Photodynamic Action of Curcumin and Methylene Blue against Bacteria and SARS-CoV-2—A Review

Coronavirus disease 19 (COVID-19) has occurred for more than four years, and the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing COVID-19 is a strain of coronavirus, which presents high rates of morbidity around the world. Up to the present date, there are no therapeutics that can avert this form of illness, and photodynamic therapy (PDT) may be an alternative approach against SARS-CoV-2. Curcumin and methylene blue have been approved and used in clinical practices as a photosensitizer in PDT for a long time with their anti-viral properties and for disinfection through photo-inactivated SARS-CoV-2. Previously, curcumin and methylene blue with antibacterial properties have been used against Gram-positive bacteria, Staphylococcus aureus (S. aureus), and Gram-negative bacteria, Escherichia coli (E. coli), Enterococcus faecalis (E. faecalis), and Pseudomonas aeruginosa (P. aeruginosa). Methods: To conduct a literature review, nine electronic databases were researched, such as WanFang Data, PubMed, Science Direct, Scopus, Web of Science, Springer Link, SciFinder, and China National Knowledge Infrastructure (CNKI), without any regard to language constraints. In vitro and in vivo studies were included that evaluated the effect of PDT mediated via curcumin or methylene blue to combat bacteria and SARS-CoV-2. All eligible studies were analyzed and summarized in this review. Results: Curcumin and methylene blue inhibited the replication of SARS-CoV-2. The reactive oxygen species (ROS) are generated during the treatment of PDT with curcumin and methylene blue to prevent the attachment of SARS-CoV-2 on the ACE2 receptor and damage to the nucleic acids either DNA or RNA. It also modulates pro-inflammatory cytokines and attenuates the clotting effects of the host response. Conclusion: The photodynamic action of curcumin and methylene blue provides a possible approach against bacteria and SARS-CoV-2 infection because they act as non-toxic photosensitizers in PDT with an antibacterial effect, anti-viral properties, and disinfection functions.


Introduction
Coronavirus disease 19 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which belongs to the subgenus sarbecovirus (previously lineage B) of genus betacoronavirus and occupies a unique phylogenetic position that is the most lethal [1].This virus is capable of human-to-human transmission through droplets or direct contact, causing outbreaks of pneumonia and spreading around the world quickly [2,3].There are no therapeutics that can avert this form of illness nowadays.Western medical approaches such as booster shots and variant-specific vaccines are used for the prevention and treatment of SARS-CoV-2 [4].However, Western medicines such as remdesivir [5] and dexamethasone [6] may cause adverse drug reactions in some patients.
Meanwhile, photodynamic therapy has been developed for COVID-19 as an antimicrobial treatment that is safe and cost-effective.Antimicrobial photodynamic therapy (aPDT) is a process utilizing a light-activating photosensitizer (PS) that is usually applied in an oxygen-rich environment [7].The energy of photons is absorbed by the photosensitizer and subsequently transferred to surrounding molecules for generating reactive oxygen species, causing damage to the macromolecules in microbes such as proteins, lipids, and nucleic acids, resulting in structural change and death [8].
Porphyrin is a common PS used to mitigate COVID-19 by preventing infections [9].Protoporphyrin (PpIX) was also used as a sensor to monitor the presence of SARS-CoV-2 in the tissue, blood, urine, or feces can map the evolution and severity of the disease or monitor the response of COVID-19 to treatment modalities.This fluorescence spectroscopy has been applied as a diagnostic tool for COVID-19 with a low cost and high sensitivity.PDT with PpIX was effective in combating COVID-19 in the acute phase [10].A series of water-soluble phosphorus(V) porphyrin molecules with OH or ethoxy axial ligands and phenyl/pyridyl peripheral substituents were used as antimicrobial agents in two Gram-negative bacteria models, such as Escherichia coli (E.coli) and Acinetobacter baumannii (A.baumannii).These porphyrins with an extremely low light dose of 5 J/cm 2 in MIC 50 values fight against COVID-19, based on the molecular interaction between phosphorus(V) porphyrin and bacterial lipid membranes [11].The photodynamic therapy with Tetrahydroporphyrin-tetratosylate photodynamic therapy (THPTS) was also used to prevent the infection of COVID-19 with a virus load-reducing effect only at a higher concentration of 3 µM upon near-infrared light irradiation (760 nm, 3.9 or 7.8 J/cm 2 ), which reduced the viral load in vivo, deactivated SARS-CoV-2 [12].Thus, tetra-p-sulphonatedphenyl porphyrin (TSPP) was able to form cationic and J-aggregates at different pH values from 1 to 4, and concentrations around 10 −5 M were suitable for the application of PDT to SARS-CoV-2 viruses [13].
Besides porphyrins, curcumin and methylene blue have also been widely used in the application of PDT against different types of microbes such as viruses, bacteria, protozoa, and fungi.Curcumin and methylene blue normally have limited adverse effect profiles and less damage to the host tissue with no resistance following multiple sessions of therapy [7].This article aimed to review and discuss (i) the background of curcumin and methylene blue, (ii) the principles of PDT, (iii) photodynamic action on diverse organisms in (a) bacteria and (b) SARS-CoV-2 from COVID-19, and its clinical studies, as well as the current approach to nanotechnology in curcumin and methylene blue for the application of PDT.

Principles of PDT
PDT is a minimally invasive therapeutic modality used for the management of a variety of cancers and benign diseases.It is achieved with the use of visible or near-infrared irradiation to activate a light-absorbing compound (photosensitizer, PS) for producing singlet oxygen and other reactive oxygen species (ROS) that destroy the unwanted cells in the presence of molecular oxygen [14].There are two types of photochemical action involved in the PDT as follows: Type I: PS is excited in a triplet state reacting with biomolecules, which transfer the hydrogen atoms to produce the free radicals and radical ions for the generation of ROS in the presence of oxygen [15][16][17][18].
Type II: PS is excited in a triplet state reacting with oxygen in its triplet ground state to produce singlet oxygen ( 1 O 2 ) that is highly reactive and cytotoxic [15].
Type I and Type II reactions spontaneously occur at the same time, and the equilibrium between these two processes depends on the nature of the PS, the concentration of oxygen and substrate, and the affinity of the PS with the substrate.
Basically, photochemical action from curcumin and methylene blue as a PS consists of both Type I and Type II reactions.Curcumin has shown great potential as a PS in PDT because of its ability to absorb blue light (425-500 nm) for producing ROS to target cells or tissues [19].Methylene blue can generate a high quantum yield of 1 O 2 via PDT to an infected area during excitation of the red light (630-680 nm) [20].
Curcumin has a broad absorption peak from 300 to 500 nm [29], and the maximum absorption peak can be divided into 408 to 430 nm in different solvent systems, such as tetrachloromethane and dimethyl sulfoxide.It is usually activated by light with a wavelength of around 425 nm to induce a strong phototoxicity at micromolar concentrations [30].Currently, curcumin has extended its usage to antimicrobial activity for human clinical trials in a variety of conditions [31], acting as an antibacterial agent and exploiting about 100 different strains of both Gram-positive and Gram-negative pathogenic bacteria [32].It has been used as a PS in the treatment of mouth pathogen microbes because of its photokilling and photo-biological abilities [29,33,34].
Curcumin is a suitable candidate as a PS in the application of PDT since it is a chemically pure substance with a specific uptake by the target tissue, minimal dark toxicity that is activated only upon irradiation, high photo-activity rate (quantum yield) to the production of ROS, and rapid clearance to avoid phototoxic side effects.It induces no toxic impact on healthy tissue and it should be able to be manipulated within short light intervals to ease treatment for outpatients [35].

Methylene Blue
Methylene blue (Figure 2) is a heterocyclic aromatic compound belonging to the phenothiazine type with the chemical structure of 7-(Dimethyl-amino)phenothiazin-3-ylidene]-dimethyla-zanium chloride (C16H18ClN3S).It consists of two benzene rings attached to one nitrogen atom and one sulfur atom and was first synthesized as a textile dyestuff by Caro in 1876 [36].Methylene blue has been used to stain the nervous tissue [37] as an analgesic [38] and antimalarial component [39].Nowadays, methylene blue is a wellknown dye in medicine and has been applied for topical treatment during photodynamic therapy (PDT) [40].
Since methylene blue has very low tissue toxicity, it has been used in medical practice for more than a hundred years, including the treatment of ifosfamide encephalopathy, methemoglobinemia, urolithiasis, and cyanide poisoning [41].It is non-toxic and can be administered in high doses to humans orally or intravenously [42].Methylene blue is possible to achieve optimal light penetration into tissue because of the strong light absorption at 620 nm wavelengths; thus, this is an excellent choice for superficial treatments of the skin or oral cavity [35].Thus, methylene blue is safe and effective for use in PDT because Curcumin has a broad absorption peak from 300 to 500 nm [29], and the maximum absorption peak can be divided into 408 to 430 nm in different solvent systems, such as tetrachloromethane and dimethyl sulfoxide.It is usually activated by light with a wavelength of around 425 nm to induce a strong phototoxicity at micromolar concentrations [30].Currently, curcumin has extended its usage to antimicrobial activity for human clinical trials in a variety of conditions [31], acting as an antibacterial agent and exploiting about 100 different strains of both Gram-positive and Gram-negative pathogenic bacteria [32].It has been used as a PS in the treatment of mouth pathogen microbes because of its photokilling and photo-biological abilities [29,33,34].
Curcumin is a suitable candidate as a PS in the application of PDT since it is a chemically pure substance with a specific uptake by the target tissue, minimal dark toxicity that is activated only upon irradiation, high photo-activity rate (quantum yield) to the production of ROS, and rapid clearance to avoid phototoxic side effects.It induces no toxic impact on healthy tissue and it should be able to be manipulated within short light intervals to ease treatment for outpatients [35].

Methylene Blue
Methylene blue (Figure 2) is a heterocyclic aromatic compound belonging to the phenothiazine type with the chemical structure of 7-(Dimethyl-amino)phenothiazin-3-ylidene]dimethyla-zanium chloride (C 16 H 18 ClN 3 S).It consists of two benzene rings attached to one nitrogen atom and one sulfur atom and was first synthesized as a textile dyestuff by Caro in 1876 [36].Methylene blue has been used to stain the nervous tissue [37] as an analgesic [38] and antimalarial component [39].Nowadays, methylene blue is a well-known dye in medicine and has been applied for topical treatment during photodynamic therapy (PDT) [40].
Since methylene blue has very low tissue toxicity, it has been used in medical practice for more than a hundred years, including the treatment of ifosfamide encephalopathy, methemoglobinemia, urolithiasis, and cyanide poisoning [41].It is non-toxic and can be administered in high doses to humans orally or intravenously [42].Methylene blue is possible to achieve optimal light penetration into tissue because of the strong light absorption at 620 nm wavelengths; thus, this is an excellent choice for superficial treatments of the skin or oral cavity [35].Thus, methylene blue is safe and effective for use in PDT because of its ability to generate singlet oxygen with high light absorption at 665 nm against several diseases [43].
Pharmaceuticals 2024, 17, x FOR PEER REVIEW 4 of 16 of its ability to generate singlet oxygen with high light absorption at 665 nm against several diseases [43].

Comparing the Photodynamic Action of Methylene Blue and Curcumin
Methylene blue is an FDA-approved PS [44]; curcumin is not approved as a PS and is only available in the United States as a dietary supplement [45].However, curcumin has been used as a photosensitizer in PDT for a long time, especially in clinical studies.
Methylene blue is a hydrophilic compound with a low molecular weight and a positive charge, which easily passes through Gram-positive and Gram-negative bacterial membranes.When being irradiated by a light source of approximately 630 nm, this compound promotes irreversible oxidation of the target cells [46].Additionally, curcumin is a hydrophobic compound.It has a high molecular weight and a neutral charge and can be irradiated by a light source of approximately 425 nm [34,47].Compared to methylene blue, it is insoluble in water with a shorter wavelength in the application of PDT.However, curcumin is a natural product without side effects.Methylene blue is a cationic dye that may cause some health problems such as asthma and eye, or skin irritations [48,49].

Photodynamic Action against Bacteria
Generally, an applied therapeutic photodynamic method involves the activation of curcumin and methylene blue with blue or red lights at specific wavelengths (425-500 nm and 630-680 nm, respectively).
Growing evidence has shown that curcumin possesses antibacterial properties in aPDT treatment against Gram-positive bacteria Staphylococcus aureus (S. aureus), and Gram-negative bacteria Escherichia coli (E.coli), Enterococcus faecalis (E.faecalis), and Pseudomonas aeruginosa (P.aeruginosa).The possible mechanism of aPDT for curcumin involves producing different types of ROS, such as hydrogen peroxide, hydroxide radical, superoxide, and singlet oxygen [50].Cytotoxic ROS react with DNA, proteins, lipids, and other components to induce cell death in organisms [51].The singlet oxygen produced has a very small radius of action, which is less than 0.02 µm, and damage only occurs in the presence of the photosensitizer and under photoactivation.Cell death of an organism is caused by the cell wall or membrane lysis and/or the inactivation of proteins or enzymes essential for microbial metabolism [52].The photodynamic action of methylene blue against bacteria is similar to that of curcumin.Actually, curcumin and methylene blue are very safe, and their photodynamic actions have a broad spectrum of antibacterial effects and no bacterial resistance [53,54], the PDT with curcumin and methylene blue has been used in antibacterial treatment (Tables 1 and 2).

Comparing the Photodynamic Action of Methylene Blue and Curcumin
Methylene blue is an FDA-approved PS [44]; curcumin is not approved as a PS and is only available in the United States as a dietary supplement [45].However, curcumin has been used as a photosensitizer in PDT for a long time, especially in clinical studies.
Methylene blue is a hydrophilic compound with a low molecular weight and a positive charge, which easily passes through Gram-positive and Gram-negative bacterial membranes.When being irradiated by a light source of approximately 630 nm, this compound promotes irreversible oxidation of the target cells [46].Additionally, curcumin is a hydrophobic compound.It has a high molecular weight and a neutral charge and can be irradiated by a light source of approximately 425 nm [34,47].Compared to methylene blue, it is insoluble in water with a shorter wavelength in the application of PDT.However, curcumin is a natural product without side effects.Methylene blue is a cationic dye that may cause some health problems such as asthma and eye, or skin irritations [48,49].

Photodynamic Action against Bacteria
Generally, an applied therapeutic photodynamic method involves the activation of curcumin and methylene blue with blue or red lights at specific wavelengths (425-500 nm and 630-680 nm, respectively).
Growing evidence has shown that curcumin possesses antibacterial properties in aPDT treatment against Gram-positive bacteria Staphylococcus aureus (S. aureus), and Gramnegative bacteria Escherichia coli (E.coli), Enterococcus faecalis (E.faecalis), and Pseudomonas aeruginosa (P.aeruginosa).The possible mechanism of aPDT for curcumin involves producing different types of ROS, such as hydrogen peroxide, hydroxide radical, superoxide, and singlet oxygen [50].Cytotoxic ROS react with DNA, proteins, lipids, and other components to induce cell death in organisms [51].The singlet oxygen produced has a very small radius of action, which is less than 0.02 µm, and damage only occurs in the presence of the photosensitizer and under photoactivation.Cell death of an organism is caused by the cell wall or membrane lysis and/or the inactivation of proteins or enzymes essential for microbial metabolism [52].The photodynamic action of methylene blue against bacteria is similar to that of curcumin.Actually, curcumin and methylene blue are very safe, and their photodynamic actions have a broad spectrum of antibacterial effects and no bacterial resistance [53,54], the PDT with curcumin and methylene blue has been used in antibacterial treatment (Tables 1 and 2).PDT may be effective against MRSA infection in a murine model of intradermal infection.
PDT treatments using methylene blue and malachite green have an antibacterial effect against E. faecalis, showing potential to be used as an adjunctive antimicrobial procedure in endodontic therapy. [78] 10 Comparing the efficacy of toluidine blue, methylene blue and curcumin in photodynamic therapy against Enterococcus faecalis Methylene blue is injected into the root canals and remained there for 120 s before irradiation, which was carried out for 60 s with an LED lamp emitting light in the red spectrum with a power peak at 630 nm and output intensity of 2000-4000 mW/cm 2 .
Colony-forming units, and scanning electron microscopy.
The adjunction of methylene blue-mediated PDT via a light-emitting diode to NaOCl irrigation has antibacterial efficacy against E. faecalis. [79]

Photodynamic Action against SARS-CoV-2
SARS-CoV-2 is a single-stranded RNA-enveloped virus that is 29,881 bp in length and encodes 9860 amino acids [80].It breaks the plasma membrane, enters human cells, and exposes its genetic material, causing severe damage to human RNA and DNA [81].The photodynamic action of porphyrin generates singlet oxygen ( 1 O 2 ), which has a diffusion distance of approximately 0.01 to 0.02 µm before quenching.Since PSs need to be closely associated with the target substrate for maximum impact, the affinity of a virus for the heme structure indicates high viral destruction.These titers are restricted to the zone of photoactivation [82].
Curcumin and methylene blue are other common PSs in the photodynamic treatment of SARS-CoV-2 (Tables 3 and 4) that attempt to inactivate the virus.They have the ability to enter the host cell and bind to the DNA, RNA, as well as protein of the virus.When binding to DNA, RNA, or protein of the virus, cross-linking mechanics could be altered to cause damage to [83] the glycoprotein, resulting in the inhibition of viral replication [81].Since SARS-CoV-2 requires an enveloped homotrimeric spike glycoprotein to interact with the cellular receptor ACE-2 [84] and the selective impairment of surface proteins, like spike glycoprotein, PDT can effectively inhibit the infectivity of SARS-CoV-2 [85].

Mechanism of Photodynamic Action against SARS-CoV-2
Methylene blue has photodynamic effects used in different illnesses, including cirrhosis of the liver, hypoxemia, and severe hepatopulmonary syndrome [92].It reduces the oxidized ferric form of hemoglobin (Fe 3+ ), which permanently binds oxygen to the ferrous (Fe 2+ ) form.This improves hemoglobin's ability to bind oxygen and, as a result, oxygen transport to tissues, which is beneficial for COVID-19 patients [93].Silent hypoxemia is the term for the low oxygen levels that COVID-19 patients frequently exhibit.These levels are usually incompatible with a life without dyspnea [94].
Curcumin can improve COVID-19 patients' prognoses by drastically reducing the length of common symptoms, hospital stays, and mortality [95].It inhibits the ACE2 receptor and the SARS-CoV-2 S protein, as well as SARS-CoV-2 proteases such as papainlike protease and main protease [96,97].Additionally, curcumin can dramatically lower inflammatory cytokine levels and stimulate anti-inflammatory pathways, which raises anti-inflammatory cytokine levels and partially restores the pro-and anti-inflammatory chemical balance in COVID-19 patients.This may help to control the cytokine storm associated with COVID-19 [96].

Clinical Study for COVID-19
Curcumin is widely found in food and it is a safe drug with broad prospects for clinical application [98].The use of nanocurcumin is a successful practice, and effectively increases O 2 saturation and reduces the severity of symptoms in COVID-19 patients [99].The minor side effects of an intake of curcumin formulations include colds, irritation, indigestibility, and nausea, which in a few cases might be attributed to adjuvants and emulsifiers [100].However, most of these studies are single-center studies with small populations and different oral doses of nanocurcumin.It is better to have more long-term, multi-center, and large-sample studies [96].
Methylene blue can also be used for COVID-19 treatment [101].However, administration of methylene blue with meticulous consideration of the dosage is necessary to prevent any untoward effects, since methylene blue is a monoamine oxidase (MAO) inhibitor and can interact with antidepressants to cause severe toxicity of serotonin [102].It has also been found to interact with dapsone to form hydroxylamine, which oxidizes hemoglobin and may cause hemolysis [103].In patients with G6PD deficiency, this can be detrimental and may cause severe hemolysis [104].Thus, this needs to be studied further to find the optimal dosage for clinical study.

Discussion
PDT, with high cure rates, minimal toxicity in healthy tissues, specific targeting and selectivity, and a low amount of side effects, shows potential for use as an adjuvant therapy when combined well with all other tumor interventions such as chemotherapy, surgery, radiotherapy, immunotherapy, or treatment of cancer [105].However, curcumin as a PS used in the clinical application of PDT has some limitations, such as poor solubility, stability, and photostability in aqueous solutions, as well as rapid metabolism and systemic elimination [106].Methylene blue is more soluble in water but its clinical use has been limited by the rapid enzymatic reduction in the biological environment [107].
These problems of curcumin and methylene blue could be overcome with the help of nanotechnology, which produces nanoscale curcumin or methylene blue; its physical specifications, chemical properties, and biological characteristics enable it to have great bioavailability, solubility, transport, and effectiveness [108].Nanotechnology improves the characteristics of curcumin and endows it with the ability to target an infected area, as well as inhibit viral replications that prevent the TMPRSS2 from interacting with disintegrin metallopeptidase domain 17 (ADAM17) to give a high level of ACE2 expression, causing lung inflammation and pulmonary oedema [109].
The nanocurcumin could inhibit natural killer cells and T helper 17 cells, as well as reduce the release of inflammatory factors such as IL-1β, IL-6, and TNF-α.Since nanocurcumin may effectively increase O 2 saturation and reduce the severity of symptoms in COVID-19 patients, the formulations might be used as a supplement to accelerate the recovery of patients, improve the symptoms, and shorten the recovery period of COVID-19 patients [99].
Additionally, methylene blue-embedded nano or microparticles could effectively deliver photo-responsive molecules to tissues and cells for translocating them into cellular compartments, thereby producing significant amounts of ROS in the target tissues.Methylene blue would maximize the therapeutic efficacy of PDT while reducing immunogenicity and side effects [110].
The solubility of curcumin and methylene blue could also be improved via the formation of nanogels, ranging from 10 to 100 nm [111].These nanogels are composed of hydrogel and synthesized through either the physical or chemical cross-linking of polymers.The cross-linked structure provides a sustainable storage and release rate [112].They are developed as carriers for curcumin and methylene blue delivery because the nanogels could absorb biologically active molecules spontaneously via the formation of salt bonds, hydrogen bonds, or hydrophobic interactions to enhance their bioavailability, such as by enhancing their solubility and stability.Thus, PDT with nano-scale curcumin or methylene blue as a potential therapy against SARS-CoV-2 is still being investigated; but much more research work needs to be carried out, especially concerning safety assessments for human clinical trials.

Conclusions
The above information demonstrates that curcumin and methylene blue are two promising photosensitizers for PDT with an antibacterial effect, anti-viral properties, and disinfection function.It presents a possible against bacteria and SARS-CoV-2 infection.The photodynamic action of curcumin and methylene blue can be enhanced with the help of nanotechnology in future developments.

Table 1 .
The photodynamic action of curcumin against bacteria.
StudyUsage of Light and Energy (J) Experiment Parameters Conclusion References 1 Antimicrobial photodynamic therapy with curcumin on methicillin-The strains are induced to form biofilm and incubated with curcumin for 20 min, irradiated with a Colony-forming units, scanning electron microscopy, confocal PDT reduces the growth of the MRSA strain biofilm, making it a relevant [55]

Table 1 .
The photodynamic action of curcumin against bacteria.

Table 2 .
The photodynamic action of methylene blue against bacteria.

Table 4 .
The photodynamic action of methylene blue against SARS-CoV-2.