ARTICLE Please do not adjust margins Please do not adjust margins ‘Received 00th January 20xx, Accepted 00th January 20xx DOI: 10.1039/x0xx00000x Lignocellulosic-biomolecules conjugate systems: green-engineered complexes modified by covalent linkers Pedro Henrique Correia de Limaa, Renato Márcio Ribeiro-Vianab, André Mathias Souza Plathc, Renato Grillo a* Lignocellulosic biomass represents an abundant and eco-friendly material widely explored in recent years. The main lignocellulosic fractions include cellulose, hemicellulose, and lignin. Nonetheless, the heterogeneity and complexity of these components pose challenges in achieving the desired properties. Conversely, their attractive functional groups can covalently link with other biomolecules, facilitating the creation and enhancement of material properties. Lignocellulosic molecules can form different linkages with other biomolecules through classic and modern methods. Bioconjugation has emerged as a suitable alternative to create new nuances, empowering the linkage between lignocellulosic materials and biomolecules through linkers. These conjugates (lignocellulosic-linkers-biomolecules) attract attention from stakeholders in medicine, chemistry, biology, and agriculture. The plural formations of these biocomplexes highlight the significance of these arrangements. Therefore, this review provides an overview of the progress of lignocellulosic-biomolecule complexes and discusses different types of covalent bioconjugate systems, considering the formation of linkers, applicability, toxicity, and future challenges. 1. Introduction Organic biomaterials substitute non-eco-friendly and hazardous substances in biomedical, food, and agricultural applications. They encompass natural or synthetic carbon structures with biological properties, capable of forming engineered complex systems with other biocompounds through novel chemical linkages.1,2 Proteins, such as chymotrypsin and bovine plasma albumin, were the first biomolecules reported in the literature to be joined in this manner, dating back to 1952. Since then, chemical modifications have been extended to many other biomolecules.3,4 Ideally, organic modifications occur in functional molecules with excellent reactivity, but the lack of chemo-selectivity in some reactions may challenge the creation of new materials.5 Covalent bioconjugation has emerged as one of the most sophisticated techniques for connecting biomolecules. A bioconjugate system forms a minimum ternary complex (involving at least one biomolecule) when intermediate molecules with reactive functional end groups bind to biomolecules under specific conditions. These intermediate molecules (linker agents, spacers, or bridges) can be single or multi-building fragments of organic non- polymeric or polymeric compounds. They connect biomolecules and may impart new properties to the system or enhance existing ones.6- 18 The primary biomolecules in bioconjugate systems include lipids, proteins, nucleic acids, and carbohydrates. 19-24 Lignocellulosic structures are notable in the carbohydrate-bioconjugate domain (Figure 1) and have garnered increased research interest in the last decade. Figure 1 – Representation of lignocellulosic bioconjugate systems. Biomolecules, linkers and attractive functional groups for bioconjugation in (a1;a2) cellulose, (c) lignin, (d) xylan, and (e) tannic acid. Lignocellulosic material is the most abundant renewable natural biological resource available on Earth, making it a substitute for fossil-based materials in constructing eco-friendly bio-nano/micro products due to their natural sources. 25 They are composed of cellulose, lignin, hemicellulose, tannins, pectin, proteins, and acids, presenting significant advantages such as i) renewability, ii) recyclability, iii) biocompatibility, iv) biodegradability, and v) attractive functional groups (Figure 1 a- e). 26-28 Functionalization and grafting are two well-known modification methods that facilitate the construction of lignocellulosic systems with other biomolecules, and bioconjugation follows the same steps. 29,30 For example, cellulose, the main compound of lignocellulosic feedstock, plays an essential role in carbon cycles and significantly impacts the global carbon budget.31 It possesses a degree of crystallinity and undergoes a. São Paulo State University (UNESP), Department of Physics and Chemistry, School of Engineering, Ilha Solteira, SP 15385-000, Brazil. b. Departamento Acadêmico de Química, Universidade Tecnológica Federal do Paraná, UTFPR-Ld, CEP 86036-370, Londrina, PR, Brazil c. Laboratory for Orthopaedic Technology, ETH Zurich, 8092 Zurich, Switzerland * Corresponding author: Dr. Renato Grillo (renato.grillo@unesp.br) Electronic Supplementary Information (ESI) available: [details of any supplementary information available should be included here]. See DOI: 10.1039/x0xx00000x ARTICLE Journal Name 2 | J. Name., 2023, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx Please do not adjust margins Please do not adjust margins heterogeneous reactions that may make specific material properties difficult, such as achieving better biocompatibility and cell-specific selectiveness. Thus, linking cellulose to biomolecules using linkers has emerged as a suitable solution. For instance, cellulose's structure (β-1,4-glycoside-linked D- glucopyranose repeating units) has been modified by opening the hexane ring (Figure 1a1) or by targeting hydroxyl points in biconjugate systems (Figure 1a2). 32-38 Another manipulated lignocellulosic material in these conjugate systems is lignin – a complex polyphenolic wood structure (previously considered useless material) exhibiting properties such as light shielding capacity and UV Light blocker.39 Despite lignin's low reactivity, it can undergo chemical modifications through its hydroxyl groups to enhance the interaction with other materials (Figure 1b). 40 The structures of hemicelluloses are more closely related to cellulose than lignin and are deposited in the cell wall at an earlier stage of biosynthesis in plants. 38 The most abundant hardwood hemicellulose is xylan - a linear molecule comprising β-1,4-linked xylose units - that offers biodegradability, biocompatibility, and antioxidant properties. However, xylan has limited solubility in water and may weaken the interaction with hydrophilic biomaterials. Therefore, bioconjugate xylan by modifying its hydroxyl groups (Figure 1c) may enhance its hydrophilic/swelling characteristics.41-43 Tannic acid (Figure 1d) is a natural polyphenol with antiviral/antibacterial properties rich in reactive hydroxyl groups that can interact with several biological components. However, this may not be ideal for drug delivery applications, and bioconjugation may provide selectivity to the material. 44-46 In this review, we summarize the progress of lignocellulosic complexes and discuss different types of bioconjugate systems, considering the formation of linkers, applicability, and the potential impact of these hybrid materials on human health and the environment. Moreover, we explore alternatives and address future challenges in producing more environmentally friendly complex materials. 2. Cellulose bioconjugate ternary systems Cellulose, derived from plants and bacteria, exhibits fibers or crystal-like shapes and bio-properties such as low toxicity, biodegradability, and cytocompatibility.31,47-52 Cellulose is the most modified lignocellulosic feedstock and can be conjugated with metallic ions, silica, and biomolecules. Peptides, proteins, and biopolymers can impart new properties to cellulose, such as cellular recognition and adhesion. 53-56 Cellulose–linkers– biomolecules conjugate systems form under different conditions, giving rise to several linkages (Table 1). In this section, we will explore the union of cellulose and biomolecules through these linkages, focusing on the employed linkers. Polyethylene glycol (PEG) is a hydrophilic polyether with cosmetics and pharmaceutical applications and can work as a linker chemically modifying biomolecules (PEGylation).57 Its terminal hydroxyl groups can be substituted by amine and carboxyl groups. For instance, Yang et al. used a carboxyl-PEG- amine (COOH-PEG-NH2) linker to form a cellulose conjugate system. The carboxyl-PEG part was ester bonded with hydroxyl biomolecules (as resveratrol (RSV) – an anticancer and anti- inflammatory polyphenol compound) in the presence of 4-Di methylaminopyridine/1-ethyl-3-(3-dimethyl aminopropyl)- carbodiimide (DMAP/EDC); where EDC interacts with carboxylic acids and generates O-acylurea intermediates, which are easily attacked by nucleophiles, and DMAP works as a nucleophilic catalyst. 15,58-62 Table 1 – Examples of biomolecules-linkers-cellulose Moreover, the Amine-PEG part can connect to carboxyl ending groups (forming amide linkages) with 2,2,6,6- tetramethylpiperidine-1-oxyl radical (TEMPO)-oxidized cellulose nanofibers (CNFs) with N-hydroxysuccinimide (NHS) - in aqueous condition because NHS is a better nucleophile than water (Figure 2a). TEMPO is selective and efficient in converting hydroxyl groups of cellulose into carboxyl groups under mild conditions. This radical is stabilized through steric hindrance, Biomolecule- Type of celulose Linker(s) Bond types Reference Tosufloxacin – CNC L-Leucine (amino acid) Ester/Amide 77 Peptides – Cellulose 1,3,5- triazine derivatives Amide 78 Antimicrobial peptides – Microcrystalline Cellulose (MCC) Thioester linker Amide 79 (2,2,6,6- tetrametil- piperidi-1- nil)oxil (TEMPO) and oxidized cellulose nanofibers and pNO2-phenyl urea moieties branched- polyethylen eimine Amide 80 Biotin-CNF Avidin Amide 81 Modified cholesterol– modified microcrystalline cellulose (MCC) Olefin – Terminated Ester and cross- metathesis 82 Methylcellulose – peptides Thiolated linker Ester and disulfide bonds 83 Lysozyme – Carboxy nanocellulose Bovine Serum Albumin (BSA) Amide 84 Journal Name ARTICLE This journal is © The Royal Society of Chemistry 20xx J. Name., 2023, 00, 1-3 | 3 Please do not adjust margins Please do not adjust margins oxidizing primary hydroxyl groups; otherwise, secondary hydroxyls become ketones. 15, 58-62 Besides NHS, molecules such as triethylamine (TEA) and 1,1ʹ-carbonyldiimidazole (CDI) also play essential roles in lignocellulosic conjugate systems. TEA assists the oxirane glycidyl methacrylate (GMA) linker to form an ester bond with rosin (an essential oil). Later, this functionalized molecule ester links to carboxymethyl cellulose (CMC). CMC is a biopolymer with excellent edible properties. Moreover, CDI participates only as an activator of CNC hydroxyl groups, facilitating their union with 2,2ʹ- (Ethylenedioxy)bis(ethylamine) (EBEA) linker.63,64 This linker bridges CNC and other biomolecules, such as phenyl sulfonates, through isothiocyanate bonds.65 Other reactions, such as click chemistry, are fast, versatile, region-specific, give high product yields, and can also functionalize cellulose. They encompass several heteroatom bonds and exhibit high thermodynamical forces, with copper- catalyzed azide-alkynes being the most notable. 66,67 In a cellulose biconjugate system, P-toluenesulfonyl chloride (TsCl)/triethylamine modify cellulose to facilitate azide functionalization (CNF-N3), while tosylation and alkyne may functionalize biomolecules such as biotin and beta- cyclodextrin (β- CD) (an oligosaccharide). For instance, alkyne- functionalized β-CD and CNF-N3 undergo a click chemistry reaction, forming a triazole bond in a hydroxide sodium (NaOH) medium (Figure 2c). 67-69 Moreover, triethyleneglycol moieties can also serve as linkers through click chemistry reactions to connect cellulose and biomolecules. Glycols with less chain repetition may be used to substitute PEGylated molecules that stimulate anti-PEG antibodies (anti-PEG immunoglobulin M (IgM)) and immune response in organisms 67,70,71 Michael addition or Schiff-base reactions play a role in assisting cellulose bioconjugate systems. For instance, lauric arginate (LAE) - a therapeutic biomolecule derived from two others (lauric acid and L-arginine) binds with antioxidant gallic acid (GA) (acting as a linker) by Michael addition. Nevertheless, this functionalized molecule can covalently or intermolecularly interact with microcrystalline cellulose (MCC) in NaOH, leading to the oxidation and oligomerization of GA. In this process, GA hydroxyl groups transform into quinones, which can react with the amino groups of LAE. Meanwhile, gallic acid can be a biomolecule conjugated to the MCC system, with GMA as the linker.72-74 Reactions with halogen molecules (chloroacetylchloride and bromoacetyl bromide) as linkers appear in cellulose-biomolecule conjugate systems due to their highly reactive with other biomolecules in dimethylformamide (DMF)-pyridine medium, and N, N–diisopropylethylamine (DIPEA) as a base. For example, ester bonds are formed when C-6 hydroxyl CNC reacts with bromoacetyl bromide; this spacer links several biomolecules through nucleophilic substitution.75,76 External parameters are essential for some lignocellulosic conjugate formations. For example, temperature plays a crucial role, and molecules like citric acid (CA) may serve as a suitable linker in thermodynamically favourable conditions. This linker forms ester bonds and works without the assistance of catalysts at temperatures near 140 degrees Celsius. In a cellulose conjugate system, this molecule links nanocrystals (CNCs) and CNF (Figure 3a).85-87 Unfortunately, citric acid covalently bonds with any hydroxyl group and becomes undesirable for a thoroughly efficient biconjugate formation,88,89 crosslinking the same type of biomolecule in the process.90 Besides citric acid, other molecules with carboxylic groups work as linkers and assemble cellulose-biomolecule systems, including oxalic acid91 and succinic acid.92,93 Lactic acid, another carboxyl biomolecule, is well-dispersed in water and forms ester bonds between CNC and fatty acids, which are carboxylic molecules (Figure 2b). 94 Figure 2 – Examples of Ternary cellulose bioconjugate systems: (a) PEG as a linker in an EDC/NHS reaction,15 (b) lactic acid as a linker, 94 (c) click-chemistry demonstration in a linker formation. 68 pH is another external parameter considered in conjugate systems. For instance, the amino acid dopamine or Levodopa (L- dopa) forms irreversible covalent bonds when it self- polymerizes in alkaline conditions and chemically modifies nanocrystal cellulose through ether bonds in a Tris ARTICLE Journal Name 4 | J. Name., 2023, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx Please do not adjust margins Please do not adjust margins hydrochloride (Tris-HCl)/alkali medium. This functionalized molecule reacts in an NHS/EDC reaction with 2-(-N-morpholino) ethanesulfonic acid (MES) as a buffer solution, which can form an amide bond with SPI in an acid medium (Figure 3b). However, it is crucial to protect one amino acid functional group while the bioconjugation reaction is happening to avoid further polymerization and to stop the reaction - this can be more expensive due to the protection and deprotection processes. 95- 98 Figure 3 – External parameters in the formation of cellulose-biomolecule conjugate systems: (a) temperature,85 (b) pH,97 and (c) UV Light.8 External parameters like UV light may also contribute to new formations. For instance, Bretel et al. esterified pristine cellulose paper with dithiodiglycolic acid in a toluene solution, and they conjugated it to cholesterol through photothiol-X reaction under UV light in a DMAP and ethyl hexanoate medium (Figure 3c).8 Overall, these covalent bioconjugate bonds can be analyzed by many characterizations, such as Ultraviolet-Visible (UV-VIS), Fourier transform infrared (FTIR), Nuclear Magnetic Resonance (NMR), X-ray Photoelectron Spectroscopy (XPS) etc. 99-101 Although only the use of one linker has been explored in this section, more complex systems employing multi-binding linkers can also be found. Thus, they will be discussed in the next section. 3. Multi-building linkers in bioconjugate systems The ability to create new linkages amplified the acquisition of more elaborate systems and brought innovation for market applications. 102, 103 The creation of bigger linkers is similar to following a complex recipe, and we will discuss the construction of linkers built in the bioconjugation process in this section, meaning cellulose binds with biomaterials through the union of fragments, which can configure more than ternary complexes. One common way to multi-build a cellulose biconjugate system is by modifying the cellulose structure with an initial linker, following other linkers’ bonds until a final attachment with the biomolecule (Figure 4a1). This is seen with Aminopropyltriethoxysilane (ATPES), a small organic molecule that does not configure new properties to cellulose complexes and works as the first fragment of a linker due to its amine functional groups. However, vicinal hydroxyls of ATPES can bind with cellulose acetate (CCA) membranes, and the amine groups of ATPES can link with molecules such as glutaraldehyde (GLA) through imine bond, forming the CCA-GLA-ATPES functionalized molecule. GLA aldehyde groups interact with biomolecules such as resveratrol (RSV) through phenyl groups. Thus, CCA-ATPES-GLA-RSV is an excellent example of a successful multi-building system under alkali conditions for improved osseointegration.104-107 Moreover, ATPES can be an initial linker for cellulose nanocrystals to construct poly(propylene imine)-cellulose dendrimers conjugate for profile release study of doxorubicin (DOX) – an anticancer drug. 108 Figure 4 – More than a ternary complex of cellulose conjugate system with (a1;a2) step-by-step reactions, and (b) a multicomponent reaction (MCR). Authors also use the union of linkers’ fragments to maximize the binding among cellulose feedstock and biomolecules. For instance, Leppänen et al. observed an increase in the binding between CNC and silk fibroin by amine-amine linked between succinimidyl-6-hydrazinonicotinate acetone hydrazone (SANH) and succinimidyl-4-formyl benzoate (SFB) moieties, but it induced a not-wanted nanocluster formation.109 Other molecules, such as the combination of tetraethylene pentamine (TEPA) and GMA, form longer linkers capable of connecting cellulose to the rhodamine chromophore (RC). In this configuration, cellulose suffers modification through the cyclohexane ring in the presence of cerium ammonium nitrate, Journal Name ARTICLE This journal is © The Royal Society of Chemistry 20xx J. Name., 2023, 00, 1-3 | 5 Please do not adjust margins Please do not adjust margins and aldehyde functional molecules such as RC attaches to cellulose – GMA – TEPA by imine reaction in DMF medium.110 Linker's fragments may also be attached to the biomolecule, configuring a new way to form a cellulose bioconjugate system (Figure 4a2). For example, Hazra et al. reacted epichlorohydrin with CNC in an ammonium hydroxide (NH4OH) solution. This base opens an epichlorohydrin epoxy cycle and creates an amino group (CNC-NH2). The intermediate amino group then links with Traut's reagent (2-iminothiolane) in the presence of EDTA, forming CNC-SH; this occurs due to the affinity among primary amine groups, creating thiol (-SH) ending groups. Finally, CNC-SH conjugates with molecules such as modified succinic anhydride transferrin (Tf - succinate) in the presence of EDC. Traut’s reagent acted as a leaving group to form amide bonds with the CNC-NH2 intermediate and Tf - succinate (Figure 4).111 On the other hand, one excellent procedure to elaborate a cellulose biconjugate system is multicomponent reactions (MCRs), when three or more components are linked in a one- pot reaction. MCRs (Figure 4b) have an outstanding vantage due to their high atom economy compared to step-by-step reactions. In this context, Ghadam et al. carried out an MCR containing cellulose, N-vinylcaprolactam (NVLC), and allylamine/acrylic acid (used as linkers) in a cerium ammonium nitrate medium. NVCL and linkers suffered polymerization, while cellulose suffered a hexane ring modification. Later, the authors reacted polyallylamine to glucuronic acid and formed amide in an NHS/EDC medium.112,113 Cellulose is the principal lignocellulosic feedstock in bioconjugate systems. However, other plant-based materials, such as lignin, tannic acid, and xylan, are also used, which will be explored in the next section. 4. Other lignocellulosic materials in bioconjugate systems Lignin is an organic wood material and the second most abundant bioresource in the world. 114.115 Lignin biconjugate systems have shown excellent biocompatibility and low toxicity and distinguished paths to be formed. For instance, Dai et al. presented a new way to form lignin microcapsules from bioconjugated lignin nanoparticles. The authors created an ester bond between alkali lignin and α-bromopropionyl bromide in DMF. Then, the intermediate structure reacted with N-isoprylacrylamide monomer in copper(I) bromide (CuBr) and N,N,Nʹ,Nʺ,Nʺ-pentamethylenediethylenetriamine (PMDETA) medium, occurring a polymerization of the biomolecule. The authors transformed this lignin bioconjugate system into nanoparticles and further converted these nanoparticles into microcapsules through an emulsion approach in palm oil. Thus, the microcapsules were capable of releasing trans-RSV in a temperature-triggered system. 39 Distinguished linkers such as diethylenetriamine (DETA) may also functionalize lignin in formaldehyde/alkali solutions and provoke a Mannich reaction, a common reaction to introduce N atoms into organic structures. Thus, lignin-DETA reacts in NHS/EDC-HCl (EDCl) medium and forms an amide bond with biomolecules such as L-Histidine - an essential amino acid for human life (Figure 5a1).116,117 Another linker is maleic anhydride – in dioxane and heating conditions – ester bonds with the lignin’s hydroxyl groups are formed. For example, Yang et al. reported nanoparticles constituted by lignin as an anchor for PEG and DOX biomolecules. The authors explored lignin bioconjugate nanoparticles as a pH-responsive system for drug release in cancer treatment. In this system, thiolated PEG bonds with maleic acid in an EDC/NHS medium through a thiol-ene click chemistry reaction - a click reaction with a rapid photoinitiation process. In another study, biomolecule (DOX) conjugates with lignin through hydrolyzable hydrazine bonds in a TEA/trifluoroacetic acid (TFA) medium (Figure 5a2).118-120 Figure 5 – Bioconjugate systems of (a) lignin117,118,, (b) xylan,124 and (c) tannic acid. 126 TEA is usually a base but can configure a linker in bioconjugate systems with xylan. Also, xylan and drugs bind through bioconjugation when a succinic anhydride linker reacts in a DCC/DMAP medium and connects xylan to curcumin and 5- fluorouracil drug.121,122 Thus, 3,3ʹ-dithiodipropionic acid (Figure 5b) connects xylan and curcumin via ester.123,124 Besides drugs, gelatin (a polysaccharide) forms a xylan biconjugate system with ethylene glycol diglycidyl ether as a bridge; this linker has cyclical ether functional groups that form ether bonds between xylan and gelatin.41 Conversely, a singular linker is a tannic acid – a natural polyphenol studied in brush and nanoparticle structures.18,125 However, in a biconjugate system, this macromolecule is an anchor to the PEG-polyaminoacid complex with phenylboronic acid as a linker. This ternary complex happens in an aqueous medium and forms boronate esters ARTICLE Journal Name 6 | J. Name., 2023, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx Please do not adjust margins Please do not adjust margins between tannic acid and the acid (Figure 5c), it also forms a nanoparticle with tannic acid in the core.45,126 5. Lignocellulosic bioconjugate applications and properties Lignocellulosic biconjugate systems play a significant role in biomedical applications. For instance, Hazra et al. created a nanocage with a cellulose-transferrin conjugate system as one of the components. They used this protein to capture tumor cells in head and neck cancer.111 Also, gemcitabine (an anti- cancer drug) was used in a novel-targeted innovative drug- delivery system constituted by a cellulose conjugate system. 113 Furthermore, cellulose-RSV conjugate was transformed into a robust, stretching hydrogel with polyvinyl alcohol (PVA) and Borax. This material was created by Yang et al. and exhibited excellent adhesion with outstanding curative properties, with over 90% of the wound being restored, compared to the control (75% with only RSV). Moreover, rats treated with the biconjugate presented more collagen and hair.15 Furthermore, cellulose acetate membranes, known for their super hydrophilicity, chemical and mechanical stability, and easy availability,127 can enhance the osseointegration properties of this material when associated with RSV. 107 Also, cellulose- linkers-biomolecules may have outstanding behavior for colon- targeted drug release, retain antimicrobial activity, or detect human immunoglobulins.77,79,81 Besides cellulose, xylan, lignin, and tannic acid, bioconjugates can be seen in delivery systems. For example, kraft lignin ternary complex with histidine nanocapsules carried with 10-Hydroxycamptothecin (HCPT) presented better results in tumor treatment than free drug (Figure 6c).117 Drugs such as wound healing curcumin and solid anti-tumor 5- Fluorouracil (5-Fu) were also used in delivery systems with xylan bioconjugate hydrogels.41 Also, protein delivery systems are related to the tannic acid-conjugate complex.45 On the other hand, Lignocellulosic conjugate systems are gaining prominence in the formulation of 3D objects (Figure 6a). Linkers, such as citric acid, may enhance the stability and contribute to high flexural strength in the resulting objects.85 Other properties are also reported in cellulose biconjugate systems. For instance, MCC and rhodamine chromophore exhibit excellent optical signals and selectivity (detection limit on Hg2+ < 9.18 x 10-7 M) upon contact with mercury, playing a notable role in detecting and removing the metal in tap water.109 Alternatively, CNC coupled with the pH-indicator dye (5 (and 6)-carboxy-2ʹ,7ʹ-dichloro-fluorescein, and l-leucine amino acid were used to construct fluorescent nanoparticles for pH-sensing, demonstrating fluorescent properties when the pH of the media was increased (Figure 6b).128 Furthermore, modified MCC with pyridone has an adsorption capacity for cationic dye applications.92 Also, these conjugate systems may be seen in food packing applications.84 Moreover, a lignocellulosic complex was developed for agricultural application, involving the controlled release of avermectin (AVM) in a carboxymethyl cellulose conjugate system (Figure 6d).63 This may indicate a beginning for these materials in agriculture as excellent materials with a length of suitable applications. Figure 6 - Lignocellulosic biconjugate applications and properties: (a) 3D objects,85 (b) cellulose biconjugate complex with fluorescent properties, 128,(c) lignin bioconjugate nanoparticles for tumor treatment, 117 (d) and cellulose pesticide delivery system. 63 6. Toxicity of lignocellulosic bioconjugates Although lignocellulose materials generally exhibit low toxicity, chemically modified lignocellulosic materials assessment is necessary post-modification for present and future applications.81 Sometimes, linkers may be harmful to the systems, while some biomolecules can reduce the toxicity of materials. However, comparing toxicity through assays is primordial to identifying the benefits of lignocellulosic complexes. Unfortunately, authors have still ignored ecotoxicity assays of these materials, and there are few studies focused on cytotoxicity assay. Nowadays, classical cytotoxicity assays, such as MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), help to understand the toxicity of cellulose biconjugate systems. For instance, Klinthoopthamrong et al. used an MTT assay to determine the cytocompatibility of their bacterial cellulose/plant-derived recombinant human osteopontin (p-rhOPN) conjugate system. 100 Furthermore, other types of in vitro assays are used, such as corneal epithelial (HCE-T) cell viability,65 and cytoplasmic lactate dehydrogenase (LDH) – a common enzyme used to indicate cytotoxicity. For example, Pandele et al. reported that cellulose acetate membranes modified with sericin have no significant difference in the LDH released from MC3T3-E1 preosteoblasts.107 Moreover, methylcellulose-biotin material had no toxic effect on Receptor Interaction Protein (RIP) cells. Tam et al. studied cells of specific organs/tissue in cellulose bioconjugate systems, although in some cases, they do not know if the bioconjugate will target this organ or tissue. 83 Journal Name ARTICLE This journal is © The Royal Society of Chemistry 20xx J. Name., 2023, 00, 1-3 | 7 Please do not adjust margins Please do not adjust margins Other examples can be found in lignocellulosic materials, such as lignin-histidine tested by MCF-7 cell viability in CCK-8 assay.117 Also, xylan bioconjugates as curcumin and 5-Fu delivery system were tested in HT-29 and HCT-15 cells by MTT assay.121,122,124 In cancer treatment, the tannic acid bioconjugate system showed less hepatoxicity than pure tannin acid.126 Therefore, it is recommended that the risk assessment be conducted in the early stages of developing these materials so that their potential impacts on human health and the environment can be better evaluated. 7. Lignocellulosic Complexes: Greenness and Future Challenges While there is a demand for new toxicological studies on lignocellulosic bioconjugates, additional future concerns arise regarding the challenges associated with using green chemicals and sustainable synthesis to produce eco-friendly complexes. Lignocellulosic materials play a significant role in the circular economy once they are sustainable and eco-friendly and pose a reduced risk of toxicity compared to synthetic polymers. 15, 69 However, using lignocellulosic materials to design new complex structures does not render the product inherently sustainable features. Overall, biomolecules linked to the lignocellulosic feedstock also originate from green sources, which possess biological properties and do not have a significant challenge regarding reaction toxicity. However, the main concerns for successful bioconjugation involve the choice of solvents, linkers, and types of reactions, some of which can be expensive and toxic. For example, step-by-step reactions are more costly than MCR following the atom economy. 112,129 Moreover, lignocellulosic conjugates showed the use of hazardous linkers, such as chloroacetyl chloride — a greener alternative such as diethylamine is recommended, even though they are less effective. 104,130 Moreover, toxic solvents such as toluene, pyridine, and DMF can be replaceable for benzene, triethylamine, and acetonitrile. 10,131 Alternatively, other solvents, such as deep eutectic132 or “greener” solvent candidates (e.g., glycerol-derived 1,2,3-triethoxypropane)133 can be used. Also, strategies can be used to increase the efficiency of reactions in water, such as amidation, amine synthesis, and transfer hydrogenation. 134 On the other hand, computational simulations can assist researchers in designing lignocellulosic biocompatibility complexes with minimal hazards using synthesis under solvent- free conditions, for example. Therefore, researchers and scientists should consider greener options because lignocellulosic conjugate systems have huge promises in several areas, especially medicine, where a hazardous material could negatively affect human health.17,117,131 Moreover, lignocellulosic biconjugate systems are becoming attractive for environmental and agricultural areas.63,110 Hence, biomolecules, linkers, and solvents can generate a potential ecotoxicological risk. 135 Therefore, future studies are expected to be carried out in line with the primary principles outlined in the Twelve Principles of Green Chemistry129 and circular economy. 8. Conclusions Recent lignocellulosic-linkers-biomolecule conjugate systems are growing in organic chemistry as new engineered tools. These systems are constructed by forming new covalent linkages and may be used in biomedical, environmental, food packing, and agricultural fields. The construction of linkers has been categorized into direct and multi-building/stepping linkages, the latter being relatively new due to its precision. On the other hand, biomolecules are attached to linkers based on the desired property or application. This review underscores the importance of green lignocellulosic feedstock and how its interaction with other organic molecules, through chemical modifications, configures a range of proprieties such as drug delivery system, adsorbent of metal, food packing, controlled release of pesticides, osteointegration, curative, etc. Moreover, toxicological assays have been reported for these complexes, indicating low toxicity in several cases. However, further toxicological studies, especially in ecotoxicological environmental issues, are needed. Furthermore, a brief discussion about their green capacity was evaluated, recommending the use of principles of green chemicals in the future synthesis of lignocellulosic complexes. Author Contributions P.H.C.L., R.M.R., A.M.S.P., and RG: Conceptualization, P.H.C.L., R.M.R., A.M.S.P., and RG: Writing-review: P.H.C.L.: Figures and tables construction, R.M.R and RG: Review, visualization, supervision, funding acquisition. Conflicts of interest There are no conflicts to declare. Acknowledgments The authors would like to thank the São Paulo Research Foundation (Grant #2022/03219-2) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brazil, CAPES – Finance Code 001. R.G. would also like to thank the National Council for Scientific and Technological Development (Grant #310846/2022- 6). References 1 T. Biswal, S. K. BadJena, and D. Pradhan, Materials Today: Proceedings, 2020, 30, 274-282. 2 L. P. Datta, S. Manchineella, & Govindaraju, T, Biomaterials, 2020, 230, 119633. 3 K. M. Burridge, R. C. Page, and Konkolewicz, D, Polymer, 2020, 211, 123062. 4 B. L. Tardy, B. D. Mattos, C. G. Otoni, M. Beaumont, J. Majoinen, T. Kämäräinen, and O. J. Rojas, Chem. 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