Carbon dots: A promising tool for viral infection treatment

Abstract

Globally, viruses have impacted health, economy, and mental well-being of all humankind. Several advanced treatments and vaccines have been developed to overcome this problem. With the advancements in nanoscience and technology in the biomedical field, nanomaterials are now widely used in detection, diagnosis, and therapy. Carbon dots (CDs) have gained significant attention because of their remarkable physical, chemical, and biological properties. The bioactive features of CDs obtained from natural sources, including their antibacterial, anticancer, antiviral, and antioxidant capabilities, have been investigated. However, a broad scope exists to expand these studies and resolve the ambiguity in understanding the associated mechanisms. Hence, it is worth gathering knowledge regarding the potential natural sources of CDs that can help fight novel viral infections. In this review, we begin with a brief introduction in the first section, followed by an overview of viral structure, life cycle, modes of host entry, and current treatment strategies in the second section. The third section discusses the emergence of novel viruses. In the fourth and fifth sections, we examine the application of nanotechnology in managing viral infections, with particular emphasis on the use of CDs and their potential as protective agents against viral diseases. Section six highlights the challenges and limitations associated with the clinical application of CDs. Finally, we summarize the key findings and discuss the future prospects of CDs in antiviral therapy. This review is intended to serve as a valuable reference for the development of innovative treatment strategies against various viral infections.

Graphical abstract

Synthesis of Carbon Dots for Antiviral Therapy.

1. Background

Viral infections remain a major global health challenge owing to their high mutation rates, which lead to drug resistance and complicated control efforts. Despite advances in vaccination and antiviral medicines, viruses such as influenza, Human Immunodeficiency Virus (HIV), and hepatitis, and new diseases such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue to spread. To address this, a multidisciplinary strategy is required, including increased diagnostics for faster outbreak responses, innovative vaccine and gene therapy delivery platforms, and novel antiviral tactics such as target-based drug development and host-targeting antivirals to overcome resistance [[1], [2], [3], [4]]. Progress in gene-editing technology has provided intriguing opportunities to inhibit viral replication [4,5]. Preventive efforts, including the development of next-generation vaccines, immunotherapy, and stronger public health initiatives, are critical for limiting viral transmission [6,7]. Furthermore, using pharmacogenomics for treatment helps personalise medicines, increase their efficacy, and reduce unwanted effects. Continued innovation and collaboration across scientific disciplines are required to effectively manage and reduce the global burden of viral infections.

Nanotechnology, particularly the utilisation of carbon dots (CDs), provides a novel solution to the limitations of existing antiviral medicines. CDs, a type of carbon-based nanoparticle, have attracted widespread attention owing to their distinct features, including size, water solubility, biocompatibility, variable photoluminescence, ease of functionalization, and low toxicity [8,9]. Because of these properties, the application of CDs has expanded beyond their initial use in bioimaging and diagnostics to a larger variety of fields, including drug delivery, biosensing, antimicrobial medicines, and, more recently, antiviral treatments. The nanoscale size of CDs (usually less than 10 nm) allows them to interact directly with viral particles and host cells, affecting critical viral life cycle activities such as attachment, replication, and release [[10], [11], [12], [13]]. CDs prevent viruses from entering host cells by attaching to viral surface proteins or breaking receptor connections, as shown in studies on HIV, herpes simplex virus (HSV), and coronavirus [14]. More importantly, several CDs can convert light into reactive oxygen species (ROS), making them potential photosensitizers for photodynamic therapy. Such CDs damage the viral components and impede their activity, making them helpful for photodynamic treatment [14,15]. Their broad-spectrum efficacy stems from their ability to disrupt various viral processes rather than targeting individual proteins, making them effective against a wide range of viruses, including SARS-CoV-2. The flexibility of CDs, paired with their adaptation to focused applications, makes them useful tools against infectious agents [13]. Furthermore, CDs can promote antiviral immunity by interacting with immune cells, which strengthens the body's defence mechanisms.

Although the CDs played a significant role in the viral treatment strategies, the application of CDs in viral infection treatments faces significant challenges related to the complex infection microenvironment and off-target effects [16]. Viral infections often create dynamic microenvironments characterised by variable pH levels, enzymatic activity, and immune cell interactions, which can alter CDs' stability and antiviral efficacy [17]. For instance, hypoxic conditions common in chronic infections may reduce ROS-dependent antiviral mechanisms, as observed in photodynamic therapy contexts where oxygen scarcity limits therapeutic outcomes [18]. Additionally, the non-specific uptake of CDs by healthy cells remains a critical concern, particularly with surface-functionalized variants. Studies show that CDs enriched with oxygen- and nitrogen-containing groups (e.g., CD_3011 (CDs with the surface carboxyl and phenol groups accompanied by nitrogen)) caused 50 % lethality in mice after repeated dosing, despite minimal organ damage in survivors, highlighting potential systemic toxicity risks [19]. This off-target accumulation could disrupt normal cellular functions, as evidenced by urea level alterations and renal tubule injuries in preclinical models [16,19]. To address these issues, researchers emphasise the need for targeted surface modifications, such as boronic acid or glycyrrhizic acid functionalization, to enhance viral specificity while minimising interactions with healthy tissues. However, the lack of detailed in vivo studies and standardised toxicity profiling protocols continues to hinder clinical translation, underscoring the necessity for deeper mechanistic insights into CDs' biodistribution and long-term safety [[20], [21], [22]].

Synergistic treatments combining CDs with conventional antiviral therapies show promise in addressing challenges like drug resistance and systemic toxicity [21,23]. For example, CDs functionalized with boronic acid ligands demonstrated dual mechanisms-inhibiting viral entry by disrupting spike protein-receptor interactions and suppressing replication by interfering with viral RNA synthesis-when tested against human coronavirus HCoV-229E [17,24,25]. Similarly, glycyrrhizic acid-derived CDs (Gly-CDs) reduced porcine reproductive and respiratory syndrome virus (PRRSV) titers by 99.999 % in vitro while stimulating host antiviral immune responses and suppressing inflammatory pathways [26,27]. These multi-targeted actions complement traditional drugs that often focus on single viral lifecycle stages, potentially reducing the likelihood of resistance. Preclinical studies also highlight the feasibility of dose optimisation: combining sub-therapeutic concentrations of tetracycline with CDs achieved a 75% reduction in E. coli viability at concentrations eightfold lower than standalone treatments [28], suggesting analogous strategies could minimise drug-related toxicity in viral infections [29]. However, challenges persist in balancing synergistic ratios-excessive CD concentrations may exacerbate non-specific cellular uptake, as seen in murine studies where oxygen/nitrogen-rich CDs caused 50% mortality despite functional efficacy [30]. Further research must prioritise in vivo pharmacokinetic profiling and computational modelling to predict optimal combinational dosing while addressing compatibility issues between CDs and biologics like monoclonal antibodies.

This review focuses on the current advancements in the development of CDs for antiviral applications, including their production, mechanisms of action, and possible therapeutic functions in treating viral infections. With sustained research and innovation, CDs may pave the way for innovative broad-spectrum antiviral drugs that are more beneficial, adaptive, and resistant to standard antiviral therapy constraints.

2. Virus: structure, life cycle, entry into host, and treatment

To develop effective treatments, it is imperative to understand the function, structure, reproduction, and interaction of viruses with their hosts. Viruses exhibit a wide spectrum of structural variations, including morphology, symmetry, and the presence or absence of envelopes. The capsid is composed of a protein shell that encases viral Deoxyribonucleic acid (DNA) and can be helical or icosahedral. Some viruses also have a lipid bilayer envelope made of the host cell membrane, which contains viral proteins that facilitate host cell invasion. The capsid protects the viral genome, which is composed of either DNA or Ribonucleic acid (RNA). In enveloped viruses, the envelope also protects the genome [[31], [32], [33]].

Cryoelectron microscopy has revolutionised the study of viral structures by providing high-resolution images of viral particles, and computational approaches have aided in the analysis of their geometry and behaviour. This is critical for the development of antiviral medication. Viral structures include various symmetries and morphologies, ranging from simple helical to complex shapes, allowing viruses to infect a wide range of hosts and adapt to changing surroundings. Their structures are dynamic, and their conformations change throughout their life cycle. Three-dimensional (3D) printable models of viruses are useful educational tools for visualising viral morphology and aiding in research. Although tremendous progress has been made in research on viral structures, there are still hurdles in completely comprehending their complexity and consequences for viral function [[34], [35], [36]].

The viral life cycle consists of a sequence of phases that a virus experiences to replicate and disseminate within a host cell. This cycle commences with the entry phase, during which the virus adheres to the host cell and infiltrates its membrane to transfer its genetic material, a mechanism that is fundamental for the initiation of infection [38,39]. Subsequent to entry, the virus harnesses the cellular machinery of the host for genome replication, a process that encompasses transcription and the synthesis of novel viral genomes [40,41]. In the context of acute infection, which is marked by a swift emergence of symptoms and viral replication, the virus capitalises on feedback loops present within the host's cellular milieu to augment its replication efficacy. The dynamics of this stage can be represented using kinetic logic, which facilitates the comprehension of the stable regimes of viral behaviour, encompassing both acute and persistent infections [42]. For certain viruses, such as human papillomavirus (HPV), the life cycle comprises distinct phases, including genome maintenance, amplification, and the subsequent assembly and release of new virions [43]. Grasping these processes is crucial for the formulation of antiviral strategies, as the targeting of specific stages in the viral life cycle can impede viral propagation without inflicting damage on host cells [44,45]. In summary, an in-depth understanding of the viral life cycle is imperative for the enhancement of therapeutic interventions aimed at combating viral infections [46].

Understanding viral entry into host cells (Fig. 1) and establishing efficient treatment options are critical for combating viral infections. Recent studies have focused on viral entry mechanisms and the development of antiviral methods that target these processes, emphasising the therapeutic potential of viral entry targeting. SARS-CoV-2 entry, for example, is mediated by the spike (S) protein, which binds to the Angiotensin-converting enzyme 2 (ACE2) receptor, a process aided by proteases such as Transmembrane protease, serine 2 (TMPRSS2) and cathepsins. Heparan sulphate, which is present on the cell surface, also functions as a cofactor. Similarly, the entry of Kaposi's sarcoma herpesvirus (KSHV) is characterised by complicated interactions with host cell factors, and interferon-inducible transmembrane proteins have been demonstrated to prevent KSHV entry in a cell-dependent manner [47,48]. The sodium taurocholate cotransporting polypeptide (NTCP) receptor facilitates the entry of Hepatitis B virus (HBV), whereas Hepatitis C virus relies on host factors such as Cluster of Differentiation 81 (CD81) and Scavenger Receptor class B type I (SR-BI). Filoviruses, such as Ebola, employ host cell components for entry, and understanding these pathways is essential for developing tailored therapy options [37,49,50].

Fig. 1.

Conventional representation of the viral entry and replication cycle with its succession of defined, consistent phases, using human metapneumovirus as an example [37].

Recent treatment techniques are evolving towards host-targeting antivirals that seek to disrupt the host cell components involved in viral entry. This broad-spectrum strategy can be beneficial for reducing drug resistance, as evidenced by the use of maraviroc, a Chemokine Receptor 5 (CCR5) antagonist, in HIV therapy [13]. SARS-CoV-2-specific treatments, such as TMPRSS2 suppression with camostat mesylate, are also under investigation. Broad-spectrum antivirals that target common host pathways used by diverse viruses show promise for the simultaneous treatment of many infections [51]. Advances in host-virus interactomics and next-generation sequencing are improving our understanding of viral entry and enabling the identification of novel treatment targets [7,50].

Although targeting viral entry presents a promising treatment avenue, problems remain owing to the complexity of viral entry processes, particularly for viruses such as KSHV. Viral evolution challenges the development of simple interventional strategies [52]. Overall, targeting viral entry is a promising method of antiviral therapy, with continuous research and technological developments leading to increasingly effective medicines.

3. Emergence of novel viral diseases

The emergence of novel viral diseases is determined by viral evolution, environmental changes, and human activity (Fig. 2). Recent research has highlighted the introduction of novel variants, such as SARS-CoV-2 JN.1, which have been related to an increase in hospitalisations, as well as the development of recombinant viruses, such as the Oropouche virus, through genetic reassortment. Human actions such as deforestation and increasing travel, combined with ecological changes, have aided the spread of zoonotic viruses such as SARS-CoV-2 and the West Nile virus [[53], [54], [55], [56], [57]].

Fig. 2.

Timeline of major disease outbreaks and recently discovered diseases in individuals living in new areas from 2003 to 2022 [62].

The evolution of the XEC strain (a recombinant variant of SARS-CoV-2) demonstrated the ability of the virus to perpetuate growth via genetic recombination and mutation. Strains such as XEC, which includes the XE variant (a hybrid of Omicron subvariants BA.1 and BA.2), show how recombination generates novel genomic architectures [58,59]. Variants such as XE, which have mutations that show continuing viral adaptability, have been reported internationally, first in the United Kingdom and then in Canada and India. Recombinant strains, such as the B.1.628 lineage (XB), demonstrate how genetic reshuffling complicates viral evolution, occasionally modifying viral characteristics and pathogenicity [60]. This advancing mutation poses public health concerns because these variations may influence transmissibility, immunological evasion, and vaccination effectiveness, emphasising the need for continuing surveillance and response efforts [61].

Similarly, the recent identification of the Langya Henipavirus (LayV) in Eastern China emphasises the need to monitor zoonotic spillover occurrences. LayV, a virus similar to Hendra and Nipah, was discovered in 35 people between 2018 and 2022 and was most likely spread by shrews. The symptoms of LayV infection include fever, tiredness, and organ dysfunction. While the risk of human-to-human transmission is unknown, it remains a biosafety class 4 disease [63,64]. Diagnostic techniques, including quantitative polymerase chain reaction (qPCR) [65], have been developed, and increased global and regional surveillance is crucial for recognising and responding to LayV and other emerging zoonotic concerns.

Following the continuing challenges presented by novel zoonotic viruses such as LayV, the resurgence of Mpox, formerly known as monkeypox, has evolved into a major worldwide public health concern. The World Health Organisation has declared Mpox a Public Health Emergency of worldwide concern, and this resurgence has been marked by a spike in cases outside the usual endemic regions because of worldwide travel and public gatherings [66]. Mpox was once endemic to specific African nations but has now expanded to 116 countries, with approximately 92,500 confirmed cases as of January 2024, including instances in Khartoum, Sudan, highlighting the need for increased surveillance and early identification [67,68].

COVID-19 has emerged as a global pandemic within a few months of its first occurrence in November 2019 in Wuhan, China. Unlike other pandemics in the history of mankind, the COVID-19 outbreak saw informed and active initiatives from people across the globe to control infection. COVID-19 becomes complicated when a person suffers from prolonged respiratory and nephrotic diseases [69]. Owing to their structural and genomic similarities to SARS, similar treatment strategies have been implemented for coronaviruses, focusing on the use of ACE-2 inhibitors, antiviral drugs, immunosuppressants/steroids, and plasma from recovered patients [[70], [71], [72], [73]]. Other drugs, such as remdesivir, have also been approved for their efficacy and ability to lead to rapid recovery [74,75]. However, its mode of action and side effects remain unclear. Similar medications, such as ritonavir and ribavirin, have been reported to cause side effects such as diarrhoea, nausea, and vomiting [76]. Steroids are used to suppress and relieve inflammation due to bloating and expansion of the lungs in the short term. Advancements in therapeutics have involved the use of serum plasma collected from recovered patients.

Continuing the trend of rising zoonotic risks, recent Nipah virus (NiV) outbreaks in South Asia (2021–2023) [77] have led to an increase in casualties, whereas novel bat-borne viruses discovered in Japan in 2021 present further concerns. The recurrence of H5N1 avian influenza since 2020 [78] has raised pandemic concerns, whereas Marburg virus outbreaks in Africa [79] have highlighted the critical need for vaccine research. These findings illustrate the importance of increased global surveillance and the readiness to address emerging public health issues.

Effective monitoring, new genetic technology, and global communication are vital for the early identification and prompt responses to epidemics. The diversity of viral mutations and environmental influences makes forecasting future epidemics difficult. Furthermore, emerging persistent diseases, such as SARS-CoV-2 Persistent Infectious Epithelial Syndrome, present novel public health issues [80]. A multidisciplinary approach that considers virology, epidemiology, and public health is required to develop effective preventive and control techniques.

4. Nanotechnology for the management of viral infections

Advances in nanoscience and nanotechnology have opened up new avenues for nanomedicine in viral therapy [45–48]. Nanotechnology offers fascinating possibilities for viral infection management by boosting antiviral medicines, optimising medication transport, and developing novel diagnostic tools (Fig. 3). Nanotechnology can overcome challenges associated with conventional antiviral therapies, such as drug resistance and delivery issues, by using the distinct characteristics of nanoparticles [81]. The structural, physical, chemical, and biological properties of nanoparticles provide a unique ability to target and penetrate abnormal cells, leading to DNA damage and gene defects. Nanoparticles can enhance the pharmacokinetic characteristics of antiviral drugs, thereby lowering their toxicity and increasing their effectiveness. For example, chitosan and gold nanoparticles improve antiviral peptide delivery by increasing solubility, stability, and selectivity [82]. In HIV therapy, nanocarriers such as liposomes, solid lipid nanoparticles, and dendrimers are being investigated to overcome drug resistance and increase drug delivery to difficult locations, such as the central nervous system and lymphatic system [83,84]. Nanomaterials, such as gold, silver, and copper nanoparticles, have virucidal capabilities because they attach to viral proteins or genetic materials and prevent reproduction [85]. For example, copper nanoparticles (Cu NPs) have demonstrated significant antiviral effects on various surfaces [84]. Nanophytovirology, which employs nanoparticles that deactivate viral components and stimulate plant defences, has shown promise for regulating plant viruses [86].

Fig. 3.

Nanoparticles and their characteristics exploited for treating viral infections [91].

In addition, nanotechnology enables the development of improved diagnostic instruments, such as nano biosensors, which have high sensitivity for detecting viral infections and can be incorporated into smartphone platforms for real-time diagnostics [87]. Dynamic cell culture systems based on centrifugal microfluidics provide physiologically appropriate models for studying viral infections and the effects of antiviral medication, as demonstrated by research on HSV- type 1 (HSV-1) [88]. Another application is infection management, which uses nano-enabled disinfectants, such as cerium-oxide nanoparticle coatings, to successfully reduce viral and bacterial loads on hospital surfaces [89]. The antibacterial characteristics of NPs are also being exploited to develop novel antiseptics and disinfectants for treating drug-resistant diseases [90]. Although nanotechnology offers several benefits, issues such as scalability, long-term efficacy, and safety must be addressed. Incorporating deep learning with nanotechnology may improve the diagnostic and predictive capacities, allowing for a more complete approach to viral infection management.

5. Current research progress on CDs for virus prevention

Fluorescent carbon nanomaterials such as CDs have gained significant attention worldwide owing to their synthesis procedures, unique properties, and wide range of applications [[92], [93], [94], [95]]. Therefore, researchers are currently devoted to converting environmental biowaste into useful CDs for various applications, and numerous reviews have described various methods of synthesis [92,[96], [97], [98], [99]]. Their distinct features, such as their ability to produce ROS and biocompatibility, make them ideal candidates for antiviral applications. Recent research has focused on their synthesis, functionalization, and use in virus prevention and therapy, demonstrating their promise as broad-spectrum antiviral medicines. Current research has mainly focused on the development of CD-based antiviral, anti-inflammatory, and antioxidant agents for therapeutic applications. Several CDs exhibit viral inhibitory activity [100]. Since the discovery of CDs, recent studies on their antiviral activity have provided encouraging results [101]. Recently, CDs derived from medicinally important plants have been used as antibacterial [101], antioxidant [102], antifungal [103], and antiviral agents. For example, Kalkal et al. reported that CDs derived from Allium sativum (AS-CDs) exhibit potent anti-inflammatory, immunomodulatory, and antiviral properties, effectively reducing cytokine storms and inhibiting SARS-CoV-2 entry, while their high biocompatibility and fluorescence enable diagnostic imaging for COVID-19 management. Studies demonstrate AS-CDs' low cytotoxicity and ability to downregulate pro-inflammatory cytokines, supporting their role as an effective theranostics agent from a natural source [104].

This review summarises the important properties of CDs and discusses their antiviral activities and mechanisms. Finally, we discuss future perspectives on CDs derived from medicinal plant material as effective antiviral agents.

5.1. Properties and antiviral activities of CDs

CDs were discovered in 2004 [105]and have demonstrated diverse and effective performance in various research areas. Investigations, modifications, and applications of CDs have been conducted to address various issues in the fields of nanomedicine, biosensing, diagnosis, and therapy [106,107]. The properties and applications of CDs are shown in Fig. 4. Typically, CDs are a class of zero-dimensional nanoparticles with a quasi-spherical shape and a size of less than 10 nm [108,109]. They exhibit either an amorphous or crystalline nature, with a crystal lattice of ∼0.34 nm or 0.24 nm, corresponding to the inter-layer spacing (002) of graphite and the in-plane lattice of graphene [100], respectively [[110], [111], [112]]. Because of these structural properties, CDs exhibit a quantum confinement effect.

Fig. 4.

Properties and applications of CDs.

Several functional groups, such as carbonyl, epoxy, amino, and hydroxyl groups, can be anchored to the CD surface [[113], [114], [115], [116]]. These surface functional groups enhance the water solubility and accessibility for functionalization with various organic compounds and biomolecules; they also significantly affect the charge and electronic structure of CDs. These features allow them to act as both electron donors and acceptors. Most notably, CDs exhibit distinctive optical properties, such as an absorption range from UV–visible to near-infrared, fluorescence, photoluminescence, and up- and down-conversion emissions [[117], [118], [119]]. CDs are advantageous in terms of photobleaching and blinking, low toxicity, and excellent biocompatibility (Fig. 4) [99,120].

The properties of CDs mainly depend on their size, shape, surface functional groups, and quantum confinement effects. Moreover, the selection of precursors and synthesis strategies is responsible for their properties. Owing to their unique properties, low cost, and ease of synthesis, they have been extensively used in various bio-applications such as drug delivery, optical sensing, bioimaging, and phototherapy [92,96,97].

The biological properties of CDs are primarily based on the selection of carbon precursors. The antiviral activities of the natural and synthetic precursors obtained from various sources using top-down and bottom-up approaches are illustrated in Fig. 5. Currently, reports on CD-based antiviral therapies are limited, although extensive studies have been conducted on CDs to explore their applications in the pharmaceutical field.

Fig. 5.

Illustration of the antiviral activities of natural and synthetic precursors by top-down and bottom-up approaches.

The study by Łoczechin et al. [121] demonstrates that CDs modified with boronic acid effectively inhibit human coronavirus HCoV-229E infection by disrupting both viral entry and RNA replication, with CDs synthesised from 4-aminophenylboronic acid achieving a tenfold higher potency (EC₅₀ of 5.2 ± 0.7 μg mL⁻¹) compared to those derived from ethylenediamine/citric acid with post-modified boronic acid ligands (EC₅₀ of 52 ± 8 μg mL⁻¹). In Fig. 6a they illustrated the dual mechanism by which CDs inhibit HCoV-229E infection, showing their ability to block the interaction between the viral spike (S) protein and host cell receptors (panel i) and to suppress viral RNA genome replication (panel ii), highlighting their effectiveness at both entry and replication stages. Recently, Ferreira et al. [3] investigated thiol-functionalized CDs fabricated using cysteamine and citric acid as a precursor for inhibiting SARS-CoV-2 by disrupting disulfide bonds in the virus's spike protein receptor-binding domain (RBD) and regulating cellular oxidative stress. CDs, with higher thiol content, achieved up to 60.4 % viral inhibition by reducing RBD disulfide bonds, as confirmed by circular dichroism, and demonstrated significant ROS scavenging and anti-inflammatory effects, suggesting a dual mechanism for mitigating viral infection and inflammation. Fig. 6b–d illustrates that thiolated CDs (CystaD 1-1 and CystaD 2-1) significantly reduce pseudo-SARS-CoV-2 infection levels, with CystaD 2-1 showing superior viral inhibition (up to 60.4 %) compared to CystaD 1-1, highlighting the correlation between higher thiol content and enhanced antiviral efficacy.

Fig. 6.

(a) Influence of CDs, fabricated by hydrothermal carbonisation, on binding of HCoV-229E virus to cells: (i) inhibition of protein S receptor interaction, and (ii) inhibition of viral RNA genome replication [121]. Copyright 2019, American Chemical Society. Viral infection levels for (b) CystaD 1–1 and (c) CystaD 2–1 treatment; (d) calculated viral inhibition of both CDs (0.1 mg/mL not shown due to the negative value on CystaD 1–1); for (A) and (B), ∗ = SS difference from untreated control (∗p < 0.05); for (C), ∗ = SS difference between CDs (∗p < 0.05) [122]. Copyright 202024, American Chemical Society.

Global vaccine advancements and research have accelerated to confront the COVID-19 pandemic while upholding the highest safety standards. CD-based vaccine delivery and adjuvant techniques are currently attracting increasing interest and are being validated. Li et al. developed a CD-based intranasal vaccine to trigger specific immune responses [123]. By employing chitosan and branching polyethyleneimine as starting materials, microwave-assisted pyrolysis was used to synthesise the CDs. Subsequently, negatively charged proteins (antigens), such as ovalbumin, were electrostatically bound to the CDs. Confocal laser scanning microscopy was used to observe the acquisition and internalization of the antigen and CD combinations by dendritic cells. Additionally, vaccination formulations based on CDs may reside at mucosal locations for an extended period with better mucosal antigen mobility, boosting antigen uptake and presentation. Mice immunised with the antigen–CD mixture showed an immunological response specific to the antigen and produced more memory T cells than those injected with the bare antigen. Enhanced antigen distribution and permeability augmentation may be responsible for the strong immunostimulatory capacity. Cheng et al. examined the practical applicability of CDs as vaccination adjuvants in a different study [124]. After the second vaccination, most hens that received the vaccine and CDs showed signs of antiviral immunity. In this study, CDs exhibited greater immunological effectiveness than conventional Freund's additives.

Several research groups have successfully synthesised CDs that can inhibit coronavirus replication. CDs synthesised using boronic acid derivatives are promising for preventing the duplication of coronaviruses [121]. CDs are assumed to enter the virus by collapsing/dissolving the outer membranes, eventually rendering them inactive for replication. The inhibition mechanism can be explained based on the water solubility of the CDs. CDs derived from curcumin, a renewable natural precursor, exhibited coronavirus inhibitory activity in model studies performed on Vero cells (Fig. 7) [125]. Interestingly, CDs may be used to modify the exterior structural features of the virus and may negatively impact its growth at various stages of its life cycle. These CDs provide the possibility for multiple-stage inhibition of the virus from the entry point to host transformation.

Fig. 7.

Impact of curcumin-derived CDs (CCM-CDs) on porcine epidemic diarrhoea virus (PEDV). (a) An indirect immunofluorescence assay was performed to analyse the impact of various CCM-CD doses on PEDV-infected Vero cells. Scale bar: 100 μm. (b) Western blot analysis measured the PEDV nitrogen protein expression level at 12 h post-immunisation in the presence of 125 μg/mL CCM-CDs [83]. Copyright 2018. Reproduced with permission from the American Chemical Society.

Studies have shown that CDs are potential inhibitors of various viruses [121]. Previous investigations have shown that the treatment is based on two typical methods. One method focuses on the entire viral structure by collapsing the exterior surface, rendering the virus unable to mutate. This may be due to time- or concentration-dependent effects. Other treatment methods target a specific part, especially the one that supports viral mutations in the host. For the treatment of COVID-19, a particular target is S1, a protein attached to the ACE-2 enzyme, which is crucial for the activity of the virus in the host [126].

5.2. Mechanism of action of CDs as antiviral agents

CDs function differently during the different phases of viral reproduction (Fig. 8). Viral infections involve four major steps: attachment, penetration, replication, and budding. CDs derived from various sources are used as antiviral agents, and their mechanisms of action are listed in Table 1.

Fig. 8.

Schematic of the mechanism of CD-based interventions against viral infections.

Table 1.

Summary of recent studies on CDs as antiviral agents.

Naturally derived CDs
Active ingredient (Source)	Synthesis approach	Antiviral action against	Mechanism of action	Ref.
Citric acid Boronic acid (vegetables & fruit)	Hydrothermal	Human immunodeficiency virus (HIV), human coronavirus (HCoV-229E)	Inhibition via syncytium formation restricts HIV-1 entry, INF type 1, and interaction with the HCoV-229E S protein.	[121]
Curcumin (Curcumin longa)	Pyrolysis	Japanese encephalitis virus (JEV)	Binds to envelope/E-protein using 4-vinyl guaiacol moieties, sabotaging the fusion and thus inhibiting viral entry into host cells.	[129]
Curcumin (Curcumin longa) Citric acid (citrus fruit)	Hydrothermal	Porcine epidemic diarrhoea virus (PEDV)	Direct action on viral entry, penetration, replication, and budding; activation of innate antiviral immunity (IL-8 and IL-6); reactive oxygen species (ROS).	[125]
Glycyrrhizic acid (Glycyrrhiza sps.)	Hydrothermal	Porcine reproductive and respiratory syndrome virus (PRRSV), PEDV, herpes simplex virus (HSV)	Exhibits antiviral activity on PRRSV by inhibiting PRRSV invasion and replication; inhibits coronavirus and Herpesviridae proliferation.	[130]
Pullulan (Aureobasidium pullulans)	Hydrothermal	Middle East respiratory syndrome (MERS)-related coronavirus, influenza A virus (IAV)	Protective effects against viral infection via interleukins (IL-1β, IL-6) and tumour necrosis factor (TNF-α)	[131,132]
Carrageenan (Chondrus sp. and Hypnea sp.)	Hydrothermal	Dengue virus (DENV), HSV, human papilloma virus (HPV), HIV, hepatitis A (HAV)	Mainly inhibits viral replication following viral internalization but before the release of the viral load.	[131,133]
Allicin (Allium sativum)	–	Coronavirus disease (COVID-19)	ROS depletion effect, anti-inflammatory properties, and strong interactions with ACE-2, impeding viral attachment.	[104]
Ricin (Ricinus communis)	Hydrothermal	–	Immunomodulation via NO and cytokine (TNF-α, IL-6) release.	[134]
Ascorbic acid (citrus fruit)	Hydrothermal	Pseudorabies virus (PRV), PRRSV	Viral inhibition by IFN-α production via IFN-stimulating gene (ISG) expression.	[135]
CDs derived from other sources
Active ingredient	Synthesis approach	Antiviral action against	Mechanism of action	Ref.
Benzoxazine monomer	One-pot Hydrothermal	JEV, Zika virus (ZIKV), DENV, porcine parvovirus (PPV)	Direct interaction with virus particles and intervening viral infectivity within host cells.	[136]
Polyethyleneimine (PEI)	Microwave-assisted pyrolysis	Kaposi's sarcoma-associated herpes virus (KSHV)	Apoptosis and proliferation of primary effusion lymphoma (PEL) cells.	[137]
Biquaternary ammonium salt (BQAS)	Hydrothermal	Virus-induced cancers	Specific immune response against viruses (CD4+ & CD8+ T cells).	[138]

5.2.1. Modification of the attachment and penetration step to inhibit viral infection

Host cell attachment is the first stage of viral infection, and prevention of this step renders the virus inactive. Most CDs that have been observed interfere with viral infection in the initial stages by modifying viral surface antigens. Japanese encephalitis virus and similar flaviviruses cannot enter host cells when they encounter CDs synthesised from benzoxazine monomers. Immunofluorescent CDs were demonstrated to significantly prevent the spread of Zika and dengue viruses in Vero cells [127]. The results of an in vitro assay performed by Barras et al. demonstrated that the direct interaction of CDs with the viral envelope, rather than the development of an antiviral response by the host cells, exacerbated the inhibition of viral infection. Treating the Japanese encephalitis virus with CDs derived from the benzoxazine monomer greatly reduced the viral interaction with host cells [128].

Modifying the cell membrane and associated proteins can restrict viral entry and invasion. Investigation of plaque reduction revealed that curcumin-derived CDs have a potent concentration-dependent inhibitory effect on the porcine epidemic diarrhoea virus. CDs synthesised from curcumin can prevent viral infection during the initial phases of viral invasion. According to Raman spectroscopy and fluorescence studies, electrostatic interference of positively charged CDs triggers viral aggregation and deactivation [125]. The study by Barras et al. demonstrated that functional CDs, particularly those modified with amine and boronic acid groups (3-AB/CDs and 4-AB/CDs), effectively inhibit HSV-1 entry into host cells by interacting with cellular receptors, preventing viral attachment and penetration. These nanostructures, synthesised via hydrothermal carbonisation, show high antiviral activity at low concentrations (80–469 μg/mL) without significant cytotoxicity, offering a promising strategy to block the initial stages of HSV-1 infection. The inhibition mechanism is independent of boronic acid interactions with glycans, likely involving amine and carboxyl groups binding to cellular receptors like nectin-1[128].

5.2.2. Restriction of viral replication to inhibit viral infection

After the virus invades the host cell, the only way to restrict viral infection is to block budding or stop reproduction. Modification of enzymes required for viral genome synthesis can restrict viral replication. The decline in the percentage of negative-stranded RNA in viruses treated with curcumin CDs compared to that in the untreated virus at different time points after infection indicated that curcumin CDs can significantly restrict RNA synthesis by the porcine epidemic diarrhoea virus. When this virus was replicated in Vero cells, the CD-treated group showed reduced viral titres and plaque counts compared to the control group [125]. One research group demonstrated the antiproliferative activity of glycyrrhizic acid-derived CDs in Verda Reno (Vero) and Porcine Kidney-15 (PK-15) cell lines (Fig. 9). Their results demonstrated antiviral effects against both RNA and DNA viruses [130].

Fig. 9.

The antiviral effects of glycyrrhizic acid-derived CDs (Gly‐CDs). A, c) Vero and PK-15 cells were independently treated with Gly-CDs at 12 and 24 h at different doses. B, d) At 12 and 24 h post-infection, immunofluorescence pictures of PRV-infected PK-15 cells or PEDV-infected Vero cells, respectively, with and without 0.30 mg mL⁻¹ Gly-CDs. The green fluorescence signal (Alexa Fluor 488-conjugated donkey anti-mouse IgG and mouse monoclonal antibody) indicates the PEDV N protein, whereas the blue fluorescence signal (DAPI) shows the nuclei. Scale bar: 100 μm [130]. Copyright 2020. Reproduced with permission from Wiley. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

5.2.3. Obstructing budding and detachment steps to prevent viral infection

The offspring of the virus are formed from the host organism as a new virus upon replication. Approaches that inhibit the budding and propagation of newly originated and extremely virulent viruses can also slow down or prevent infection. ROS are overexpressed in some viral infections and promote DNA damage through signalling pathways that regulate apoptosis. CDs derived from curcumin can prevent the production of ROS caused by coronavirus infections [125]. Although these are the primary antiviral mechanisms of CDs against viral infections, other studies have demonstrated their antiviral efficacy without specifically describing the antiviral mechanism. A thorough investigation is required to examine all the potential operational strategies for CDs to suppress, destroy, or neutralise viruses.

6. Challenges and limitations of CDs in clinical applications

CDs have emerged as promising nanomaterials for clinical applications due to their distinctive properties, including adjustable fluorescence, significant biocompatibility, and multifaceted functionalization. Nonetheless, the application of these materials in clinical settings encounters various challenges and limitations that require thorough investigation to fully exploit their potential. These challenges encompass concerns related to synthesis, stability, safety, and efficacy within biological contexts. The subsequent sections elucidate these challenges and limitations.

The fabrication of CDs frequently entails intricate methodologies that can compromise their reproducibility and scalability. Achieving uniform quality and consistent properties during large-scale production continues to pose a considerable obstacle [139,140]. Although CDs may be synthesised through environmentally friendly techniques, optimising the cost and ecological ramifications of large-scale production is imperative to render them feasible for widespread clinical implementation [141]. It is essential to guarantee the prolonged stability of CDs within biological environments to facilitate their application in clinical settings. Alterations in their properties over time may significantly influence their performance in therapeutic and diagnostic roles [139]. While the functionalization of CDs is readily achievable, ensuring the stability of these functional groups in vivo presents considerable challenges. This instability can adversely affect their targeting capabilities and overall efficacy in drug delivery systems [142].

Although CDs are predominantly regarded as biocompatible, the long-term toxicity and potential repercussions on the immune system remain inadequately understood. Comprehensive toxicological assessments are essential to ascertain their safety across clinical applications [142,143]. The interaction of CDs with the immune system constitutes a pivotal factor that may influence their safety and efficacy. Gaining insight into and mitigating any negative immune responses is crucial for their successful clinical utilisation [142]. High target specificity is vital for effectively deploying CDs in drug delivery and imaging modalities. CDs must be engineered to selectively target pathological tissues while minimising off-target interactions [144]. CDs must exhibit sufficient bioavailability and the capacity to traverse biological barriers, such as the blood-brain barrier, to achieve effectiveness in clinical applications. Augmenting these properties continues to represent a significant challenge [139,144]. While CDs present substantial promise for clinical applications, it is imperative to address these challenges to ensure their successful incorporation into healthcare practices. Ongoing research endeavours aim to enhance CDs' synthesis, stability, and safety, alongside augmenting their targeting capabilities and efficacy. As these challenges are systematically addressed, CDs are anticipated to be more prominent in personalised medicine and advanced therapeutic strategies.

7. Conclusions and future perspectives

This review comprehensively explores the potential of CDs as a transformative nanoplatform for combating viral infections, highlighting their unique physicochemical and biological properties that make them promising candidates for antiviral therapy. We have detailed the mechanisms by which CDs inhibit viral infections, including interference with viral attachment, penetration, replication, and budding, as demonstrated in studies targeting viruses such as SARS-CoV-2, HIV, and PEDV. CDs derived from natural precursors, such as curcumin, glycyrrhizic acid, and Allium sativum, exhibit potent antiviral, anti-inflammatory, and immunomodulatory effects, often with low cytotoxicity and high biocompatibility. These properties, combined with their ability to generate ROS for photodynamic therapy and their tunable fluorescence for diagnostic imaging, position CDs as versatile theranostic agents. Furthermore, we have discussed the synergistic potential of CDs when combined with conventional antiviral drugs, which can reduce drug resistance and systemic toxicity, as evidenced by studies showing enhanced efficacy against human coronavirus HCoV-229E and PRRSV. Despite these advancements, challenges such as scalability, long-term stability, and off-target effects in complex biological microenvironments remain significant hurdles. The variability in CD synthesis methods and the lack of standardised toxicity profiling protocols complicate their clinical translation. However, these limitations underscore the need for continued research to optimise CD design and application.

Future research should prioritise several key directions to harness the potential of CDs in antiviral therapy fully. First, developing scalable, reproducible, and eco-friendly synthesis methods using natural precursors with inherent antiviral properties is critical to ensure cost-effective production and consistent quality. Advanced functionalization strategies, such as targeted surface modifications with boronic acid or thiol groups, should be explored to enhance viral specificity and minimise off-target interactions, as seen in studies achieving up to 60.4 % inhibition of SARS-CoV-2 with thiol-functionalized CDs. Second, in-depth in vivo studies are essential to elucidate CD biodistribution, pharmacokinetics, and long-term safety profiles, addressing concerns like the 50 % lethality observed in mice with oxygen/nitrogen-rich CDs. Computational modelling and machine learning could aid in predicting optimal CD formulations and synergistic drug combinations, reducing systemic toxicity while maintaining efficacy.

Beyond therapeutics, CDs hold immense promise in diagnostic and preventive applications. Their fluorescence properties enable high-sensitivity bioimaging and real-time viral detection, while their integration into nano-coatings for PPE, such as N95 respirators and antiviral sprays, could prevent viral transmission. CD-based vaccine adjuvants, as demonstrated in studies enhancing immune responses in mice and hens, offer a novel approach to improving vaccine efficacy against emerging viral threats like the XEC strain of SARS-CoV-2 and Langya Henipavirus. Additionally, combining CDs with gene-editing technologies, such as CRISPR, could target viral genomes with unprecedented precision, opening new avenues for combating persistent infections like HBV and HIV.

In conclusion, CDs represent a multifaceted nanoplatform with the potential to revolutionise antiviral therapy, diagnostics, and prevention. By addressing current challenges through innovative synthesis, targeted functionalization, and rigorous in vivo validation, CDs could lead to the development of broad-spectrum, resistance-resistant antiviral strategies. Their integration into personalised medicine, coupled with advancements in nanotechnology and interdisciplinary collaboration, will pave the way for effective management of both existing and emerging viral diseases, significantly reducing their global health burden.

CRediT authorship contribution statement

Gangaraju Gedda: Writing – original draft, Writing – review & editing, Methodology, Funding acquisition, Conceptualization. Chandra Lekha Putta: Investigation, Data curation, Writing – original draft. Anamika Verma: Data curation, Writing – original draft. Sasvat Sayee Ram Ramesh: Validation, Writing – original draft. Wubshet Mekonnen Girma: Validation, Data curation, Writing – original draft, Writing – review & editing. Yoo-Jin Park: Formal analysis, Validation, Data curation. Aravind Kumar Rengan: Writing – review & editing, Supervision. Myung-Geol Pang: Supervision, Resources, Funding acquisition.

Declaration of competing interest

The authors declare that there is no conflict of interest regarding the publication of this article.

Data availability

No data was used for the research described in the article.