal keratinocytes (HaCaT) was investigated using wound scratch assay.

[0050] The scratch width, as defined by the distance between the edges of the scratch, of Sm and Psm were 789 µm ± 18 µm and 236 µm ± 17 µm, respectively, on day 1. On day 2, the wound was completely closed on PSm, whereas the wound scratch was still open in the case of Sm and control. The scratch widths between different groups and time points showed statistically significant differences, clearly demonstrating the better healing ability of PSm compared to Sm. The results indicate that on the PSm coatings the cells exhibited significantly higher migration levels than on Sm and control after 2 days. This is in agreement with the MTT results. The capacity of the PSm coatings to enhance skin cells migration is a valuable property, indicating potential for enhancing wound healing rate.

[0154] Overall, our investigations into the antibacterial, antifouling, biocompatibility, and wound healing properties of PSm reveal that the materials generated the process described herein possess dual functionality. As bacterial infections are a primary factor in poor wound healing, the PSm coatings promise to improve clinical outcomes by simultaneously inhibiting bacteria and promoting wound healing.

[0155] The data provided herein demonstrates that the bioactive coatings produced according to the methods described herein have antibacterial activity against Staphylococcus aureus and Pseudomonas aeruginosa pathogens. These pathogens are commonly found in chronic diabetes-related foot ulcers (DFUs). Moreover, the coatings exhibit compatibility with macrophages (key immune cells), induce an anti-inflammatory response by reducing interleukin-6 production, and promote keratinocytes proliferation and migration. These are key properties required for an ideal chronic DFU dressing.

[0156] Example 2 – Animal studies

[0157] Small animal studies were carried out to demonstrate the efficacy and safety of the coatings aswell as understanding the effects on healing of infected (non-chronic) wounds utilising an infected wound model in rats (non-diabetic). (Figure 20) Wounds of 15mm diameter were generated on a rat and contaminated with methicillin-resistant Staphylococcus aureus (MRSA). An open wound control and a commercial wound dressing were used as controls. Two types of Spirulina biomass were deposited on the commercial dressing, namely Spirulina maxima and Spirulina platensis, without (Sm and Sd) and with plasma treatment (P.Sm and P.Sd), to demonstrate applicability of the technology to different algal biomass. Although all wounds healed by day 21, a remarkably faster rate of healing was observed with all coated dressings, and in particular in the case of P.Sd at earlier timepoints (day 7). Early collagen deposition and CD31 expressing cells were significantly enhanced, particularly strongly in the P.Sd group, indicating improved healing and vascularisation, the latter being of great benefit / relevance for chronic DFUs, which have an ischaemic component around 50% of the time. On another hand, inflammatory markers such as COX-2 were lower in all bioactive coatings demonstrating promising effects on healing of infected wounds.

[0158] Conclusion

[0159] The present inventors have developed a novel Ar-APJ process which can transform S. maxima biomass into a bioactive ultrathin coating for treating wound infections and enhancing wound healing. We found that our Ar-APJ system is sufficiently delicate to break down cell walls of microalgae while enabling the retention of most of the bioactive components of S. maxima. Subsequently, the fragmented cell wall was reformed to create a contiguous layer which acts as a robust and stable ultrathin coating. This treatment promoted the sustained release of bioactive compounds such as C-phytocyanin. The Ar- APJ assisted coating was found to exhibit enhanced antimicrobial properties compared to the already high antibacterial rate of untreated S. maxima. Ar-APJ-assisted coating was found to be biocompatible with THP-1 and HaCaT cells. Furthermore, a reduction in the expression of pro-inflammatory cytokines suggests a favourable reduction of inflammation. The results presented herein provide support for the potential wound healing properties of Ar-APJ-assisted S. maxima coating, attributed to its diverse bioactive components. However, it is important to note that other components in S. maxima may also contribute to these effects, warranting further advanced experiments to elucidate the underlying phenomena. Overall, this study demonstrates the potential of the Ar-APJ technology described herein to generate bioactive coating from S. maxima and thus overcome current processing challenges.

[0160] The reference to any prior art in this specification is not, and should not be taken as, an acknowledgement or any form of suggestion that such prior art forms part of the common general knowledge.

[0161] It will be understood that the terms “comprise” and “include” and any of their derivatives (e.g. comprises, comprising, includes, including) as used in this specification, and the claims that follow, is tobe taken to be inclusive of features to which the term refers, and is not meant to exclude the presence of any additional features unless otherwise stated or implied.

[0162] In some cases, a single embodiment may, for succinctness and / or to assist in understanding the scope of the disclosure, combine multiple features. It is to be understood that in such a case, these multiple features may be provided separately (in separate embodiments), or in any other suitable combination. Alternatively, where separate features are described in separate embodiments, these separate features may be combined into a single embodiment unless otherwise stated or implied. This also applies to the claims which can be recombined in any combination. That is a claim may be amended to include a feature defined in any other claim. Further a phrase referring to “at least one of” a list of items refers to any combination of those items, including single members. As an example, “at least one of: a, b, or c” is intended to cover: a, b, c, a-b, a-c, b-c, and a-b-c.

[0163] It will be appreciated by those skilled in the art that the disclosure is not restricted in its use to the particular application or applications described. Neither is the present disclosure restricted in its preferred embodiment with regard to the particular elements and / or features described or depicted herein. It will be appreciated that the disclosure is not limited to the embodiment or embodiments disclosed, but is capable of numerous rearrangements, modifications and substitutions without departing from the scope as set forth and defined by the following claims.REFERENCES

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Claims

CLAIMS 1. A method for forming a coating directly from biomass using plasma operated either at atmospheric or at low pressure.

2. The method according to claim 1, comprising providing a solution comprising biomass material, coating a substrate with the solution comprising biomass material, drying the coating on the substrate to form a biomass coated substrate, and exposing the biomass on the biomass coated substrate to a plasma formed from a gas at atmospheric pressure or sub-atmospheric pressure to produce a bioactive coating on the substrate.

3. The method according to claim 2, comprising coating the substrate with the solution comprising biomass material to achieve a dry weight density of at least 0.05 mg cm−2.

4. The method according to claim 3, comprising coating the substrate with the solution comprising biomass material to achieve a dry weight density of from about 0.1 mg cm−2to about 0.1 kg cm-2.

5. The method according to claim 4, comprising coating the substrate with the solution comprising biomass material to achieve a dry weight density of about 0.1 mg cm−2.

6. The method according to any one of claims 2 to 5, comprising exposing the biomass coated substrate to atmospheric plasma for from about 1 sec cm−2to about 1000 min cm−2.

7. The method according to claim 6, comprising exposing the biomass coated substrate to atmospheric plasma for 100 sec cm−2or 200 sec cm−2.

8. The method according to any one of claims 2 to 7, wherein the gas is selected from one or more of the group consisting of argon, helium, nitrogen, oxygen and air.

9. The method according to claim 8, wherein the gas is an inert gas.

10. The method according to claim 9, wherein the inert gas is argon.

11. The method according to any one of claims 2 to 10, wherein the plasma is formed at a gas flow rate of from about 0.01 L / min to about 1000 L / min.

12. The method according to claim 11, wherein the plasma is formed at a gas flow rate of 10L / min.

13. The method according to any one of claims 2 to 12, wherein the plasma is located from about 1 mm to about 500 mm above the surface of the biomass.

14. The method according to claim 13, wherein the plasma is located about 30 mm above the surface of the biomass.

15. The method according to any one of claims 1 to 14, wherein the biomass is selected from one or more of the group consisting of algae, seaweed, and other biologically derived material.

16. The method according to claim 15, wherein the biomass comprises Spirulina maxima.

17. The method according to claim 15, wherein the biomass comprises Spirulina platensis.

18. The method according to any one of claims 1 to 17, wherein the coating formed substantially preserves the bioactive components of the biomass.

19. The method according to any one of claims 1 to 18, wherein the coating has antibacterial properties.

20. The method according to any one of claims 1 to 19, wherein the coating has antifungal properties.

21. The method according to any one of claims 1 to 20, wherein the coating has tissue healing properties.

22. The method according to any one of claims 1 to 21, wherein the coating has anti-inflammatory properties.

23. The method according to any one of claims 2 to 22, wherein the substrate material is selected from one or more of the group consisting of metals, ceramics, polymers, and composites.

24. The method according to any one of claims 1 to 23, wherein the coating is deposited on material of complex shapes.

25. The method according to any one of claims 1 to 24, wherein the coating has a tuneable degree of crosslinking.

26. A bioactive coating formed by the method of any one of claims 1 to 25.

27. A method for extracting compounds of interest from biomass comprising exposing biomass to aplasma operated either at atmospheric or at low pressure.

28. The method according to claim 27, wherein the biomass is selected from one or more of the group consisting of algae, seaweed, and other biologically derived material.

29. The method according to claim 28, wherein the biomass comprises Spirulina maxima.

30. The method according to claim 28 wherein the biomass comprises Spirulina platensis.

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