Development of citric acid-based biomaterials for biomedical applications

Abstract

The development of bioactive materials with controllable preparation is of great significance for biomedical engineering. Citric acid-based biomaterials are one of the few bioactive materials with many advantages such as simple synthesis, controllable structure, biocompatibility, biomimetic viscoelastic mechanical behavior, controllable biodegradability, and further functionalization. In this paper, we review the development of multifunctional citrate-based biomaterials for biomedical applications, and summarize their multifunctional properties in terms of physical, chemical, and biological aspects, and finally the applications of citrate-based biomaterials in biomedical engineering, including bone tissue engineering, skin tissue engineering, drug/cell delivery, vascular and neural tissue engineering, and bioimaging.

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Shihao Zhang

Shihao Zhang was born in Henan Province, China. He received the M.S. degree in Biomedical Engineering from East China University of Science and Technology in 2023. He is currently pursuing his PhD degree at East China University of Science and Technology, under the supervision of Prof. Yulin Li. The main research direction is the controllable synthesis of medical polymer material and medical hydrogel dressing.

Cailin Liu

Cailin Liu was born in Sichuan Province, China. She received her bachelor's degree in environmental Engineering from Panzhihua University in 2022. Currently, she is pursuing her master's degree at East China University of Science and Technology under the guidance of Prof. Shiyong Wu. Her research topic is the synthesis of biodegradable elastomers.

Meng Su

Meng Su was born in Hebei province, China. She received her master's degree from Mudanjiang Medical University. She is currently engaged in the development of absorbable medical devices at Shanghai University Wenzhou Research Institute.

Dong Zhou

Dr. Dong Zhou is a postdoctoral researcher at the East China University of Science and Technology. Dr. Zhou received his Master's degree in Engineering from Hubei University in China in 2020 and PhD degree in chemistry from Nanchang University in 2024. Dr. Zhou has published 20 peer-reviewed journal papers, and his research focuses on absorbable medical polymers and organic/inorganic composites, with a specific focus on the application of these materials in promoting tissue regeneration.

Ziwei Tao

Ziwei Tao was born in Hubei Province, China. She graduated from East China University of Science and Technology in 2023 with a master's degree in Materials Science and Engineering under the supervision of Prof. Yulin Li.

Shiyong Wu

Shiyong Wu is a professor at East China University of Science and Technology. His research interests include gasification and liquefaction of solid fuels such as coal, biomass and petroleum coke to liquid fuel technology and equipment technology. Waste gas and organic solid waste resource disposal and other purification technology and equipment technology; He has published more than 120 papers (more than 80 SCI papers) in domestic and foreign academic journals, and more than 10 authorized patents. He has won one first prize and one second prize of Shanghai Science and Technology Progress, one second prize of innovation and Entrepreneurship of China Invention Association, one appraisal achievement of China Renewable Energy Society and China Rural Energy Industry Association, member of editorial Committee of Gas and Heat, director of Coal Chemical Industry Committee of Liaoning Chemical Industry Society, Secretary General of Applied Insulation Engineering Committee of Shanghai Energy Society.

Lan Xiao

Dr Lan Xiao is a lecturer at School of Medicine and Dentistry, Griffith University. Dr Xiao obtained her bachelor's degree and master’s degree in Dentistry from Wuhan University, China, and PhD degree in Biomedical Engineering at QUT in 2017. Dr Xiao has published 80 peer-reviewed journal papers and attracted over $900,000 research funding. Her research mainly focuses on targeting immune system via engineered approaches to boost tissue regeneration.

Yulin Li

Yulin Li is a professor of East China University of Science and Technology. His research interests focus on the development of medical polymer materials and bioabsorbable medical devices. He has presided over 15 key R&D projects from the Ministry of Science and Technology, the Science and Technology Commission of the Military Commission, and the National Natural Science Foundation of China. He has published more than 100 papers as the first/corresponding author in Chem Rev, Adv Funct Mater etc. He has applied for 25 domestic and foreign invention patents. He has successfully made a product translation on a series of PLA, PLGA, PLCL medical-grade raw biomaterials, and obtained a Medical Device Registration Certificate on medical hydrogel from National Medical Products Administration for China. His research outcome was awarded the First Prize of Scientific Progress of Shanghai Municipality in 2023.

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1. Introduction

Globally, the incidence of tissue and organ damage caused by various diseases is increasing. Tissue engineering is a promising strategy to address this problem because of its potential in regenerating or replacing the damaged tissues and organs.^1–3 Tissue engineering integrates different disciplines such as life sciences, materials science and biomedical engineering. As the field of tissue engineering continues to expand, there is a need for biomaterials to achieve tailored physio-chemical properties and bio-functionality.^4,5

Most tissues in the human body are soft and elastic with structural stability during tissue growth. An ideal tissue engineering scaffold should be able to restructure these physical properties of natural tissues.⁶ In addition, it should be biocompatible, biodegradable, with adjustable porosity and sterilizability and appropriate mechanical properties. Moreover, it should promote cell adhesion, proliferation and differentiation and integrate well with surrounding tissues.^7–12 Citric acid (CA) is an intermediate of the tricarboxylic acid cycle and plays an important role in metabolism, calcium sequestration, hydroxyapatite formation, and regulation of structural thickness of bone apatite.^13,14 Since CA has three carboxyl groups (COO–) and one hydroxyl group (–OH), it actively participates in hydrogen bonding interactions with other polymer networks and improves their properties.¹⁵

Modification of polymers with CA produces antioxidant and anti-inflammatory properties, and the residual carboxyl and hydroxyl groups can be further modified functionally by converting hydroxyl polymers into active functional polymers through cross-linking reactions, i.e., citrate-based biomaterials (CBBs). Compared with conventional biodegradable synthetic polymers, CBBs have controllable mechanical properties, good biodegradability and excellent biocompatibility, which are the foundation for their biomedical applications. The mechanical and degradation properties of CBBs can be adjusted by controlling the cross-linking temperature (of the initial monomers during synthesis), cross-linking time, vacuum and molar ratio. More importantly, CBBs can be completely degraded in phosphate-buffered saline within 6 months and more rapidly due to the action of various enzymes in the body. Furthermore, in contrast to other exogenous adhesion protein coatings, CBBs mediate non-specific adsorption of proteins to promote cell adhesion and do not cause chronical inflammation in vivo. Importantly, CBBs has a mild cross-linking temperature and normal pressure during synthesis, which can effectively maintain the activity and stability of the encapsulated drug or protein. Therefore, CBBs have many advantages such as simple synthesis, controllable structure, good biocompatibility, biomimetic viscoelastic mechanical behaviour, controllable biodegradability, further functionalization, etc., and have a broad application prospects in the regeneration of soft and hard tissues and nano-drug design,^16–18 which attracting the attention of researchers in different scientific and engineering fields.

This review focuses on the synthesis of CA, the development of CA-based biomaterials, and discusses the favorable physiochemical properties of tunable CA-based copolymers in biomedical applications, therefore providing guidance for further research. Finally, we highlight the applications of CA-based biomaterials in biomedical engineering such as bone tissue engineering, skin tissue engineering, drug delivery, vascular and neural tissue engineering, and bioimaging, aiming to foster the biomaterial innovation to meet diverse biomedical needs (Fig. 1).

Fig. 1 Functional CA-based biomaterials for biomedical engineering applications.^19–24 The PCCGA hydrogel can restore uniform articular surface and cartilage ECM levels, as well as inhibit cartilage resorption and matrix metalloproteinase-13 levels;²⁵ Copyright 2023, John Wiley and Sons. PC and PC-PPCN facilitate macrophage polarization from M1 to M2, inhibit bacterial infection, promote skin cell migration, and stimulate angiogenesis;²⁶ Copyright 2023, John Wiley and Sons. The pSCDs synthesized via citric acid (CA) and 1-(3-aminopropyl) imidazole (API) can facilitate DOX loading via hydrophobic interactions (loading efficiency: 78.55%). The DOX-loaded pSCDs collapse at tumoral pH (pH ∼ 6.5) due to protonation of the imidaz;²⁷ Copyright 2019, American Chemical Society. The SF/citric acid (CA)-based biomimetic composite coatings exhibit well-balanced antibacterial properties, biocompatibility, and osteo/angiogenic functionalities;²⁸ Copyright 2024, Elsevier Ltd. All rights reserved. Bioactive polycitrate nanoclusters (POCG-PEI600) could significantly enhance myoblast mitochondria number and proliferation, significantly improve the myogenic differentiation of myoblasts through p38 MAPK γ signaling pathway;²⁹ Copyright 2020, Elsevier Ltd. All rights reserved. Citric acid carbonized polymer dots (RCPDs) with red/NIR emission for LNs-targeted imaging, Lymphocyte homing has been demonstrated as the cellular mechanism of CPDs-targeted LNs imaging;³⁰ Copyright 2023, Elsevier Ltd. All rights reserved.

2. Development of CA-based biomaterials

2.1 Preparation of CA

The discovery of CA was in 1784 by Scheele. In 1937 CA was found as an important metabolic intermediate in the tricarboxylic acid cycle (by Krebs).³¹ CA is an organic compound, a tricarboxylic hydroxy acid, with three carboxylic functional groups. It is a triprotic compound that undergoes three constant dissociations, which allows it to form three types of salts and exhibit buffering properties (Fig. 2(A)).³² CA forms crystalline mono-, di-, and tri-basic salts with various cations. From a technological perspective, the most important are calcium citrate, potassium citrate, and sodium citrate.

Fig. 2 (A) Chemical structure of CA;³² Copyright 2023, Elsevier Ltd. All rights reserved. (B) CA production processes with A. niger;³³ Copyright 2024, MDPI. (C) biosynthesis of citramalate in Escherichia coli expressing the cimA gene coding citramalate synthase. Copyright 2022, John Wiley and Sons.³⁴

CA can be produced through microbial fermentation or it can be extracted from a natural source. Chemical syntheses of CA are possible but uneconomical because the starting chemical is usually more expensive than the desired product-citric acid. The production of CA by microbial fermentation has many advantages compared to direct extraction from citrus fruits and therefore is the most widely used one to produce CA. Compared with other methods, fermentation method has the advantages such as simple control, time-saving, labour-saving production procedure as well as higher yield obtained in the end,³⁵ and can be performed at a low temperature, moderate pH, and high substrate concentration.³⁶

At present, submerged fermentation using Aspergillus niger is the mainstream technology in current use.³⁷Aspergillus niger microorganisms offer several advantages, including their ability to quickly adapt and grow on various substrates, regulate and control metabolic pathways, and regulate the secretion of citric acid from both mitochondria and cytosol. This contributes to CA accumulation and prevents its degradation in the Krebs cycle. Moreover, cultures using Aspergillus niger are characterized by high production efficiency and homofermentative citric acid biosynthesis.³⁸Aspergillus niger strains have been recognized as safe, as they do not produce ochratoxin under controlled cultivation conditions and do not elicit strong allergic reactions in humans (Fig. 2(B)).³³

Currently, the methods of preparing ca by microbial fermentation are also developing and progressing. For example, Rzechonek et al. used Yarrowia lipolytica strains overexpressing Gut1 and Gut2 to produce CA under cheap crude glycerol pH 3 to establish aseptic conditions for the synthesis of organic acids, and the results showed that over expression of GUT1/GUT2 could produce organic acids aseptically and that crude glycerol was a suitable substrate for the efficient production of isocitric acid.³⁹ Wu et al. prepared the E. coli citrate synthase was engineered to contain point mutations intended to reduce the enzyme's affinity for acetyl–CoA, but not eliminate its activity. Cell growth, enzyme activity and citrate production were compared for several variants in shake flasks and control fermenters. Citrate synthase GltA[F383M] not only promoted cell growth in the absence of glutamate, but also increased citrate yield by 125% in batch fermentations compared to a control strain containing native citrate synthase (Fig. 2(C)).³⁴ In the post-treatment process of CA production, Ye et al. used CO₂ to convert calcium citrate into citric acid and CaCO₃ by controlling the reaction parameters (ratio of reactants, temperature and pressure), and the CaCO₃ produced during the conversion process can be further used in the fermentation industry to produce citric acid. Under the optimal experimental conditions, the highest conversion rate of 94.7% was achieved. The method is simple, easy to separate CA and environmentally friendly, and can be used as a potential alternative downstream processing route for CA production by fermentation.⁴⁰

2.2 Synthesis of citrate-based biomaterials

CA is a highly bio-friendly and cost-effective material. It can be obtained from citrus fruits through a relatively simple process and is approved by the U.S. Food and Drug Administration for a variety of biomedical applications. The tetrafunctional nature of the CA monomer provides both dangling carboxyl and hydroxyl groups, and the presence of three carboxyl and one hydroxyl groups allows the polymerisation of CA with diols via a simple thermal condensation reaction, which in turn forms ester bonds and facilitates hydrolytic degradation. Meanwhile, effective retention of the carboxyl and hydroxyl groups during polymerisation contributes to further network cross-linking of the polymer post-polymerisation, which is useful for the subsequent design of biodegradable polymers with antioxidant, antimicrobial and fluorescent properties.⁴¹

In 2004, Yang et al.⁴² synthesized the first CA-based biodegradable elastomers, poly(2-hydroxyethylene citrate) (POC). They used CA and 1,8-octanediol as starting materials, via a simple and economical one-pot polycondensation reaction under mild conditions without the addition of catalyst or cross-linking reagent. Subsequently, a series of novel degradable citrate-based biomedical elastomers, including poly(hexamethylene citrate) (PHC), poly(octamethylene citrate) (POC), poly(decamethylene citrate) (PDC) and poly(dodecamethylene citrate) (PDDC), were synthesized using CA and diols by melt polymerisation and vacuum thermal cross-linking techniques.

The main crosslinking approaches of citrate-based polymers include thermal crosslinking (esterification and hot-spot knockdown reactions) and room-temperature crosslinking (HDI crosslinking, double-bond crosslinking and mussel-excited crosslinking). During synthesis, the mechanical and degradation properties of citrate elastomers can be modulated by controlling the cross-linking temperature, cross-linking time, vacuum, and molar ratio of the initial monomers. For example, increasing the cross-linking temperature or cross-linking time can increase the cross-linking density of CA elastomers, improve their tensile strength and Young's modulus, and decrease the elongation at break and degradation rate, offering CA-based biodegradable elastomers with controllable mechanical properties, good biodegradability and excellent biocompatibility.⁴³

Citrate-based materials include not only conjugates of CA and diols, but also biomaterials in which CA is used as a cross-linking agent. Cross-linking reagents, also known as cross-linkers, are compounds that have two or more reactive ends which can chemically connect to functional groups present on the polymers such as primary amines, sulfhydryls, carboxylic ends and others. CA is capable of forming polymeric networks by crosslinking one, two or more biopolymers. CA has been extensively utilized as a cross-linker for a wide range of carbohydrates such as starch, cellulose, chitin, hemicellulose, chitosan and pectin. Despite having three carboxyl groups in total, a single CA molecule can only attach one or two polymer chains, whether they are different or the same polymer chains. Because the centre carboxylic group of CA only makes it possible for one of the other carboxylic groups to receive a proton during the twice-occurring esterification reaction, tri-esters cannot establish cross-linking bonds.⁴⁴ The process of cross-linking is based on covalent intermolecular di-ester interactions between the –COO of the CA and the –OH groups of the biopolymer or polysaccharide.⁴⁵ The most accepted cross-linking mechanism for CA-mediated cross-linking is via esterification which works through the formation of an anhydride intermediate (Fig. 3).³²

Fig. 3 Mechanism of citric acid crosslinking with different biopolymers.³² Copyright 2023, Elsevier Ltd. All rights reserved.

Carboxymethylcellulose (CMC)–poly(ethylene glycol) (PEG) based hydrogel were prepared via a chemical cross-linking route (CA as cross-linker) for delivery of poorly soluble drugs. The most reactive CMC C₆–OH groups (OH groups attached to the C₆ of the anhydro glucose unit) and PEG terminal OH groups can easily engage in the esterification process with CA, resulting in the formation of CMC–PEG hydrogels.⁴⁶ The utility of CA as a cross-linker for hydrogel development opens up a plethora of biomaterial advancement opportunities; the carboxyl and hydroxyl groups from citrate molecules on polymer chains can be employed for bio-conjugation for the development of biofunctional materials. Sabzi et al. developed pH-sensitive drug delivery hydrogels from PVA with CA and Ag nanoparticles.⁴⁷ Chen et al. developed a CA-modified chitosan hydrogel containing an antimicrobial drug, which is promising for wound healing.⁴⁸ Risley et al. prepared poly(sebacate glyceryl citrate) (PGSC) elastomers using CA as a cross-linking agent and investigated the effects of different citrate content and curing time on mechanical properties, elasticity, degradation and hydrophilicity. The results showed that PGSC has rapid thermal cross-linking and tunable elasticity properties, making it a potential candidate for large-scale biomedical applications.⁴⁹ Mendonça et al. prepared a crosslinked polymer hydrophilic gel using glyceryl oleate reacted with citric acid, which showed satisfactory water absorption and reducing properties.⁵⁰

2.3 Properties of citrate-based biomaterials

2.3.1 Biochemical properties. The specificity of the structure of the CA monomer, as well as the hydroxyl and carboxyl groups of the side chains during polymerisation make it possible to subsequently design citric acid-based biomaterials with antimicrobial, antioxidant, adhesive and fluorescent properties, which can then be widely used in bioengineering.
2.3.1.1 Antimicrobial properties. Bacterial and fungal infections are major problems in wound closure and repair using surgical devices or implants, leading to prolonged healing time, wound dehiscence, abscesses and even sepsis formation. Wound infections are particularly susceptible to large burns and trauma for diabetic or other immunocompromised patients.⁵¹ CA is not only biocompatible and haemocompatible, but also recognized as a highly bactericidal chemical.⁵² This is because CA, as an organic acid, flows through bacterial cell membranes and lowers the intracellular pH. Low intracellular pH damages enzyme activity, proteins, DNA and the extracellular membrane, leading to microbial death.⁵³ For example, Kong et al.⁵⁴ found that CA inhibits the oxidation of nicotinamide adenine dinucleotide (NADH) after lowering pH, which leads to bacterial death. Another possible reason is that CA chelates reactive ions in the cell wall after altering the local pH environment, which may inhibit microbial uptake of required nutrients and alter the permeability of the cell wall, resulting in damage that leads to cell death, a cause that is particularly specific to Gram-negative bacteria. Therefore, unlike other biodegradable polymers such as PLA and PLGA, CA in citrate-based co-polymers not only acts as a feedstock monomer but also exerts potential antimicrobial properties.
2.3.1.2 Antioxidant properties. In biomaterials-induced inflammatory responses, immune cells secrete pro-inflammatory cytokines that induce the production of reactive oxygen species (ROS, including hydrogen peroxide (H₂O₂), hydroxyl radicals (OH⁻), and superoxide anion radicals (O²⁻)⁵⁵) from resident inflammatory cells, which can impair the normal cellular functions by damaging the DNA, proteins, and lipids. During the inflammatory phase, excessive accumulation of ROS defeats the inherent antioxidant system and fuels the inflammatory response, thus delaying wound healing.⁵⁶ It has been pointed out that secondary events triggered by traumatic brain injury (TBI) are key factors affecting the recovery of patients, where excessive release of ROS and local inflammation at the injury site are the main obstacles to the healing process.⁵⁷ Polycitric acid diols have intrinsic antioxidant properties, which can be enhanced by material design. For example, human exfoliated deciduous teeth stem cell-derived exosomes (SHED-Exo) have been found to promote functional recovery of TBI⁵⁸ by ameliorating neuroinflammation.⁵⁹ However, this effect is limited by the unsustainable release of exosomes and the presence of oxidative stress (which exacerbates secondary damage in TBI). To obtain the long-term anti-inflammatory effect of SHED-Exo and to alleviate oxidative stress, Lei et al.⁶⁰ prepared gallic acid-grafted poly(citric acid) (PGEGa) with the aldehyde Pluronic F127 (DF-F127) in a Schiff's reaction to produce a multifunctional poly(citric acid-gallic acid) hydrogel (FPGEGa). FPGEGa hydrogel is thermosensitive, injectable, self-healable and highly efficient in antioxidant capacity, and showed significant therapeutic potential for TBI in a rat model.
2.3.1.3 Fluorescence. In recent years, most of the reported fluorescent materials are synthesized from conjugation or encapsulation of organic dyes or quantum dots, however, certain problems exist, such as lower anti-photo-bleaching of organic dyes and higher toxicity of inorganic quantum dots, which significantly limiting the application of these biodegradable fluorescent polymers. Therefore, more and more attention are paid to the design of fully degradable and biocompatible fluorescent polymers.⁶¹ Yang et al.⁶² synthesized a series of biodegradable photoluminescent polymers (BPLPs) based on CA via one-pot polycondensation using various essential α-amino acids reacted with CA and aliphatic diols to introduce intrinsic photoluminescent properties. Unlike conventional aromatic fluorescent polymers, non-degradable organic dyes and quantum dot fluorophores, the BPLPs exhibit excellent photoluminescent properties, including high quantum yields up to 62.33%, tunable fluorescence emission from 303 to 725 nm and remarkable photostability. Moreover, by selecting different amino acids, BPLPs can display fluorescence properties from blue to red.⁶³
2.3.1.4 Adhesion. Existing surgical adhesives are not ideal for many surgical applications due to the material toxicity or poor mechanical and adhesive strength under wet conditions. The surgical adhesives with strong wet tissue adhesion, degradability, controlled mechanical properties and good biocompatibility are preferred. To improve adhesion under wet and mechanically dynamic conditions, researchers, inspired by the adhesion mode employed by some marine organisms (e.g., the blue mussel Mytilus edulis),⁶⁴ have developed an adhesive that can adhere to non-specific surfaces in an aqueous environment. Despite attractive wet tissue adhesion properties, existing mussel-inspired adhesion polymers are inherently non-degradable,⁶⁵ thus greatly limiting their potential use in various medical applications such as tissue engineering and drug delivery. As a result, Yang et al.⁶⁶ synthesised a novel family of injectable CA-based mussel bioadhesives (iCMBA) via a one-step polycondensation reaction using CA, polyethylene glycol (PEG), and catechol-containing monomers, such as dopamine or levodopa. iCMBA's wet tissue adhesion strength is 2.5–8.0 times stronger than clinically used fibrin glue, and has controlled degradability and tissue-like elastomechanical properties, which exhibits excellent cell/histocompatibility both in vitro and in vivo. Sutureless wound healing experiments in rats (2 cm length × 0.5 cm depth) demonstrated that iCMBAs were able to stop bleeding immediately and seamlessly, whereas the existing gold standard, fibrin glue, could not achieve this due to weak wet tissue adhesion. Moreover, the new bioadhesive promotes wound healing and can be completely degraded and absorbed without causing a significant inflammatory response. In conclusion, iCMBA is a novel adhesive material with high wet adhesive strength, controllable degradability, improved biocompatibility, and significantly reduced manufacturing cost. Xu et al.⁶⁷ prepared a series of citrate-based tissue adhesives and explored the effects of hydrophobic and hydrophilic segment units in the skeletal structure on the adhesion, and the results showed that in this amphoteric citrate-based soft-tissue adhesive system, co-coagulation could be formed through hydrophobic interaction when the feed ratio of the hydrophilic segment was lower than 0.7, which can form a hydrophobic effect similar to that of the efficient underwater mussel-like adhesive system.

2.3.2 Mechanical properties. The average molecular weight (M_w) of the POC prepolymer was 1085 g mol⁻¹ (M_n = 1085 g mol⁻¹), and then post-polymerisation was carried out under vacuum or atmospheric pressure for 1 h ∼2 weeks at 120 °C, 80 °C, 60 °C and even 37 °C, respectively, to obtain the POC bioelastomer. Tensile tests confirmed that the POC elastomers do not undergo permanent deformation and have good tensile strength and Young's modulus (6.1 MPa and 0.92 ± 0.02–16.4 ± 3.4 MPa, respectively), which are comparable to those of the elastin in bovine ligaments (tensile strength and Young's modulus of 2 MPa and 1.1 MPa, respectively). Maximum elongation at break was 265 ± 10% of the initial length, similar to that of arteries, veins (up to 260%), and elastin (up to 150%), and the tensile strength, elongation at break, and modulus of elasticity were all tunable over a range. As the crosslinking temperature and time increase, the tensile strength and modulus of the material increase and the elongation at break decreases. Since low cross-linking temperatures favour the stability and activity of the drug or protein encapsulated in the material, the prepolymers obtained by low-temperature cross-linking (80 °C, 60 °C, 37 °C) offer the possibility of using POC as drug delivery carriers. Also cross-linking POC at these temperatures leads to low cross-linking degree and relatively high molecular weight between cross-links, conferring high elongation and low modulus to the elastomer. It was demonstrated that the introduction of MEDA into the crosslinked network not only achieves high tensile strength and Young's modulus, but also significantly increases the degradation rate of the material.⁴¹

To further modulate the mechanical properties of CA-based biomaterials, He et al.⁶⁸ combined CA with glycerophosphate, β-glycerophosphoric acid disodium salt (β-GP-Na) and glycerophosphoric acid calcium salt (GP–Ca), respectively, by a simple and convenient one-pot condensation reaction to obtain POC-βGP-Na and POC-GP-Ca with tensile strengths as high as 28.2 ± 2.44 MPa and 22.76 ± 1.06 MPa, respectively. The initial modulus ranges from 5.28 ± 0.56 MPa–256.44 ± 22.88 MPa. Meanwhile, it is feasible to control the mechanical properties of the POC-GPs by changing the type of salt and the salt feeding ratio.

In addition, Dey et al.⁶⁹ doped polyurethane chain segments within the polyester network of POC to obtain a novel prepolymer, urethane–paraben polycitric acid glycol ester (UPE), which was crosslinked to the elastomer as CUPE. Polyester network was doped with polyurethane chain segments, the hydrogen bonding force in the polymer network was enhanced, and the mechanical strength of the polymer was significantly increased, with the tensile strength of CUPE reaching up to 41.07 ± 6.85 MPa, and the corresponding elongation at break was 222.66 ± 27.84%. The mechanical properties and degradation rate of CUPE can be controlled by varying the selection of the diols used for synthesis, the polymerisation conditions, and the concentration of the polyurethane bonds in the polymer.

2.3.3 Biodegradability. Biodegradability is a highly desirable property of biomaterials. The material degradation pattern and rate, as well as the possible toxicity during the degradation products, collectively influence the application potential of the material. In contrast to conventional non-degradable polymers, CA copolymers do not require subsequent surgical removal after in vivo implantation, expanding their range of biomedical applications such as tissue engineering, drug/gene delivery and bioimaging.⁶⁹ The surface erosion is degradation of the matrix surface only, and bulk erosion is uniform degradation of the matrix surface and interior.⁷⁰ Lu et al.⁷¹ systematically studied the in vivo and in vitro degradation mechanisms of CA-based copolymers, and found that the copolymers degradation behaviour in vivo was similar to the in vitro degradation results. The degradation is mainly through bulk erosion. The inflammatory response induced by degradation was comparable or even less pronounced than that of PLA. Wan et al. developed a novel citrate-based polyester elastomer with artificially regulatable degradation rate, that the degradation rate of the citrate-based polyester can be regulated by breaking the S–S in its crosslinking network using glutathione. The development of citrate-based polyester with artificially regulatable degradation rate was expected to facilitate the individualized therapy and precision remedy of biodegradable implants in clinical treatment.⁷²

2.3.4 Biocompatibility. The biocompatibility of any additional building blocks in the polymer network for further modification or functionalization should be considered at the outset of material design. In the early 1960s, it was discovered that the citrate molecule accounts for approximately 5 wt% of the total organic composition of bone, and that more than 90% of the total citrate content of the human body is within the skeletal system.⁷³ Citrate is maintained at a constant concentration in human and animal plasma. Normal plasma citrate concentration in humans ranges between 100–150 μM, and any significant change in plasma citrate concentration may indicate a pathological condition and can be used as a diagnostic biomarker.⁷⁴ All these data underline the compatibility of CA in vivo and support the wide range of applications of CA-based biomaterials such as POC, which has shown potentials in tissue engineering (bone, skin, blood vessels), drug delivery, and bioimaging. In contrast to other exogenous adhesive protein coatings, POC elastomers mediate non-specific adsorption of proteins, promote cell adhesion and do not cause chronic inflammation in vivo.

3. CA-based biomaterials in biomedical engineering

3.1 Bone tissue engineering

Bone tissue is a layered structure composed of organic proteins (mainly collagen I), inorganic minerals (mainly calcium phosphate) and a variety of cells (osteoblasts, osteoclasts, stem cells, etc.).⁷⁵ The ECM of bone mainly consists of cross-linked collagen fibres, with bone mineral (calcium phosphate crystals) distributed in and around the collagen fibres.⁷⁶ The composition and layered structure of bone tissue gives it unique mechanical properties and physiological functions.⁷⁵ Ideally, bone tissue engineering scaffolds should have multifunctional properties, including biomimetic structural properties, controlled biodegradation, good biocompatibility and antimicrobial activity, as well as good osteoconductivity and osteoinductivity.^77–81

In the early 1960s, it was found that citrate molecules accounted for approximately 5% of the total organic components of bone, revealing that citrate is a critical component of bone.^73,82 Citrate binds to bone inorganic minerals and plays an indispensable role in stabilizing mineral crystals,⁸³ controlling crystal size,⁸⁴ and crystallinity.⁸⁵ As a powerful and multifunctional monomer, citrate chemistry can form three-dimensional cross-linked network structures¹³ and provide abundant carboxyl and hydroxyl groups when reacted with different diols. Previous studies have shown that citrate in human bone tissue plays an important role in metabolism, calcium sequestration, hydroxyapatite formation, and regulation of bone apatite structure thickness.^86–88 Therefore, citrate-based polymers are promising candidates for bone tissue engineering (Fig. 4 and Table 1).

Fig. 4 (A) The possible mechanism of bone mineral absorption;⁸⁹ Copyright 2018, Elsevier Ltd. All rights reserved. (B) Calcium carbonate and citric acid were used as foaming agents to improve the physicochemical properties and osteogenic ability of MPC;⁹⁰ Copyright 2023, Elsevier Ltd. All rights reserved. (C) Schematic diagram of the possible mechanisms for the synthesis of PLA-CD-HA-72 and its biocompatibility and bioactivity.⁹¹ Copyright 2018, Royal Society of Chemistry.

Table 1 Functionalized CA-based biomaterials for bone tissue engineering

Materials	Component	Properties	Tissue engineering	Ref.
GT/PCS/EPL	Gelatin; PCS; ε-polylysine	Bionic elastomer behaviour; controlled preparation; controlled degradation rate	Broad-spectrum antimicrobial activity; enhanced osteogenic bioactivity	92
Cit-Zn-Hap NPs	HAp; citrate; zinc	Altered phosphate environment and reduced surface charge; bionic hydroxyapatite nanoparticles	Synergistically induces osteogenesis in BMSCs; regulates aspects of bone metabolism and promotes osteogenic differentiation	93
BPLP-Ser/HA	CA; 1,8-octanediol; L-serine; HA	Highly plastic; hydration-induced mechanical strength; adjustable degradation rate; low swelling ratio; coagulation; haemostatic sealing	Highly biocompatible; bone promoting; in vivo fluorescence monitoring; osteogenic and angiogenic	94
POCG-PEI600	CA; 1,8-octanediol; polyethylene glycol	Biocompatibility; biosafety; bioactivity; in vivo degradation and elimination	Accelerated myoblast proliferation; myogenic differentiation and skeletal tissue regeneration	95
PEGMC/HA	Poly(ethylene glycol); CA; maleic anhydride	Highly porous microstructure; adjustable mechanics; controlled degradation	Ideal injectable cell carrier; increasable ALP production and calcium deposition	96
PLA/CC	Calcium citrate; PLA	Uniform pore size distribution; appropriate mechanical properties	Synergistic degradation; very high mineralisation activity and ability to direct apatite formation	97
TCP-4Sr	α-Calcium sulfate hemihydrate; TCP; CA; chitosan	High biocompatibility; good mechanical properties; anti-collapse and rapid hardening properties	Better biodegradability and osteogenic properties; ability to promote new bone tissue growth and bone stimulation potential	98
CA-PEOz @E2	HOOC-PEOz-PCL; CA	Very strong stability; good biocompatibility; bone targeting	Reduces bone loss; promotes drug release in the bone resorption microenvironment	99
CTBCs	Tannic acid; hydroxyapatite; POC	Antimicrobial properties; enhanced biomineralisation; increased cell adhesion and proliferation	Enhanced osteoconductivity and osteoinductivity; cell adhesion; biomineralisation	19
PPM	PCS; PCL; miRNA	Control of miRNA loading and release	Enhanced bone regeneration	100
PEC-GS/BG	POC; BG	Improved angiogenesis; osteoblast differentiation	Enhanced bone regeneration	101
PPCN-g	Polyethylene glycol citrate-co-nisopropylpropylacrylamide (PPCN); gelatin; stem cells	Thermo-responsive gels; cell carriers	In vivo induction of bone formation	102

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Bone graft is the second most commonly transplanted tissue in the world after blood transfusion. More than 2.2 million bone graft surgeries are performed annually worldwide, among which 50% are spinal fusion surgeries. The ideal bone substitutes should be osteoconductive, osteoinductive, biodegradable, non-immunogenic, non-transmitting, easy to use, mechanically robust, and cost-effective. Recently, it has been recognised that citrates play an integral role in enhancing osteoconductivity and osteoinductivity to promote bone formation. Tang et al.¹⁰³ synthesised citrate-based polymers with mechanical robustness and rapid degradation by incorporating N-methyldiethanolamine (MDEA) into poly(1,8-octanediol citrate), and prepared POC-M-click-HA scaffolds by forming a composite material with HA, which were used for interbody fusion in a rabbit model. The results of the study demonstrated that the POC-M-click-HA scaffold promoted new bone formation, achieved higher spinal fusion rates (11.2 ± 3.7 and 80 ± 4.5 at weeks 4 and 8, respectively), and maximum loads and stiffnesses of the scaffold-fused vertebrates were 880.8 ± 14.5 N and 843.2 ± 22.4 N mm⁻¹, respectively. Therefore, CA-based scaffold has the potential to serve as promising bone graft for spinal fusion applications.

Guo et al.¹⁰⁴ developed a citrate-based bionic biphasic scaffold to replicate the native structure of cortical and cancellous bone, which could provide immediate structural support and long-term tissue regeneration for large bone defects. Citrate improves biocompatibility and promotes bone formation and provides pendant carboxyl groups that chelate with HA particles up to 65 wt%. The addition of citrate into the biphasic scaffold design better mimics the bimodal distribution of highly porous cancellous bone and dense compact cortical bone, which provides structural support immediately after implantation, and allows for fine-tuning of the scaffold structure and mechanical properties to meet the needs of different sites. He et al.⁶⁸ utilised the activity of CA to prepare POC-GP by incorporating two types of glycerophosphates into citrate-based materials to further enhance the bioactivity of citrate materials and promote osteogenic differentiation of stem cells. The results showed that GP incorporation greatly improved the mechanical properties of the polymers. The degradation rate of the resulting polymers could be easily adjusted by varying the salt type and feeding ratio, and the results of the rabbit femoral condylar defect model demonstrated that the osteoconductive capability of GP-doped POC was significantly improved. Guo et al.¹⁹ synthesized tannic acid modified HA (AgTHA) followed by subsequent reaction with POC prepolymer yielding citrate-based and tannin-bridged bone composites (CTBCs). CTBCs compression strengths up to 323.0 ± 21.3 MPa and possess tunable degradation profiles, which obtained favorable biocompatibility, enhanced biomineralization performance, increased cell adhesion and proliferation, and showed considerable antimicrobial activity. In vivo study of porous CTBCs using a lumbar fusion model further confirmed CTBCs’ osteoconductivity and osteoinductivity, suggesting the potential of promoting bone regeneration in clinical practice.

A proper pH microenvironment is essential for the regenerative function of biomaterials. Mao et al.¹⁰⁵ introduced the alkaline fragment BHEp into citrate-based biodegradable elastomers to neutralise acidity and reduce the inflammation due to rapid release of CA, and then fabricated poly(citric acid-1,8-octanediol-1,4-bis(2-hydroxyethyl)piperazine (BHEp)) (POPC)/β-tricalcium phosphate (β-TCP) porous scaffolds using 3D printing. The results showed that the alkaline fragment BHEp could effectively correct the acidic environment, improve the biocompatibility and cell affinity of POPC, therefore promoting cell adhesion, proliferation, and the expression of osteogenesis-related genes.

Injectable bone implants have been widely used in bone repair such as treating comminuted bone fracture (CBF). Xie et al.²⁰ developed a novel injectable citrate-based mussel-inspired bioadhesive hydroxyapatite (iCMBA/HA) bone substitute for CBF treatment. It is notable that iCMBA/HA can be set within 2–4 minutes and the as-prepared (wet) iCMBA/HA possesses low swelling ratios, compressive mechanical strengths of up to 3.2 ± 0.27 MPa, complete degradation in 30 days, suitable biocompatibility, and osteoinductivity. This is also the first time that citrate supplementation in osteogenic medium and citrate released from iCMBA/HA degradation have been demonstrated to promote the mineralization of osteogenic differentiated human mesenchymal stem cells (hMSCs).

3.2 Skin tissue engineering

The skin consists of an outer epidermal layer and an inner dermal layer, which have specific functions such as protection against dehydration, barrier against trauma, sensory perception, vitamin D synthesis and immune surveillance. The outer layer of the epidermis consists mainly of keratinocytes (nearly 90%), which eventually proliferate from the basal layer and differentiate into the terminal layers of the epidermis to form the epidermis. Due to the presence of stem cells, the epidermis is a highly regenerative skin layer that contributes to skin homeostasis and ultimately aids in wound repair.^106,107 Skin is exposed to constant damage, therefore is prone to have defects (wounds) due to its elastic and soft nature.¹⁰⁸ Although human skin can spontaneously self-repair to restore its structural and functional integrity, wound care remains essential to prevent infection and dryness, relieve pain, protect open areas, accelerate the healing process, and avoid scar formation, especially for large, open wounds or burns.^109,110 Wound healing depends on several factors such as the type of wound (epidermal, deep skin, full thickness), tissue damage from burns (first, second and third degree) or physical trauma, moisture status around the wound, inflammation, secondary infections, etc.^111,112 Wound healing is a gradual process consisting of (1) an inflammatory phase characterised by macrophage or leukocyte infiltration and cytokine secretion, (2) a proliferative phase involving removal of damaged tissue and formation of granulation tissue at the wound site, (3) a maturation phase in which the extracellular matrix produced by proliferating tissues becomes clear, and (4) the formation of scar tissue indicating completion of the wound healing process.^107,113,114 The process is affected by various external factors such as bacterial infection, temperature, pH and sugar levels.¹¹⁵ Previous studies have shown that various factors can effectively improve wound healing, such as good haemostasis, removal of pathogenic microorganisms, inhibition of inflammation, modulation of the wound microenvironment, reduction of scar formation and promotion of vascularisation.^116,117 To improve healing, an ideal wound dressings should have excellent tissue adhesion and porosity, controlled mechanical properties, haemostatic capacity, antimicrobial activity, anti-inflammatory properties and sustained release of bioactive molecules.¹¹⁸

Citrate-based polymers have excellent biocompatibility, antimicrobial properties, and angiogenic capacity, with potential applications in wound healing, as shown in Fig. 5 and Table 2. In skin tissue engineering, inhibition of inflammation and promotion of early angiogenesis are of great interest. Xie et al.¹¹⁹ reported the potential anti-inflammatory mechanism of poly(octanediol-citrate-polyethylene glycol) (POCG) copolymers in modulating skin wound repair. It was found that POCG could down-regulating the expression of pro-inflammatory cytokines (tumour necrosis factor-α (Tnf-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6)) and polarising macrophages to an anti-inflammatory (M2) phenotype. This increased the expression of angiogenic factors (vascular endothelial growth factor (Vegf) and cluster of differentiation 31CD31) and promoted endothelial cell angiogenic differentiation. In vivo studies have shown that POCG can accelerate skin wound repair by inducing early angiogenesis and inhibiting inflammation via regulation on macrophage polarisation (Fig. 5(B)). Liu et al.¹²⁰ reported an anti-inflammatory polycitrate–polyethyleneimine–ibuprofen multifunctional FEA-PCEI hydrogel dressing, and PCEI effectively increased the number of anti-inflammatory M2 macrophages and inhibited the expression of inflammatory factors. The FEA-PCEI hydrogel was found to have an injectable, self-healing, anti UV, haemostatic and antimicrobial properties. It showed highly anti-inflammatory activity in vivo, effective enhancement of skin wound healing and appendage regeneration, and reduction on scar formation. This study provides a strategy to repair wounds by modulating the immune cell phenotype through the design of bioactive multifunctional biomaterials.

Fig. 5 (A) Carboxymethyl starch/polyvinyl alcohol/citric acid (CMS/PVA/CA) hydrogels containing silver nanoparticles (AgNPs) with multiple functions such as antibacterial activity, non-toxicity, and good mechanical properties;¹²¹ Copyright 2020, Elsevier Ltd. All rights reserved. (B) Schematic illustration for showing the chemical structure of POCG polymer and its potential function in skin wound healing and skin regeneration; Copyright 2022, John Wiley and Sons.²⁶

Table 2 Functionalized Citric acid-based biomaterials for skin tissue engineering

Materials	Component	Properties	Tissue engineering	Ref.
POC/PLA NFMs	CA; 1,8-octanediol; PLA	High elasticity; controlled degradation rate; high hydrophilicity; swelling properties	Accelerated wound healing	122
CNDs	CA; urea	Bactericidal; antioxidant; anti-inflammatory properties	Promote wound healing	123
GPDF	GelMA; PCG; Fe^3+	Injectability; self-healing; antioxidant properties; photothermal properties.	Postoperative tumour recurrence and tissue regeneration	21
POC/PAA	POC polymers; poly(acrylic acid)	ECM; antimicrobial activity; growth factor carrier; enhanced adhesion and proliferation of skin fibroblasts	Treatment of infected wounds	124
PCE/PCL	ε-Polylysine; POC	Excellent tensile modulus of elasticity; antimicrobial; good hydrophilicity	Accelerated wound healing; angiogenesis	125
				126
PPCP	PCS; PLLA; curcumin; dopamine	Good antioxidant; anti-inflammatory; broad-spectrum antibacterial properties	Promotes chronic wound healing	127
iCMBA	CA; PEG; dopamine	High adhesion strength; controlled mechanical and degradation properties	Haemostatic adhesives and wound closure	128
iCMBA-EPE/MgO	CA; PEG–PPG–PEG diol; magnesium oxide	High adhesive strength; low swelling; good antimicrobial and haemostatic properties	Promotes wound closure and potential bone regeneration	129
H-HKUST-1	PPC; HKUST-1 NPs	Sustained release of copper ions; antioxidant properties; promotes collagen deposition and angiogenesis	Accelerated diabetic wound healing	130
PC	D,L-panthenol; CA; polyethylene glycol glycerol	Thermo-responsive; oxidative; antibacterial; anti-inflammatory; Ppomotes angiogenesis	Accelerated wound closure; improved quality of regenerated tissue	26
PVA/ZnO	ZnO; PVA; CA	High swelling ratio; antimicrobial activity	Anti-acne action	26
AbAfiCs	CA, 10-undecenoyl acid (UA); ZnCl2, polyethylene glycol; dopamine	Wet tissue adhesion; excellent in vitro cytocompatibility; rapid degradability; strong antibacterial and antifungal properties	Bioadhesive; wound closure	131

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Microbial infections are major medical factors affecting wound healing. CA has been widely used in dental rinses and root canal irrigants for antimicrobial cleansing and even for bactericidal purposes in concentrations of up to 25–50%. As an organic acid, the potential antimicrobial effect of CA may be due to its pH effect, which reduces the intracellular pH of bacteria, leading to reduced enzyme activity, DNA damage¹³² or inhibition of nicotinamide adenine dinucleotide (NADH) oxidation.¹³³ In addition, the relatively acidic environment created by CA is otherwise favourable for wound healing.¹³⁴ Xie et al.¹³⁵ developed an in situ moulded biodegradable hydrogel (iFBH) system consisting of a combination of a newly developed biodegradable poly(ethylene glycol) citrate maleate (PEGMC) and poly(ethylene glycol) diacrylate (PEGDA). The in situ molded hydrogel system adapts to the shape of the wound, thereby providing complete coverage and preventing bacterial invasion. iFBH wound dressings are also conjugated and functionalized with antimicrobial peptides. In vivo and in vitro evaluations against bacteria in rats showed that the peptide-containing iFBH wound dressings provided excellent bacterial inhibition and promoted wound healing. Luo et al.²¹ report a multifunctional bioactive therapeutics-repair-enabled citrate-iron hydrogel scaffold (GPDF) for efficient post-surgical skin cancer treatment. GPDF scaffolds possess the inherent injectable self-healing antioxidative photothermal UV-shielding capacities to achieve the purpose of inhibiting tumor recurrence and enhancing wound repair at the same time. The polycitrate-dopamine enabled the pronounced antioxidation performance of GPDF, which greatly promoted the wound repair and regeneration through decreasing the inflammation response and improving angiogenesis. Taguchi et al. developed a novel tissue adhesive consisting of collagen and a CA derivative, which was prepared by modifying the three carboxyl groups of citric acid with N-hydroxysuccinimide in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, which introduces three reactive ester groups in a single citric acid molecule. The maximum bond strength of this adhesive was similar to the tear strength of approximately 80% of the original soft tissue, it was biocompatible, and it biodegraded within 7 days after subcutaneous injection.¹³⁶

In a recent study, Wang et al.¹³⁷ developed a novel compound and biomaterial building block, panthenol citrate (PC). It has interesting fluorescent and light-absorbing properties, and it was shown that PC could be used to prepare thermo-responsive hydrogel dressings to improve the delayed wound healing in diabetic patients. PC possesses antioxidant, antibacterial, anti-inflammatory, and angiogenesis-promoting properties, and promotes migration and proliferation of keratinocytes and dermal fibroblasts. It can improve re-epithelialisation, granulation tissue formation and neoangiogenesis in a diabetic wound model, and also reduce inflammation and oxidative stress in the wound environment, and can improve the quality of regenerated tissue. Chang et al.¹³⁸ developed the first CA crosslinked sphingan WL gum (WL) hydrogel film, and the covalently crosslinked and solubilised WL–CA hydrogel was hydrolytically stable, highly porous (>60%), moderately tissue-adhesive, and well rheological, and the loading of the drug ciprofloxacin (CIP), WL–CA–CIP hydrogels exhibit sustained drug release properties, long-lasting antimicrobial activity and good biocompatibility for potential wound dressing applications.

3.3 Drug/cell delivery

Drug delivery systems (DDS), especially nanoparticle (NP)-based DDS, have great potential to improve drug diagnostics and therapeutic efficacy. The field of drug delivery has progressed tremendously in the last few decades due to improvements on nanoscale DDS. These NPs (e.g., liposomes) have been investigated for the delivery of drugs or pharmaceutically active compounds to the intended location to improve drug solubility, reduce toxicity and extend circulation time.^139–141 Ideal delivery vectors should have multifunctional properties including high loading rate, retention of biological activity, controllability and microenvironmental responsiveness (e.g., pH, magnetism, and reactive oxygen species), sustained release, targeted delivery, and good biodegradability and biocompatibility, which ensure the safety of the vectors in vivo and enhance the therapeutic efficacy of the drug/gene.^142,143 Citrate-based polymers have biodegradable, controllable mechanical properties and biocompatibility, and various scaffolds of citrate-based materials including amphiphilic micelles, porous elastomers, nanofibers, and hydrogels based on POC polymers have been extensively investigated for the delivery of drugs and genes, as shown in Table 3 and Fig. 6.

Table 3 Citrate-based functionalized polymers for drug/gene delivery

Materials	Component	Properties	Tissue engineering	Ref.
CA-cl-SSH	CA; salvia spinosa seeds	Thermal stability; stimulus responsiveness; biocompatibility	Zero-order kinetic sustained-release drug delivery system	144
NCH	PEG-200; maleic acid; CA	pH response; excellent swelling and mechanical properties; cytocompatibility; antimicrobial properties	Adjuvant to skin cancer treatment and wound healing	145
IONPs-DOX	CA; iron oxide magnetic nanoparticles; DOX	Excellent settling stability and strong magnetic properties; long-term stability; biocompatibility; targeted drug delivery	Tumour therapy	146
MMBs	Celastrol (CST); Fe3O4; CA; polyethylene glycol; PLGA	Good imaging properties; good biosafety; improved drug uptake in solid tumours; pH-sensitive properties; targeted drug delivery	Tumour therapy	147
BC-T	CA; tragacanth gum (TG); α-arbutin (AR); bacterial cellulose	Good hydrophilicity; excellent mechanical properties; high biocompatibility; controlled release	Whitening drug delivery	148
CA-PEOz	HOOC-PEOz-PCL; CA; β-estradiol (E2)	Bone targeting; reducing bone loss	Osteoporosis (PMOP) treatment	99
cAp-Tb-DF	Diclofenac; citrate; apatite (Ap)	Good cytocompatibility; controlled release; luminescence changes localise inflammation	Bone-localised delivery of anti-inflammatory drugs	149
PCA/DOX NPs	Poly(methacrylic acid); CA; DOX	pH-sensitive properties; chelating Cu^2+ effect; anti-tumour efficacy; low toxicity	Tumour therapy	150
CA-NP	CA; carboxymethyl cellulose; HA-NPs	Rapid penetration; efficient transport; efficient oral absorption; more effective bacteraemia treatment effect.	Oral delivery of peptide drugs	151
PCD-zeolite	Zeolite; CA; cyclodextrin (CD)	Improved solubility and bioavailability	Loading and release of ibuprofen	152
The crosslinked ceiba pentandra fibers	Kapok cellulose; CA; chlorhexidine diacetate (CHX); ceiba pentandra	Biocompatible; controlled release.	Drug delivery systems	153

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Fig. 6 (A) The CA modification significantly increased the bone targeting of this drug delivery system, and the delivery system was able to achieve rapid drug release under bone acidic conditions;⁹⁹ Copyright 2023, Elsevier Ltd. All rights reserved. (B) The surface of MSNs was surface modified with CA and then heated with Lys to form the DES-MSNs system. The system drives the nanoparticles into deeper skin via “Drag” effect. transdermal delivery of the nanoparticles into body circulation was achieved;¹⁵⁴ Copyright 2022, Elsevier Ltd. All rights reserved. (C) Novel drug delivery system based on PVA/CA/Ag NPs was developed the CA crosslinker endowed pH-responsiveness and antibacterial activity to the PVA;¹⁵⁴ Copyright 2020, Elsevier Ltd. All rights reserved. (D) Hyperbranched polyesters as drug delivery systems, cisplatin was loaded by these copolymers and P(CAx-G)-CDDP drug delivery systems were obtained;¹⁵⁵ Copyright 2013, John Wiley and Sons.

Conventional DDS has a number of limitations and is unable to meet the growing demands of current clinical practices. Scientists are seeking truly achievable targeted and personalised therapies. Poor localisation remains the most serious problem, and most drugs are poorly targeted, thus usually leading to a range of side effects.¹⁵⁶ In order to overcome the drawbacks of conventional nanomaterials and to prolong the drug circulation time by avoiding recognition by the mononuclear phagocyte system (MPS), cellular DDS came into the limelight, especially for erythrocytes, immune cells, stem cells, and so on. Drug delivery based on DDS of these cell types has attracted much attention. Ye et al.⁶⁰ designed a hybrid hydrogel (FPGEGa) based on the bioactive antioxidant poly(citrate–gallate) to encapsulate SHED-Exo for the treatment of traumatic brain injury. This thermosensitive, injectable, self-healing and antioxidant FPGEGa provides an ultra-long sustained release of SHED-Exo (21+ days) and significantly reduces ROS production in CNS microglia. FPGEGa carrying SHED-Exo (FPGEGa@SHED-Exo) exhibited improved anti-inflammatory potential on microglia by promoting M2 (anti-inflammatory) polarisation and inhibiting M1 (pro-inflammatory) polarisation. FPGEGa@SHED-Exo improved neural regeneration, rescued motor function and regenerated damaged cortical tissues in traumatically brain-injured rats. Repair and recovery of motor function after spinal cord injury (SCI) remains a worldwide challenge. Inflammatory microenvironment is one of the major obstacles inhibiting recovery from spinal cord injury. Wang et al.¹⁵⁷ prepared an injectable adhesive anti-inflammatory agent, F127-poly(citrate-poly(ethyleneimine)) hydrogel (FE), which possesses sustainable and long-lasting release of extracellular vesicles (FE@EVs), and can be used to improve the recovery of motor function after SCI.

The gene-based therapies have become one of the very attractive approaches for restoring tissue function (tissue regeneration) and treating diseases such as cancer. Zhang et al.¹⁵⁸ developed a safe and efficient anti-tumour nanoplatform (M@NPs/miR365), which consists of poly(citrate-peptide) (PCP), miRNA365 mimic, and MC38 cancer cell membranes (M), and can effectively deliver the miR365 mimic into MC38 cancer cells, promote apoptosis of MC38 tumour cells and regulate the expression of Bcl2 and Ki67 in vitro. Tumour cell implantation mice model showed that the injection of M@NPs/miR365 via tail vein could effectively inhibit tumour development. Zhang et al.¹⁵⁹ developed an anti-obesity nanosystem consisting of poly(citrate)-glycerol-polylysine (PCG-EPL) and miR33 agonist by self-assembly (PCG-EPL/miR33agonist). PCG-EPL could effectively load, protect and delivered miR33 agonist into adipocytes and reduced the expression of obesity-related IL-1β in adipocytes in vitro, which effectively reduced body weight in rats by promoting lipid metabolism and reducing the expression of inflammatory factors (IL-1β, TNF-α and IL-6). Ganguly et al. prepared a novel in situ gel for sustained drug delivery and targeting. This in situ gel system consists of chitosan-glyceryl monooleate (GMO) and citric acid in. The release of the drug from the gel follows matrix diffusion control mechanism. The novel in situ gel system can be used for sustained delivery of the drug by oral and parenteral routes.¹⁶⁰

Yuan et al.¹⁶¹ prepared an injectable citrate-containing polyester hydrogel that sustained the releases of citrate as a cellular modulator and the encapsulated growth factor mydgf along with hydrogel degradation, which significantly reduced scar formation and myocardial infarction area, increased myocardial wall thickness and neovascularisation, and improved cardiac function. Alpaslan et al.¹⁶² synthesised novel biocompatible homopolymer and copolymer microparticles based on maleic acid and CA for absorption/release of thiamine model drug, which have excellent ability to absorb and release thiamine in addition to biocompatibility, antioxidant and antibacterial properties.

Local tumor therapy through injectable biodegradable hydrogels with controlled drug release has attracted much attention recently, due to their easy operation, low side effect and efficiency. Wang et al.¹⁶³ developed a multifunctional injectable biodegradable-visible citrate-based self-healing scaffold with microenvironment-responsive degradation and drug-release functionality for safe and efficient cutaneous oncology treatment (FPRC hydrogel). The scaffolds have multifunctional properties including thermal sensitivity, injectability, self-healing, photoluminescence and pH-responsive degradation/drug release. The FPRC scaffold with strong red fluorescence has good photostability, tissue penetration and biocompatibility, which allows it to be tracked and monitored to assess the degradation of the scaffold in vivo. In addition, the FPRC scaffold showed pH-responsive DOX release, which effectively reduced A375 cancer cell proliferation in vitro and inhibited tumour growth in vivo. A feasible method for hyperbranched polyesters consisting of CA and glycerol monomers was described by Adeli et al.¹⁵⁵ The ability of the synthesised hyperbranched polyester to load and transfer cisplatin as an anticancer drug was investigated. It was found to have a high loading capacity, and the prepared drug delivery system was stable in saline for several months. Cisplatin loaded in polyester showed lower IC50 compared to free cisplatin values, confirming the efficacy of the synthesised citrate–glycerol hyperbranched polyester as a carrier for transporting anticancer drugs (Fig. 6(D)). Microgels fabricated based on microfluidic technology with monodisperse properties and controllable morphology have shown attractive prospects for controlled drug delivery, tissue engineering, magnetic separation, contrast agents, thermotherapy and thermal ablation, Moharramzadeh et al. prepared alginate microgels containing citrated magnetic nanoparticles (MNPs) using a microfluidic system with citrate groups attached. The hydrodynamic size of MNPs changed from 142 nm to 826.7 nm, which improved dispersibility and stability of aqueous phase, as well as high biocompatibility and ability to promote cell proliferation.¹⁶⁴

3.4 Vascular and neural tissue engineering

Cardiovascular diseases (CVDs) have become the leading cause of death worldwide. The growing burden of CVD has become a major public health problem, and how to treat CVD efficiently and reliably has become an urgent global need.^165,166 Considering the structure and function of natural cardiovascular tissues, cardiovascular tissue engineering strategies have focused on the development of biomaterial scaffolds to mimic or replace natural cardiovascular tissues, which provide the necessary microenvironment for cell proliferation/differentiation and effectively maintain normal physiological activities. Ideal cardiovascular grafts should possess multifunctional properties, including bionic elastomer mechanical properties, appropriate biodegradation, excellent biocompatibility, inhibition of thrombotic and inflammatory responses, and promotion of endothelialisation.^167,168 One tissue engineering strategy to overcome these challenges is the use of synthetic polymers, but this strategy also suffers from limitations due to the inherent challenges of blood-polymer interactions. The design of bioactive polymers for cardiovascular tissue engineering could address some of these challenges.

Depending on the application type, different biomolecules can be bonded via pendant carboxyl functional groups in citrate-based biopolymers (CBBs) to enhance their biological functions. Among the CBBs used for cardiovascular tissue engineering, POCs have been the most extensively investigated (Table 4 and Fig. 7). Xu et al.¹⁶⁹ fabricated a scaffold with encapsulated vascular endothelial growth factor (VEGF) and β-fibroblast growth factor (bFGF) via POC and PLGA particles, which significantly improved endothelial progenitor cell proliferation, haemocompatibility, platelet activation, and inflammatory response. Yang et al.¹⁷⁰ used POC to modify the inner lumen of expanded polytetrafluoroethylene (ePTFE) grafts. The POC interface conferred hydrophilicity to the ePTFE grafts, reduced the rate of thrombosis, promoted endothelialisation of the grafts in vitro, and reduced macrophage infiltration on the grafts. Due to its bionic mechanical properties and good haemocompatibility, POC is an excellent coated biomaterial and vascular implant for cardiovascular tissue engineering. Ware et al.²³ optimized a CA-based bioresorbable photopolymer ink (B-Ink) for high-speed 3D printing of bioresorbable vascular scaffolds (BVSs) with increased mechanical strength. 3D printed BVSs were non-cytotoxic and completely degradable through base accelerated hydrolysis. Qian et al. developed a titanium dioxide (TiO₂) covalently immobilized CA nanohybrid coating, which is fabricated on nitinol (NiTi) braided FDs by liquid phase deposition and dip-coating, to inhibit thrombosis and promote re-endothelialization. The CA molecules are covalently bound onto the pre-deposited TiO₂ nanoparticulate coating. The coating has a unique homogenously nanostructured morphology as well as super-hydrophilicity. Both in vitro and in vivo results verify that the coated samples inhibit platelets and fibrinogen adhesion, delay coagulation, and concomitantly promote re-endothelialization (Fig. 7(B)).¹⁷¹ Ding et al. fabricated an absorbable vascular scaffold (BVS) using photopolymerisable citrate-based biomaterials and a high-precision additive manufacturing process with a scaffold thickness of 62 μm, which meets clinical requirements. The BVS was successfully delivered into porcine coronary arteries using a customised delivery system. The safety and efficacy of the BVS was similar to the commercial XIENCE™ DES drug-eluting stent in terms of maintaining vessel patency for 28 days.¹⁷²

Table 4 Citrate-based functionalized polymers for cardiovascular tissue engineering

Materials	Component	Properties	Tissue engineering	Ref.
POC-PCL	POC; PCL	Biocompatible; promotes cell adhesion and proliferation	Cardiac Tissue Engineering (CTE) of cardiac patches	173
PCMO-CysAm-NO	CA; maleic acid; 1,8-octanediol	Releases NO; regulates vasodilation; promotes cell proliferation and angiogenesis; regulates inflammation and immune response	Vascular tissue engineering	174
POCR-ePTFE	POC; Eptfe; atRA	Reduces macrophage and leukocyte infiltration; inhibits endothelial formation; accelerates endothelialisation	Prosthetic vascular grafts	175
POC-PES	POC; PES	Enhanced haemocompatibility; fibrinogen adsorption; reduced platelet adhesion	Blood purification field	17
PITCO	Dimethyl dicarboxylate; triethyl citrate; 1,8-octanedanol	Small molecule permeability; appropriate elastic properties; promotes cell adhesion and proliferation	Vascular tissue engineering	176
mPDDC	PDDC; acetyl methacrylate	Customisable; compressible; self-expanding; bioabsorbable; antioxidant	3D printed vascular scaffolds	23
PICO	POC; DMI; OD; TEC	Adjustable elasticity; controlled gel time	Heart tissue patch	18
POC-PDDC	POC; PDDC	Biphasic tubular simulation of blood vessels; shortening cell co-culture time	Small diameter vascular stents	177
POC	CA; 1,8-octanedanol	Blood compatibility	Vascular tissue engineering	178
MCPC	CA; MCPC	Enhancement of osteogenic function of osteoblasts and angiogenesis of vascular endothelial cells	Angiogenesis and osteogenic differentiation	179

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Fig. 7 (A) Proposed structure of POCA elastomer generated by thermal cross-linking from POCA prepolymer. POCA elastomer coating number was adjusted by a modified spin-coating method to prepare small-diameter ePTFE vascular grafts similar to natural vessels;¹⁸⁰ Copyright 2021, Royal Society of Chemistry. (B) The proposed anticoagulation and pro-endothelization process of TiO₂–CA coated NiTi braided mesh;¹⁷¹ Copyright 2023, John Wiley and Sons. (C) Engineering antioxidant poly(citrate–gallic acid)-exosome hydrogel with microglia immunoregulation for traumatic brain injury-post neuro-restoration;⁶⁰ Copyright 2022, Elsevier Ltd. All rights reserved.

The nervous system includes central nervous system (CNS, which includes the brain and spinal cord) and peripheral nervous system (PNS, which includes sensory and motor neurons). Nervous system plays an important role in transmitting various types of information in the body.¹⁸¹ The natural regenerative potential of the nervous system is largely inefficient for severe neurological injuries caused by trauma or disease. Therefore, artificial nerve grafts are required to prevent the loss of nervous system function. Previous studies have shown that various factors can effectively promote nerve regeneration and re-establish nerve function, such as maintenance of endogenous neuronal cell attachment and proliferation, electrical stimulation, nerve growth factor and vascular endothelial growth factor.^182,183 Therefore, neural tissue-engineered scaffolds have been extensively investigated to promote nerve repair. Ideal neural regenerative scaffolds should have multifunctional properties, including bionic mechanical properties, controlled biodegradation and porosity, and excellent biocompatibility.^184,185 The mechanical properties of the scaffolds allow them to effectively resist to physiological loading in vivo. Controlled biodegradation of the scaffolds can effectively match the regrowth rate of nerve, and the porosity can ensure the transport of nutrients and gases.

It has been shown that neuronal function in the CNS is supported and regulated by a citrate-rich microenvironment.¹⁸⁶ The effect of citrate-based biomaterials (CBBs) in peripheral nerve regeneration is due to their elasticity, biocompatibility, strength, and resistance to physiological tension, all of which is attributed to the citrate effects on the chemical and mechanical behaviour of the polymers.⁴¹ Ye et al.¹⁸⁷ developed a hybrid hydrogel of bioactive antioxidant poly(citrate–gallate) loaded with SHED-Exo (FPGEGa@SHED-Exo). The thermosensitive, injectable, self-healing, and antioxidant FPGEGa was able to provide an ultra-long sustained release of SHED Exo (more than 21 days), which significantly reduced ROS production in CNS microglia and exhibited better anti-inflammatory potential. FPGEGa@SHED-Exo improved neural regeneration, rescued motor function, and regenerated damaged cortical tissues in traumatic brain-injured rats. Wang et al.¹⁵⁷ prepared an injectable adhesive anti-inflammatory agent termed F127-poly(citrate-poly(ethyleneimine)) hydrogel (FE). It possesses sustained and prolonged release of extracellular vesicles (FE@EVs) and can be used to improve the recovery of motor function after SCI. The FE@EVs hydrogel's Orthotopic injection wraps extracellular vesicles around the injured spinal cord, thereby synergistically inducing effective integrative modulation by inhibiting fibrotic scar formation, attenuating inflammatory responses, and promoting myelin re-formation and axonal regeneration. Tran et al. fabricated a bionic, multi-channel, crosslinked POC polyester tissue engineered (CUPE) neural rail, which mimics the natural microtubule and dermal nerve structure for neural tissue repair. The CUPE neural conduit has precisely controllable channels it has precisely controllable number and diameter of channels, porosity, and mechanical properties, with a final peak stress of about 1.38 MPa and an elongation of about 22.76%, which is comparable to natural nerves. In addition, CUPE nerve rails showed fibre numbers and densities equivalent to those of nerve autografts after 8 weeks of treatment for 10 mm sciatic nerve defects in rats, suggesting its potential in neural tissue engineering.¹⁸⁸ Liu et al.¹⁸⁹ used CA as a cross-linking agent to design and fabricated a macroporous chitosan-based hydrogel with excellent cytocompatibility and antioxidant properties. The hydrogel showed positive effects on cell survival, adhesion and proliferation as well as antioxidant properties, and could inhibit oxidative stress after nerve injury.

3.5 Bioimaging

Modern biomedical imaging techniques have revolutionised life science by providing anatomical, functional and molecular information on biological species with high spatial resolution, deep penetration, temporal responsiveness and chemical specificity. In recent years, these imaging techniques have been increasingly used to characterise biomaterials and probe their interactions with biological tissues. This, in turn, has driven tremendous advances in engineering properties of materials to accommodate different imaging modalities.¹⁹⁰ Biodegradable fluorescent biomaterials have been extensively used to develop carriers for tissue engineering. Ideal fluorescent biomaterials should have multifunctional properties, including controlled and stable fluorescence, excellent fluorescence penetration, good biodegradability and biocompatibility, and simple synthesis pathways.¹⁹¹ CA-based polymers exhibit good photoluminescence properties after further functional modification due to their special chemical structures as shown in Table 5 and Fig. 8.

Table 5 Citrate-based functionalized polymers for bioimaging

Materials	Component	Properties	Tissue engineering	Ref.
GT-PCS-EPL	PCS; ε-poly(L-lysine); collagen; gelatin	Bionic elastomer behaviour; tunable photoluminescence; broad-spectrum antimicrobial activity; osteogenic bioactivity capacity	In vivo real-time non-invasive imaging tracking for tissue regeneration in multidrug-resistant bacterial infections	126
PCGA	CA; polyethylene glycol; L-arginine	High haemocompatibility; low cytotoxicity; excellent photostability	For live cell attachment and proliferation imaging; targeted bioimaging	192
PPFR	Folic acid; CA; 1,8-octanediol; polyethylenediamine; rhodamine B	Stable photoluminescence; haemocompatibility; cytocompatibility	For targeted tumour imaging and gene therapy	24
PCGA	POC; PEG; arginine	Water soluble; photoluminescent	Bioimaging	24
BPLP-Cys/ECM-Hep	POC; cysteine; ECM; heparin	Photoluminescence; reduction of platelet adhesion and endothelial proliferation; antioxidant activity	Vascular tissue engineering	193
CHPO-ET/PEG	PHC; cysteine/serine; thiol acid; multi-arm PEG	Injectability; strong and tunable fluorescence properties; sustained release of drugs	Fluorescence imaging in vivo	194
PSC-based	POC; AS; CSNW/SN/BGN	Controlled mechanical properties and biodegradation; tunable fluorescence emission; photostability	Bioimaging and tissue regeneration	195
PCE	POC; EPL	High elongation and recovery rate; antibacterial	Bioimaging	196
RCPD	CA; benzoylurea; NH4F	Excellent red/NIR emission properties	Targeted imaging of lymph nodes	30

---

Fig. 8 (A) Synthesis of citric acid-based CDs using three different nitrogen-containing precursors;¹⁹⁷ Copyright 2016, American Chemical Society. (B) A novel carbonized polymer dots (RCPDs) with red/NIR emission for LNs-targeted imaging was prepared by citric acid and benzoylurea;³⁰ Copyright 2023, Elsevier Ltd. All rights reserved. (C) C-dots were synthesized from citric acid following citric acid with tris (hydroxymethyl) methyl aminomethane (CATris), and exhibit favorable biocompatibility, an easily modifiable surface, strong blue fluorescence emission and low cytotoxicity;¹⁹⁸ Copyright 2023, MDPI. (D) Schematic of functionalities (fluorescence imaging, PA imaging, and conductivity that promote cell communications) of cross-linked BPLPATs;¹⁹⁹ Copyright 2018, John Wiley and Sons.

Nitrogen-doped carbon dots synthesized from CA as a carbon precursor have recently been considered to contain fluorescent derivatives of citrazinic acid, which contribute to their emission in the blue spectral range. Schneider et al. synthesized three samples employing CA and three different nitrogen sources: ethylenediamine, hexamethylenetetramine, and triethanolamine. By analysing the nitrogen content and its coordination by X-ray photoelectron spectroscopy, FTIR spectra, and systematically comparing absorption, steady-state emission, and photoluminescence decays of each kind of carbon dot, we derive the influence of the molecular precursors and gain further understanding of the complex structure of carbon dots, highlighting the strong impact of molecular fluorescence in the samples produced with ethylenediamine and hexamethylenetetramine¹⁹⁷ (Fig. 8(A)).

A red/near infrared (NIR) emission CPDs (RCPDs) with one and two-photon bioimaging based on CA and benzoylurea (BU) are prepared. Notably, the RCPDs are capable of targeting LNs for imaging³⁰ (Fig. 8(B)). Leveraging the advantages of citrate chemistry, Shan et al. developed a multifunctional citrate-based biomaterial platform with both imaging and therapeutic capabilities (BPLPATs) utilizing a facile and efficient one-pot synthesis. BPLPAT nanoparticles are able to label cells for fluorescence imaging and perform deep tissue detection with PA imaging¹⁹⁹ (Fig. 8(D)).

Bioimaging is an important component of therapeutic systems, because biomaterials capable of in situ imaging can assess material degradation, track drug delivery, and identify specific diseased tissues for the treatment of cancer and degenerative diseases. The increasing number of degenerative diseases, defective tissues and cancers has increased the demand for advanced biomaterials with multifunctional therapeutic capabilities but minimal design complexity. Li et al.²⁰⁰ prepared photoluminescent poly(citrate-siloxane) nanoparticles (PCSNPs) via an oil/water emulsion method. The PCSNPs possessed a uniform particle size distribution (average diameter of 120 nm) and highly stable dispersion (more than 30 days) in various physiological media, as well as excellent fluorescence. PCSNPs have good cellular biocompatibility, which can be effectively internalised by cells and selectively image cellular lysosomes with high photostability. Compared to commercial Cell Tracker™ Green and Cell Tracker™ Red, PCSNPs can stably label adipose MSCs or human liver cancer cells for more than 14 days during cell growth and development (7 passages) for long-term targeted subcellular structural imaging, in vivo tumour tracking, and therapeutic applications. Davoodikia et al.²⁰¹ synthesised a novel CA-based dendritic polymer-G2-alendronate nanoradio-tracer, which showed no significant cytotoxicity against HEK-293 at different concentrations, and higher accumulation of the radiotracer in bone compared to the commonly used bone scanning agent 99mTc-MDP. Compared with 99mTc-MDP, the accumulation of radiotracer in bone is higher, and its biological distribution pattern is similar to that of 99mTc-MDP.

Conventional fluorescent biomaterials include inorganic quantum dots, rare-earth-based nanoparticles, fluorescent dyes, and proteins, which have various drawbacks for biomedical applications. Rare earth-based nanoparticles are very prone to accumulate in the body due to their non-biodegradable properties, which may lead to long-term toxicity.²⁰² Biodegradable polymer biomaterials with intrinsic photoluminescent properties have attracted attention for their potential advantages in tissue regeneration and non-invasive bioimaging. Du et al.²⁰³ developed elastomeric poly(silicone citrate) (PSC) hybrid polymers with controlled biodegradability and mechanical properties, tunable intrinsic fluorescence emission (up to 600 nm), and high photostability (more than 180 min under UV light and more than 6 months in natural light), as well as high cytobiocompatibility and minimal in vivo inflammatory response in vivo. The development of elastic PSC polymers provides a new strategy for the synthesis of novel inorganic–organic hybrid photoluminescent materials for tissue regeneration and bioimaging applications.

Carbon-based dots (CDs) have received much attention due to their unique optical properties. However, the exact origin of its optical properties remains controversial.^204,205 CA and some amino-containing small molecules have been considered as ideal precursors for the synthesis of highly luminescent CDs by various heat treatment processes. Fang et al.²⁰⁶ chose CA and four small amino-containing molecules as a model for the synthesis of CD, and systematically investigated its photoluminescence (PL) properties. The results indicate that the photoluminescence properties of CD are synergised by the contained luminescent pyridine derivatives and defective states, which is of great significance for understanding the concentrative properties of CD as well as the preparation of highly concentrative CDs with tunable emission spectra.

Targeted tumor imaging and efficient specific gene delivery in vivo are among the main challenges in gene-based cancer diagnosis and therapy. Wang et al.²⁴ engineered a CA-based polymer with intrinsic photoluminescence and gene loading capacity to achieve targeted delivery of siRNA and tumor imaging in vitro and in vivo. The multifunctional platform was formed from the self-assembling of poly(CA)–polymine conjugated with folic acid and rhodamine B (PPFR). PPFR showed stable photoluminescent ability and could effectively bind and protect the siRNA against RNase degradation. PPFR also exhibited good blood compatibility and cell compatibility against C2C12, MCF-7 and A549. Compared with commercial transfection agent Lipofectamine™ 2000, PPFR had a high cellular uptake, equivalent transfection efficiency and effectively down-regulated intracellular p65 expression in A549 cancer cells. Importantly, PPFR could efficiently accumulate and label the tumor tissue through the fluorescent imaging, selectively deliver siRNA into tumor tissue in vivo in the tumor-bearing nude mice model. This work may provide a facile strategy to synthesize multifunctional biocompatible biomaterials for targeted tumor imaging and gene therapy.

3.6 Other applications of citric acid-based biomaterials

The search for an ideal biomaterial with appropriate mechanical properties and biocompatibility has been a hot topic in the field of soft tissue engineering. Gyawali et al. reported a novel photocrosslinked biodegradable elastomer derived from CA termed poly(octamethylene maleate citrate) (POMC), which retains pendant hydroxyl and carboxyl functional groups after crosslinking for potential conjugation of bioactive molecules. The development of POMC enriches the family of CA-derived biodegradable elastomers and expands the range of biodegradable polymers to meet the diverse needs of biomedical applications.²⁰⁷ Zhang et al. developed a novel polyester elastomer (CUPOMC) by reacting a photocrosslinkable POMC prepolymer with 1,6-hexamethylene diisocyanate followed by a photocrosslinking polymerisation reaction, CUPOMC has tunable mechanical properties and supports cell adhesion and proliferation. Unlike other prepolymers of crosslinked elastomers, CUPOMC prepolymers are highly processable, and the development of CUPOMC will expand the choice of biodegradable elastomers for a variety of biomedical applications such as soft tissue engineering.²⁰⁸

To date, most tissue-engineered products require surgeries in clinical use, which has limited their use in areas such as cardiac tissue engineering because of the various risks and possible complications associated with open-heart surgery. Therefore, combining tissue engineering with minimally invasive delivery would greatly enhance the prospects for widespread adoption of tissue-engineered products. Montgomery et al. developed novel shape memory scaffolds using citrate-based polymers (POMaC), which are photocrosslinkable, biodegradable, non-toxic and have minimal inflammation. Through microfabrication and photocrosslinking, the material can deliver fully functional tissues without major surgery in the heart, liver and aorta of a large animal (pig), as well as in subcutaneous injections in mice and rats.²⁰⁹ Tran et al. also demonstrated that POMaC has elastomeric properties, as cardiac tissues cultured on anisotropic elastic POMaC scaffolds (10 × 10 mm) were able to deform through a 1-mm opening and regain the scaffolds’ original shapes without damaging the tissue. In a rat model of myocardial infarction, implantation of the POMaC scaffolds significantly improved cardiac function compared to controls with only myocardial infarction.²¹⁰

Photocrosslinked bioelastomers are widely used in tissue engineering applications due to their good physical, chemical, mechanical and biocompatibility. Huyer et al. synthesised poly(itacolate-citrate-octanediol) (PICO) polyester elastomers using CA, 1,8-octanediol and itaconic acid, which showed tunable elasticity via free radical cross-linking of itaconic acid esters in a viscous polyester gel polymer backbone network formation. By varying the reaction time and the molar composition of the monomers, materials with a tunable elasticity range (36–1476 kPa) can be generated, and finally the materials are used as scaffold supports for cardiac tissue patches, which can show significant tissue structure and viability.¹⁸ On this basis, Savoji et al. prepared a series of novel fast photocrosslinked bioelastomer prepolymers using PICO. Tensile tests confirmed their elastic properties with Young's modulus between 11–53 kPa. These materials supported the culture of viable cells and enabled the adhesion and proliferation of human umbilical vein endothelial cells.¹⁷⁶

4. Conclusions

In this paper, we review the development of multifunctional citrate-based biomaterials for biomedical applications, and summarize the multifunctional properties of citrate-based biomaterials in terms of their physical, chemical, and biological aspects, including mechanical and biodegradation, as well as biocompatibility, adhesion, antimicrobial, antioxidant, anti-inflammatory, and fluorescent properties for biomedical applications. Finally, the applications of citrate-based biomaterials in biomedical engineering are summarised, including bone tissue engineering, skin tissue engineering, drug/cell delivery areas, vascular and neural tissue engineering, and bioimaging. It is worth noting that citrate-based biomaterials have good properties for a variety of biomedical applications; however, further detailed studies on material preparation, functionalisation, properties and biomedical applications are needed. However, further detailed studies on material preparation, functionalisation, properties and biomedical applications are needed. Future challenges and prospects based on functional citrate-based biomaterials include the following: (1) to prepare high molecular weight citrate-based polymers and expand their application in biomedical engineering. (2) To further study in detail the molecular mechanism of the function of citrate-based biomaterials in tissue repair. (3) To study the specific metabolic pathways of citrate-based biomaterials in vivo to achieve more targeted functional modifications. By addressing the above issues and then cross-fertilising regenerative medicine with materials science, we believe that citrate-based biomaterials will be transformed into more clinically applicable products in the biomedical field.

Data availability

The authors declare no conflict of interest. Some or all data during the study are available from the corresponding author by request. All images used in the paper have been copyrighted.

Conflicts of interest

The authors declare no conflict of interest.

Acknowledgements

The research was supported by National Key R&D Program of China (2022YFC2405802) and National Natural Science Foundation of China (51973060).

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Footnote

† Co-first authors.

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