Biomaterials

### **Chapter 3**

## Silk Fibroin Nanoparticles: A Biocompatible Multi-Functional Polymer for Drug Delivery

*Faith H.N. Howard, Zijian Gao, Hawari Bin Mansor, Zidi Yang and Munitta Muthana*

### **Abstract**

The versatility of nanomedicines allows for various modifications of material type, size, charge and functionalization, offering a promising platform for biomedical applications including tumor targeting. One such material, silk fibroin (SF) has emerged, displaying an excellent combination of mechanical and biological properties characterized by its high tensile and breaking strength, elongation, stiffness and ductility. High stability allows SF to maintain its chemical structure even at high temperatures (around 250°C) and compared with other biological polymers like polylactide (PLA), poly(lactic-co-glycolic acid) (PLGA), and collagen, SF shows excellent biocompatibility and lower immunogenic response making it a very suitable material for drug delivery and tissue engineering. Here we describe the structure, synthesis and properties of SF nanoparticles**.** We evaluate its emergence as a multi-functional polymer for its utility as a nanocarrier to deliver cancer therapies directly to tumors together with considerations for its clinical use.

**Keywords:** silk fibroin, polymer, nanomedicine, nanocarrier, drug delivery

### **1. Introduction**

As stated by the World Health Organization, cancer is the second leading cause of death globally, accounting for an estimated 9.6 million deaths, or 1 in 6 deaths, in 2018 [1]. GLOBOCAN also predicts an increase in cancer rates, with more than 20 million new cancer cases expected annually by 2025 [2]. Over time, as our understanding and knowledge of molecular and tumor biology has increased, the cancer treatment paradigms have notably changed, particularly during the past 20 years. Previously, cancer was listed and treated based on its origin or its unique histomorphologic characteristics. However, in 2002, Schiller et al. reported that third generation chemotherapy administered to non-small-cell lung cancer showed almost similar survival curves [3]. Although the results are only limited to lung cancer, this indicates that cancer treatment using general (non-specific) cytotoxic chemotherapies have reached a therapeutic plateau. Since then, this research area has evolved based on two focus

areas: tumor molecular profiling and molecular targets. Together, these efforts have further realized two recent revolutions in cancer research. Firstly, genotype-directed precision oncology which focuses on personalized therapies to treat specific genomic abnormalities regardless of the cancer type. Secondly, the targeting of particular components in the tumor microenvironment. As a result, the discovery of an abundance of anticancer drugs have showed a promising early step for cancer eradication. However, most of these therapeutics agents have undesirable characteristics, limiting their clinical usage and invalidating further drug research [4].

Recent studies have suggested that the use of nanoparticles as a drug carrier may be one of the best alternatives to improve the therapeutic effect of anticancer drugs. Nanotechnology, or in this case, nanomedicine, is the use of materials usually in nanometer scale in the fields of medicine and health [5]. Nanoparticle-based drug delivery systems have shown remarkable progress in overcoming the limitations of conventional drug delivery or drug therapy method. The unique characteristics that usually accompany these potential carriers include nanoscale size, high surface-tovolume ratio, auspicious physical and chemical properties and most importantly, endless possibilities for modifications that support cell targeting, gene delivery etc. [5].

Nanoparticles fall into two different categories, namely soft/organic and hard/ inorganic nanoparticles. Soft nanoparticles are based on organic material, typically prepared from polymers or molecules that can self-assemble (coacervation) into large particles. The materials can range from full synthetic polymer to natural materials such as silk proteins [6]. Hard nanoparticles, on the other hand, are inorganic and usually insoluble, e.g. silver, gold nanoparticles, and carbon nanotubes [7, 8]. The benefits of organic nanoparticles as drug nanocarriers have been reported numerously in recent years, citing desirable characteristics including, biodegradability, nonantigenic and superior biocompatibility [9]. Thus, the delivery system's advancement holds the promise of future precision medicine, which would greatly improve cancer survival rate by treating each cancer patient with the most effective drugs in the most efficient ways [10].

One such natural polymer which promises great potential as a drug delivery system is silk fibroin (SF). Silk has been recognized as a valuable natural material for the fabric industry for centuries, but during the last few decades, it has attracted immense attention as a promising biopolymer for biomedical and pharmaceutical applications [11]. Silk from the domesticated silkworm *Bombyx mori (B. mori)* is well characterized and has been approved as a safe biomaterial by the US Food and Drug Administration (FDA) [12]. This chapter focuses on the use of *B. mori* derived SF as a functional material for cancer drug delivery.

### **2. Silk fibroin**

### **2.1 Structure**

Most SF utilized for biomedical and commercial applications are derived from cocoons of *B. mori* domestic silk moths. Constructed from a continuous fiber strand comprised of two cores of fibroin protein held together by sericin protein [13, 14]. The primary structure of *B. mori* SF mainly consists of glycine (Gly) (43%), alanine (Ala) (30%) and serine (Ser) (12%) [15]. The secondary structure of SF exists in three different structural forms including silk I, silk II and silk III. Silk I consists of α-helix domains which is in a water-soluble state and easy to convert to silk II

*Silk Fibroin Nanoparticles: A Biocompatible Multi-Functional Polymer for Drug Delivery DOI: http://dx.doi.org/10.5772/intechopen.109642*

**Figure 1.** *Cross-sectional and structural composition of silk fibers. Created with BioRender.*

structure once treated with organic solvents, electromagnetic fields, or physical spinning environments. Unlike silk I, silk II contains an antiparallel β-sheet/crystal molecular model which has hydrogen side chains from glycine and methyl side chains from the alanines resulting in higher stability and both water and solvent insolubility. Silk III prevails at the water/air interface (**Figure 1**) [16–18].

### **2.2 Properties**

In the past centuries, silk has been used as a natural material for the fabric industry, but recently SF nanoparticles have been considered as a potential alternative carrier for anticancer drug delivery because of its physicochemical, mechanical, and biological properties [11]. Compared with other materials, SF is characterized by its high tensile and breaking strength, elongation, stiffness and ductility [19]. High stability allows maintenance of SF chemical structure even at high temperatures (around 250**°**C) [20]. In addition, compared with other biological polymers like polylactide (PLA), poly(lactic-co-glycolic acid) (PLGA), and collagen, silk fibroin shows excellent biocompatibility and lower immunogenic response [21–23] making it a very suitable material for drug delivery and tissue engineering. It is commonly used as a suture material, demonstrating comparable immunogenicity and biocompatibility to other natural and synthetic suture materials [24]. Moreover, it has consistently shown to be non-toxic and fully resorbable, this includes any degradation products [25]. Surprisingly, SF has been proved to have an intrinsic anti-inflammatory ability which could be used in the treatment of inflammatory bowel disease [26].

Degradability is another important property of silk fibroin. The degradation rate of SF is related to the molecular weight, the degree of crystallinity, morphological features and crosslinking [27]. This affords a tunability from seconds to years, another unique feature of SF. As a result of these properties, SF has been used in different nanosystems, including films, sponges, hydrogels, tubes [28]. Here, we focus on silk nanoparticles.

### **3. Silk nanoparticles**

### **3.1 Synthesis of silk nanoparticles**

Silk nanoparticles are an excellent candidate as a carrier for drugs, targeted therapies and contrast agents. They are synthesized from regenerated SF through a variety of methods based on their self-assembly behavior. Among all of the approaches, desolvation (**Figure 2**) is the most common method for SF nanoparticle synthesis, in which dissolving agents including ethanol, acetone, dimethyl sulfoxide (DMSO) and methanol could be used to dehydrate and package the silk chain, leading to the change from silk I to silk II structure and forming SF nanoparticles [29]. During the desolvation method, lipophilic active drugs (e.g. curcumin and 5-fluorouracil) can be easily dissolved in the organic solvents allowing nanoencapsulation of anticancer drugs [30]. The main challenge of the desolvation method is to find the ideal SF/dissolving agent ratio and ensure adequate mixing during SF nanoparticle formation which plays an important role in the control of the nanoparticle size.

### **Figure 2.**

*Representation of the desolvation and salting-out method for the production of silk fibroin nanoparticles. Created with BioRender.*

*Silk Fibroin Nanoparticles: A Biocompatible Multi-Functional Polymer for Drug Delivery DOI: http://dx.doi.org/10.5772/intechopen.109642*

Salting-out (**Figure 2**) is another widely used method in the preparation of protein-based nanoparticles by removing the water barriers between protein molecules and increasing the interactions between proteins, leading to aggregation and precipitation from the solution [31]. The salt, pH, and ionic strength are major factors influencing the yield, particle morphology, zeta potential, and nanoparticle stability [30]. The salting-out method has advantages in avoiding the usage of toxic solvents, therefore maintaining the activity of the protein. However, the synthesized particle size is relatively large (500–2000 nm), and the high amounts of salts are difficult to remove [29].

The electrospraying method uses electrical forces to obtain liquid atomization where the liquid flowing out of a capillary nozzle is dispersed into small droplets by an electric field [32, 33]. Similar to the desolvation method, the main limitation of electrospraying is the organic solvents used during the synthesis that may damage the bioactivity of their cargo (e.g. enzymes, genes, and cell vitality) [33]. Other silk nanoparticle synthesis methods which are not used widely used include supercritical fluid technologies [34], mechanical comminution [35], capillary-microdot technique [36], and microemulsion [37].

### **3.2 Functionalization**

Many drug delivery systems lack a targeting mechanism, resulting in poor accumulation at tumor targets. To achieve efficacious concentrations at the tumor, saturating drug doses are often administered which can lead to non-specific or toxic effects. Nanoparticles have been designed to overcome this problem by delivering the drug to specific tissue instead of a more generalized treatment. Currently, four different targeting mechanism have been explored; passive targeting, targeted recognition, triggered release and guided delivery (**Figure 3**). Historically, the enhanced permeability and retention (EPR) effect (a unique phenomenon of solid tumors) has been utilized whereby nanoparticles can extravasate through the leaky blood vessels of the tumor tissue without the need for surface modification [5, 38, 39]. Some surface modification that affects circulation time may also indirectly affect passive targeting such as PEGylation of the nanoparticles. This is due to EPR effect increases proportional to the circulation time [40]. However, some tissues may also contain fenestrated blood vessels resulting in a similar effect on nanoparticles, causing them to accumulate there [5]. Additionally, the tumor microenvironment varies depending on the tumor type and passive targeting may not be as efficient in that particular condition.

Targeted recognition involves the use of targeting molecules as an attachment to the drug-loaded nanoparticles. Targeting molecules such as ligands have high specificity to receptors and other cancer-specific target molecules available on the surface of cancerous tissue such as glycans [4]. Conjugation of nanoparticles with ligands such as transferrin, folic acid, enzymes, antibodies and other macromolecules has demonstrated enhanced uptake of nanoparticles by cancer cells [5, 39]. However, they still rely on passive accumulation at the tumor site.

Traditional cancer treatment methods suffer from a lack of specific regional and temporal activation leading to off-target effects. The concept of smart nanosystems uses the intrinsic and environmental differences between normal cells and cancer cells to trigger activation or release of drugs at a tumor site [41]. Intrinsic activation strategies include using the differences in pH, enzyme expression level, the concentration of membrane proteins and soluble molecules between healthy cells and cancer cells to trigger drug release at a specific location in the

**Figure 3.** *Schematic of different drug targeting approaches for nanoparticles. Created with BioRender.*

body [5, 41–43]. Extrinsic activation includes using ultrasound, magnetic field, light and photodynamic therapy to provide an activatable system with less toxic, safe, and minimal adverse effects [44–46]. At the moment, the development of nanoparticles activation strategy is not only satisfied with a single treatment but also presume a multimodal trigger system. Therefore, the synthesis and analysis of multifunctional nanomaterials have been extensively researched *in vitro* and *in vivo* for cancer [47, 48].

The above strategies, however, lack a navigational force to the desired target as well as the ability to penetrate tumors beyond diffusion limits. Magnetic tumor targeting, using magnetic carriers and an external magnetic field is demonstrating promise for enhanced tumor accumulation of chemotherapy [49] and virotherapy [50] following their systemic administration. In the presence of a magnetic field, peptide-functionalized magnetic silk nanoparticles demonstrated increased cellular uptake of an anticancer agent (ASC-J9) by HCT116 colorectal cancer spheroids [51]. Additionally, in an orthotopic model of breast cancer, magnetic targeting enriched Doxorubicin-loaded magnetic SF nanoparticles at the tumor site with a concomitant suppression of uptake by the liver, resulting in a significant decrease in tumor volume and survival [52]. The provision of an external driving force expands therapeutic use to a wide variety of tumors, independent of specific receptor expression.

*Silk Fibroin Nanoparticles: A Biocompatible Multi-Functional Polymer for Drug Delivery DOI: http://dx.doi.org/10.5772/intechopen.109642*

### **3.3 Nanocarrier for cancer**

### *3.3.1 Chemotherapy delivery*

Due to excellent stability in the change of temperature [53], humidity [54], and pH [55], silk-based nanocarriers have been widely studied for the delivery of numerous chemotherapeutic substances such as doxorubicin, cisplatin, paclitaxel, 5′-fluorouracil, and floxuridine for cancer treatment (**Table 1**). In addition, silk nanoparticles can also be used in the delivery of natural plant-derived therapeutics including curcumin [65], celastrol and triptolide [70], which are limited by their poor water solubility. To reduce systemic toxicity and adverse side effects of chemotherapeutics, targeted delivery can be achieved by conjugation of various targeting ligands to the silk material (as described above), recognizing overexpression of particular epitopes on the surface of target cells [71]. For example, Lei Huang et al. [63] designed a folate (FA) conjugated silk nanoparticle double loaded with doxorubicin (FA-SFPs-DOX-DOX), which provided a pH-dependent targeted drug release, lasting for over 30 hours. This


*RSF = regenerated silk fibroin, 5-FU = 5-fluorouracil, F = flank, OT = orthotopic, and X = xenograft. All in vivo models performed in mice.*

### **Table 1.**

*Silk nanoparticles as nanocarriers for chemotherapy.*

study demonstrated the importance of the target ligand, with FA-SFPs-DOX-DOX inducing greater cytotoxicity against HeLa cells compared with SFPs-DOX-DOX.

### *3.3.2 Peptide and protein delivery*

Silk-based nanocarriers can also bind with peptides and proteins improving their *in vivo* stability. Lactoferrin is one such protein showing anti-cancer properties, whereby apo-bovine lactoferrin loaded silk nanoparticles induces significantly higher internalization and cytotoxicity towards the MDA-MB-231 and MCF-7 breast cancer cell lines [72]. Peptide-based cancer vaccines are another important therapeutic agent in cancer treatment. However, peptide vaccines suffer from short *in vivo* stability caused by proteolytic degradation and rapid clearance from the bloodstream [73]. A silk nanoparticle delivery system is an effective way to improve the bioavailability and stability of peptide tumor vaccines [74]. Using engineered spider silk nanoparticles a peptidebased vaccination resulted in successful activation of cytotoxic T-cells, without unspecific immune responses [75]. How these antigens are delivered can also influence the developing vaccination response. It is thought that controlled, persistent antigenic signals elicit stronger responses than transient bolus vaccine exposure [76–78], such as that seen with microneedle skin patches. Microneedle vaccines exploit the skin's accessibility, both in terms of ease of administration as well as access to densely populated areas of antigen presenting cells. Silk microneedles therefore represent an attractive prospect due to their tunable release kinetics of encapsulated cargo as well as their overall biodegradability. This system demonstrated a > 10-fold increase in ovalbumin (OVA)-specific T cell and humoral responses in C57/Bl6 mice when compared with parenteral immunization [79], warranting further investigation.

### *3.3.3 Gene delivery*

Viral vectors are traditional carriers for gene delivery, however, their drawbacks in inducing high systemic toxicity and immune responses limit their application in cancer treatment [22]. Thus, non-viral vectors have emerged to address challenges surrounding improving transfection efficiency, target specificity and cytotoxicity [80]. Among various materials, silk-based nanocarriers have been reported to provide biodegradability, biocompatibility, high transfection efficiency, and DNase resistance in gene delivery [9]. Through genetic engineering, the transfection efficiency of silk nanoparticles could be further improved. Numata et al. [81] combined silk protein-based nanocarriers with poly(L-lysine) (PLL) for gene delivery, resulting in improved transfection efficiency of pDNA in human embryonic kidney (HEK) cells. Additionally, to further enhance target specificity of the silk-based gene delivery system, they included tumor homing peptides (THP) [80]; F3 peptide (specifically targeted towards nucleolin expressing tumor and endothelial cells) and Lyp1 peptide (shows target specificity towards the p32 receptor overexpressed in tumor cells) [82, 83]. The use of cationic polymers with silk-based nanocarriers is another popular strategy due to their high cellular uptake efficiency, good water solubility, excellent transferability and easy synthesis [84]. Polyethyleneimine (PEI) is one of the commonly used cationic polymers which easily assembles with gene therapies and demonstrates improved cellular uptake due to their positive charge [85]. Song et al. [65] designed magnetic-silk/PEI core-shell nanoparticles for targeted delivery of c-myc antisense oligodeoxynucleotides (ODNs), which had high uptake efficiencies and significantly inhibit the growth of MDA-MB-231 cells.

*Silk Fibroin Nanoparticles: A Biocompatible Multi-Functional Polymer for Drug Delivery DOI: http://dx.doi.org/10.5772/intechopen.109642*

### *3.3.4 Diagnostics and theranostics*

In addition to therapeutic delivery, silk nanoparticles are also promising as non-invasive imaging components and provide an opportunity to augment existing imaging modalities for diagnostic purposes. These modalities are often limited by inadequate contrast between healthy and diseased tissue, contributing to failure to detect signs of illness, particularly early signs. Silk nanoparticles can be used as a vehicle for loading magnetic resonance contrast agents, overcoming agglomeration limitations of magnesium oxide nanospheres [86]. Alternatively the use of fluorescent dyes and carbon dots for modification of the silk fibroin itself has applications for live cell imaging or visualizing degradation of silk-fibroin implants [87]. The production of fluorescent silk nanoparticles can be created using simple dyes, chemical modification of the fibroin, conjugation or entrapment of fluorescent proteins and even doping the silkworm larvae's diet with fluorescent dyes such as rhodamine and fluorescein [88]. Additionally, carbon quantum dots (CQDs) generated from SF are strongly fluorescent, resist photobleaching, can be further functionalized [89–91] and in comparison to other colloidal materials, avoid the need for toxic heavy metals. However, this process does require controlled and pressurized heating of the fibroin for carbonization into CQDs.

By combining these imaging modalities with their role as a drug carrier, silk nanoparticles are becoming an important theranostic device. Theranostics is an approach that combines cancer treatment and diagnosis, in which efficient imaging guidance of therapy is necessary for detecting the drug loading, targeted delivery, and release123. For example, Levodopa (a PTT agent) and manganese dioxide particles (a contrast agent) were formulated with silk sericin from *B. mori* cocoons to create a one-step method for MRI-guided photothermal therapy [92]. The composition of spheres made of spider silk and iron oxide nanoparticles have also demonstrated drug loading and release capacity with potential to be used in both hyperthermia and magnetic resonance imaging (MRI) applications combined with drug delivery against tumor cells [93].

### **4. Conclusion**

Nanoparticles used for drug delivery require desired physicochemical properties including size [94], shape [95], structure [96], rigidity [97], and surface modification [98]. Translation and application of nanoparticles, including silk, to the clinic must first overcome a number of challenges including their heterogeneity, reproducibility and upscale production. Silk derived from different sources will possess different amino acid sequences and morphology, whilst LPS contamination of recombinant silk is a major obstacle for progression to clinic, requiring careful characterization of its toxicity and immunogenicity. Additionally, traditional nanoparticle preparation methods involving breaking down of large particles, nanoprecipitation, or self-assembly of monomers, suffer from wide size distribution and large batchto-batch variability [99]. To obtain more stable and controllable nanoparticles, microfluidics has emerged for manipulating tiny fluids (1 × 10<sup>−</sup><sup>9</sup> L–1× 10<sup>−</sup>18 L) in micro-channels with dimensions of tens of micrometers [100]. Several flow patterns including laminar flow, turbulent flow and droplet flow could be achieved under microfluidic control with potential to enhance fluid mixing, reduce reagent consumption and batch-to-batch variations [101, 102]. Interestingly, the introduction

of superparamagnetic magnetic nanoparticles (used to provide magnetic targeting capabilities) during the SF formation process provided artificial regulation of this process as well as drug entrapment, preventing agglomeration of SF and resulting in uniform, spherical nanoparticles [52]. Ultimately, SF nanoparticles provide many attractive properties for multi-functional drug delivery strategies but future use relies on reliable, reproducible manufacture to ensure appropriate comparisons can be made for their translation.

### **Acknowledgements**

Financial support provided by Cancer Research UK (CRUK grant reference: C25574/A24321) and Public Service Department Malaysia (JPA).

### **Author details**

Faith H.N. Howard\*† , Zijian Gao† , Hawari Bin Mansor† , Zidi Yang and Munitta Muthana University of Sheffield, Sheffield, UK

\*Address all correspondence to: f.howard@sheffield.ac.uk

† Joint first authors.

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Silk Fibroin Nanoparticles: A Biocompatible Multi-Functional Polymer for Drug Delivery DOI: http://dx.doi.org/10.5772/intechopen.109642*

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## Emerging Selenium Nanoparticles for CNS Intervention

*Jonaid Ahmad Malik, Jeba AjgarAnsari, Sakeel Ahmed, Archana Rani, Shabana Yasmeen Ansari and Sirajudheen Anwar*

### **Abstract**

Central nervous system (CNS) diseases have seriously impacted human wellness for the past few decades, specifically in developing countries, due to the unavailability of successful treatment. Due to the blood-brain barrier (BBB) and blood-cerebrospinal fluid barrier transport of drug and treatment of CNS disorders has become difficult. Nanoscale materials like Selenium nanoparticles (SeNPs) offer a possible therapeutic strategy for treating brain diseases like Alzheimer's, Frontotemporal dementia, Amyotrophic lateral sclerosis, Epilepsy, Parkinson's disease, and Huntington's disease. After being functionalized with active targeting ligands, SeNPs are versatile and competent in conveying combinations of cargoes to certain targets. We shall pay close attention to the primarily targeted therapies for SeNPs in CNS diseases. The objective of this paper was to highlight new developments in the exploration of SeNP formation and their potential applications in the management of CNS diseases. Furthermore, we also discussed the mechanisms underlying management of CNS disease, several therapeutic potentials for SeNPs, and the results of their preclinical research using diverse animal models. These methods might lead to better clinical and diagnostic results.

**Keywords:** selenium, nanoparticles, CNS diseases, management of CNS disorders, Alzheimer's disease, Parkinson's disease, Huntington's disease, epilepsy

### **1. Introduction**

The central nervous disordersare progressive degeneration of neurons in the central nervous system, which results in altered brain cellular function. The major symptoms begin from degeneration of neurons to loss of coordination and memory and ultimately result in complete loss of function in healthy individuals. The three crucial neurodegenerative disorders (NDs) have been recognized as Alzheimer's disease (AD), Parkinson's disease (PD), and Amyotrophic lateral sclerosis (ALS) [1], affecting millions of individuals worldwide. AD is one of the most prevalent NDs and has affected mostly 30% of the aged people [2]. The second most common neurodegenerative disorder is PD, which affects 10–15 individuals per 100,000 people yearly [3]. It is estimated that PD cases will increase worldwide and may cross 12 million

cases in 2050 [4]. Similarly, cases of ALS are also increasing with 1–2.6 new cases per 100,000 persons. The average age of onset of this disease is 59–60 years, and the average time from diagnosis to death is 3–4 years [5]. Besides these disorders, Gliomas, and glioblastoma, intrinsic brain tumors arise from neuroglial cells [6].

Other disorders include Huntington's disease (HD), depression, anxiety, autism spectrum disorders, seizures, etc. [7]. The establishment of innovative NDs treatments are urgently needed as the WHO has predicted that within the next 20 years NDs will surpass cancer and will become the second most common cause of death [8]. Natural products may offer great promises compared to classical therapies available to improve the symptoms but cannot prevent their progression. Therefore, researchers are continuously searching for new natural products that can potentially treat NDs without compromising the patient's health. However, all-natural products are not always safe; because natural products as drugs may have more adverse effects than their benefits as they are derived from various biological sources, their conversion into therapeutic formulations may face many hurdles such as safety issues, difficulty in identifying active ingredients because natural products contain various active phytochemicals such as flavonoids, alkaloids, etc. And it becomes difficult to identify which components of the herb have maximum therapeutic potential, low stability and high degradation, and difficulty crossing the blood-brain barrier [9]. However, till now, there are no effective treatments available that can alter the main symptoms of autism or which can improve the cognitive and deficit symptoms of schizophrenia; the majority of the people who have epilepsy, depression, brain injury, and posttraumatic stress disorder have acquired a few satisfaction from the current therapy available. The discovery of new effective medication for NDs has proven difficult compared to other diseases. Most pharmaceutical companies have shifted their interest from the field of neurology to other fields [10]. Chances of failure of clinical trial rate in the last stages are higher for a neurological and psychiatric disorders as compared to other diseases; due to the complex physiology of the brain, there are fewer animal models available that can effectively predict the safety and efficacy of the drug for the disorder that mainly affects the cerebral cortex [11]. Drug discovery is a costly and risky process, while drugs used to treat neurological or

### *Emerging Selenium Nanoparticles for CNS Intervention DOI: http://dx.doi.org/10.5772/intechopen.109418*

psychiatric disorders not only require a longer time to complete all stages of a clinical trial but also take much longer to complete the process of approval from a regulatory authority. The higher risk and high expenditure related to drug discovery and the development of neurological disorders are directly linked to scientific challenges. Several obstacles faced by pharmaceutical companies faces in discovering new drugs include: (a) Slow process of target identification and validation, (b) lack of appropriate animal models, (c) long duration of clinical trials paucity of knowledge regarding the disease's etiology [12]. One of the biggest hurdles in the establishment of the treatment of CNS disorder is because of the blood-brain barrier [13]. BBB restricts the permeation of therapeutic drugs to reach to the target site in the CNS therefore more than 98% of small molecular drugs remain ineffective in treating the CNS disorder [14].

Transportation of drugs through the BBB is a complex process requiring very sophisticated and nanosized particles. As nanoparticles have the potential to deliver drugs in various diseases, selenium nanoparticles (SeNPs) have been found to play a significant role in neurodegenerative diseases [15]. Various studies revealed that selenium nanoparticles have better bioavailability, improved antioxidant properties, and less toxicity than selenium-containing compounds. Thus selenium nanoparticles have free radical scavenging properties and improve behavior abnormalities and neurochemical alterations. Therefore, selenium nanoparticles have the potential to improve the impairment of memory and can be used as a potential therapy for NDs [16]. This book chapter provides a thorough overview of recent discoveries in the fields of investigation and use of nanoformulations for treating NCDs such as Parkinson's, Alzheimer's, ALS, and Huntington's, as well as the use of SeNPs for diagnosis and treatment.

### **2. Selenium nanoparticles in the management of CNS disorders**

Due to the BBB and BCSFB, which prevents drug transport, treating ailments of the CNS is notoriously problematic. A promising clinical methodology for the intervention of some common NDs like, frontotemporal dementia, ALS, PD, and HD is provided by nanotechnology-based drug delivery methods, one of the new tactics to get around these obstacles and delivering medications to the CNS [17]. SeNPs could be a novel approach for treating such CNS disorders. Nanotechnology has emerged as an intriguing and promising new tool for treating NDs with considerable potential to solve issues with conventional methods. Nanostructured materials could traverse the BBB, target specific cells or signaling pathways, react to endogenous stimuli, transport genes, help axonal regeneration, and promote cell viability, among other specialized activities [18].

### **2.1 Role of SeNPs in treating CNS diseases**

On the brain and neurons, selenium exhibits a direct antioxidant impact [19]. Low or moderate doses of selenium suppress cancer progression and have therapeutic benefits on NDs, including AD. Elevated levels of selenium increase the development of cancer cells and exhibit neurotoxicity. In *in vivo* and *in vitro*, selenium's antioxidant and anti-inflammatory activities have been established [20]. Selenium is a co-factor in the enzyme glutathione peroxidase, a scavenger. The catalytic properties of GSH-Px cause hydrogen peroxide (H2O2) to be transformed into water [19, 21–23]. A growing body of research shows that memory loss in AD patients is directly related to selenium deficiency in serum and hair samples. In animal studies and AD patients, selenium

supplementation has reportedly been shown to reduce the likelihood of cognitive issues [20, 24–27]. The exploration of selenium and selenoproteins in neurological disorders, such as AD, has attracted much interest. Proteins known as selenoproteins include selenium as the amino acid selenocysteine. Since antioxidant mechanisms are crucial for delaying the emergence and spread of AD, these are primarily expressed in human brain tissue [28].

Additionally, certain recently developed selenoproteins and SeNPs with exceptional physiological characteristics exist. These particles may replace traditional therapeutic medications in managing AD because of their great efficiency and low toxic effect [20, 29]. SeNPs may be a promising therapeutic molecule for treating AD, according to the Nazrolu et al. report [20]. Using the whole-cell patch clamp method, Yuan et al. investigated the effects of SeNPs on sodium influxes and the excitation of DRG (dorsal root ganglion) neurons [30]. According to their study, SeNPs appeared to reduce sodium influx in a concentration and time-dependent way, raising the possibility that SeNPs may be neurotoxic [20, 31, 32]. Epigenetic, chronic stress, metabolic, and dietary factors all have a role in developing HD, an untreatable condition that causes a gradual loss of brain functionality. According to studies, selenium (Se) concentration in the brain are inadequate for HD illness, but restoring Se regulation there may lessen neuronal death and functioning. Most research showed that nano-Se reduced peroxidation, prevented huntingtin protein aggregation, and suppressed the production of histone deacetylase family members at the mRNA level. Nano-Se offers great promise as a treatment for HD. Therapy for HD disease will derive from nano-Se NPs' ability to heal neuronal processes and shield against degradation under stress [33]. Recent research has crucially demonstrated Se's beneficial impact on HD. For instance, sodium selenite may reduce mutant huntingtin clumping and rates of oxidized glutathione in HD mouse brains [34]. SeNPs were reported to offer neuroprotection by upregulating Nrf2 and HO-1, suppressing the inflammatory process, and apoptotic pathway, and avoiding the emergence of oxidative stress. Upon the onset of epileptic seizures, SeNPs can counteract alterations in the concentration and functionality of neuromodulators. SeNPs have strong antioxidant, anti-inflammatory, and neuromodulatory properties that make them a potential candidate for use as an anticonvulsant medication [35].

Selenium has been investigated as a screening tool for several neurological disorders, including epilepsy, AD, and PD. Se is available in high levels in the grey matter areas and the glandular sections of the brain. It participates in several neurotransmission and dopaminergic pathways [36, 37]. A few elements identified as potential influences for the involvement of Se in Alzheimer's pathogenicity include its antioxidant, neuroprotective effects, influence on the regulation of cytoskeletal elements assembly, affinity for several neurotoxic metals, and competence to mitigate Aβ accumulation and tau proteins hyperphosphorylation [34, 36, 38]. Se has been worn to prevent dopaminergic neurons by many selenoproteins, supporting its ability to fend off PD. Se levels were also linked to aggressive behaviour, anxiety, and mood swings. Se application in neurological diseases may be beneficial for individuals with profound Se insufficiency and/or mutants in genes related to Se transport or selenoproteins synthesis. At the same time, brain Se levels are usually low, and high Se levels might be detrimental (**Figure 1**) [39, 40].

SeNPs have shown great potential in managing various CNS disorders through particular mechanisms. The cellular signaling pathways that regulate the metabolic activity of neurons (TSC1/TSC2, p-mTOR, mTORC1), antioxidant (FoxO1, β-catenin/Wnt, Yap1), and inflammatory system (jak2/stat3, Adamts-1), *Emerging Selenium Nanoparticles for CNS Intervention DOI: http://dx.doi.org/10.5772/intechopen.109418*

### **Figure 1.**

*Selenium nanoparticles in the management of several neurodegenerative diseases.*

autophagy and induction of apoptosis (Mst1, ULK1, Bax, Caspase-3, and Bcl-2), and the preservation of hippocampal neurons (rictor/mTORC2) [41] these are the molecular mechanisms related with the neuroprotective activity of SeNPs. MST1 regulates neuronal cell death via Casp3 Signaling Pathway [42]. The JAK-STAT pathway prevents apoptosis in neurons. STAT3 is upregulated and stimulated selectively in regenerated neurons after axon damage. This pathway plays a direct role in forming glial scar tissue across lesions and neuronal restoration. After a CNS injury, STAT3 stimulation is required for the production of glial scars and the control of the spread of inflammation, both of which are essential for astrogliosis [43]. Numerous investigations have functionalized SeNPs with particular molecules, like sialic acid and epigallocatechin-3-gallate, to improve their penetrability toward the BBB. SeNPs are shown to minimize accumulation and stimulate their fragmentation to act as an antioxidant in the brain, either effectively or as part of GPx [20, 44] (**Figure 2**). Additionally, SeNPs were investigated in conjunction with substances that have demonstrated anti-disease Alzheimer's effects, such as resveratrol (Res) [45], curcumin (Cur) [46], chiral D-penicillamine (DPen) [47], and chlorogenic acid (CGA) [36, 48].

### **2.2 Types of SeNPs in the treatment of CNS disorders**

SeNPs in conjugation with several molecules acts as an antioxidant and neuroprotective agent and has also been reported for various neurological diseases. Resveratrol's antioxidant and neuroprotective abilities, which counteract Aβ-aggregation and its oxidative consequences, have shown promise in the defense against AD. It has been demonstrated that Res-SeNPs selectively attach to Aβ through N-donors found in amino acids, forming a Se-N bond and enhancing Ressuppression on Cu2+-induced Aβ aggregation. Res-SeNPs are non-toxic to neuroblastic cells (PC12 cells) and protect them against oxidative stress by reducing the apoptosis caused by Aβ, suggesting a potential synergism between SNPs and Res [45]. Curcumin also has demonstrated potential synergy with SeNPs in the therapy of AD. The antioxidant,

**Figure 2.**

*Anti-inflammatory mechanism of SeNPs in management of central nervous system related diseases.*

anti-Aβ inflammation and anti-Tau hyperphosphorylation capabilities of these NPs allowed them to pass through the BBB and bind to Aβ. It has been demonstrated that curcumin binds Aβ by hydrophobic contacts at the nonpolar regions of Aβ, enhancing their anti-inflammatory and antioxidant activities alongside Se [46]. As a result of their ability to bind to the N-donors of Aβ-proteins and form a Se-N bond, which prevents Aβ from aggregating them, CGA-SeNPs were shown to minimize Aβ-generated ROS in a dose-dependent manner, hindering their neurotoxicity and, consequently, lowering the rate of apoptosis. This was transcribed into a synergistic effect among CGA and SeNPs [49]. When given to transgenic mice 5XFAD with the mutation, the curcumin-loaded selenium-PLGA nanospheres developed by Huo et al. demonstrated a reduction in Aβ plaque production and inflammation [46]. Using an anti-Tfr receptor monoclonal antibody (OX26) as a functionalizing agent, PEG-SeNPs were also applied to treat stroke. OX26-PEG-SeNPs have been shown to play a function in preventing stroke in neuronal cells by minimizing the cellular edema brought on by aberrant Na+ ion influx. When the middle cerebral artery was blocked in Wistar rats, SeNPs could reduces the infarction volumes, reduce the number of necrotic cells, increase the myelinated areas, and prevent the loss of axons in the hippocampal region [41]. SeNPs have also demonstrated promise in the battle against Huntington's disease. In HA759 mutant nematodes, SeNPs, in a dose-dependent manner, lowered neuronal death by reducing protein aggregates associated with HTT genetic variants and ROS, suppressing histone deacetylase mRNA, axonal degeneration, and improving reflexes [50]. Work on selenium-doped carbon quantum dots (Se-CQDs) has demonstrated their capacity to reduce ROS, and they have been successfully used to reduce secondary damage in TSCI. The findings showed that Se-CQDs had bioactivity and had a notable preventive role on astrocytes and PC12 cells toward H2O2-induced oxidative stress [51].

### *Emerging Selenium Nanoparticles for CNS Intervention DOI: http://dx.doi.org/10.5772/intechopen.109418*

A frequent neurological condition called cerebral ischemia sets off a series of pathophysiological processes that include a drop in glucose and oxygen levels, an uncontrolled emission of glutamate, a fast rise in cellular calcium levels, and the production of free radicals. These occurrences consequently cause the endoplasmic reticulum and mitochondria's functions to be disrupted, which in turn causes the death of brain cells through apoptosis or necrosis [41, 52–54]. SeNPscan cross the BBB, builds up in the brain, and stops cell death from occurring. SeNPs are known to promote the production of BDNF and lower levels of Aβ and IL-6 in the hippocampus. Increased BDNF production reduces oxidative stress and prevents the degeneration of GABAergic neurons, particularly vulnerable to hypoxia and ischemia [55–57]. The mechanisms of the protective role of a modest dose of SeNPs on brain cells during OGD/R were examined by Turovsky et al. [58]. Additionally, the Bcl-2 family of proteins, the mechanisms of calcium homeostasis repair, suppression of mitochondrial and ER stress mechanisms, and eventually silencing of caspase-3 and inhibition of apoptosis are all part of SeNPs protective measure [58, 59].

Fei Gao et al. created selenium-chondroitin sulfate nanoparticles (CS@Se) as part of a multitargeted therapy for AD. Amyloid-ß (Aß) accumulation was successfully prevented by CS@Se, and SH-SY5Y cells were shielded against cytotoxicity brought on by Aß1–42. In SH-SY5Y cells, okadaic acid-induced actin cytoskeleton disruption was dramatically reduced by CS@Se. The ROS and MDA levels were reduced by CS@ Se, while the amounts of GSH-Px were elevated. This research shows that CS@Se might reduce tau protein hyperphosphorylation, ameliorate oxidative stress, prevent Aß from aggregating, and lessen cytoskeleton disruption. CS@Se is an effective multifunctional drug for the management of AD [60]. Xian Guo developed SeQDs, which have a multitarget therapeutic impact and can easily enter the BBB, to improve the therapeutic effect of pharmaceuticals through the BBB. SeQDs' unique fluorescence properties could be used to diagnose and manage AD. SeQDs are highly effective at scavenging free radicals and shielding cells from oxidative stress. By down-regulating PHF1 and CP13, the SeQDs can dramatically reduce tau protein phosphorylation and further neutralize free radicals, rebuild metabolic activity, preserve nerve cell solidity, and defend nerve cells from oxidative damage. These effects preclude Aß-mediated cytotoxicity and Aß aggregation, preventing the AD cascade reaction. Compared to conventional single-target medications, usingSeQDs in AD therapy has many benefits and offers a fresh approach to the co-managementof neurological illnesses [61]. The mechanism of SeNPs is depicted in **Figure 3**.

**Figure 3.**

*SeNPs against various neurodegenerative disorders.*

### **3. Therapeutic application of selenium nanoparticles in CNS disorder**

### **3.1 Alzheimer's disease**

AD is a progressive neurodegenerative disease; the pathological hallmark of this disease is the deposition of Aβ plaques. Various attempts have been made to develop therapies that potentially inhibit its deposition in the brain; in this regard, nanoparticles have shown promising results due to their distinctive physiochemical properties of small size and large surface area. Se is one of the most important mineral nutrients having a wide range of pharmacological actions. Studies have found that selenium has a neuroprotective effect. Due to the high stability of SeNPs, it has potential effects on the neurotoxicity of Aβ42 in primary cultures of murine hippocampal neurons [62]. In human clinical trials, curcumin has been found as a highly efficacious compound without exerting any adverse effect, even when taken at a higher dose of 4 g/day. Curcumin forms intermolecular hydrogen bonds and binds effectively with Aβ plaques in AD. In AD, curcumin's drug delivery properties were modified by encapsulating SeNPs and changing the surface of the poly-lactide-co-glycolide (PLGA). In preclinical studies of memory impairment in mice models, it was found that the drug delivery system of curcumin-loaded SeNPs has the potential to decrease the aggregation of Aβ plaque in Alzheimer's disease mice model [46]. Moreover, selenium-chondroitin sulfate nanoparticles were also found to decrease the aggregation of amyloid-β and reduce the tau protein hyperphosphorylation by targeting the GSK-3β [63]. Another research revealed that chitosan-coated SeNPs (ChSeNPs) could enhance the effectiveness of stem cell-based therapy to attenuate the neurotoxicity in the streptozotocin-induced model in rats [64].

### **3.2 Parkinson's disease**

PD is the second most prevalent CNS disorder after AD. Oxidative stress is considered one of the major factors responsible for this disease, causing neuronal death and apoptosis. Therefore, behavioral abnormalities in PD can be improved by decreasing the level of oxidative stress. As selenium possesses antioxidant properties, in preclinical studies, the neuroprotective effect of glycine-nano-selenium on oxidative stress was evaluated in PD rat model, and oxidative stress is induced by using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in the rat. It was found that oxidative stress in PD rat model was reduced by administering intragastric glycine nano-selenium, which ultimately reduced the neurobehavioural abnormalities in rats [65]. The administration of selenium in humans and animals has shown a rise in the level of glutathione and glutathione peroxidase, thus slowing the degeneration of neurons and preventing the depletion of dopamine levels. Hence it is regarded as an important micronutrients in Parkinson's disease as well [66].

### **3.3 Huntington's disease**

Various studies have also been conducted for Huntington's disease by using selenium nanoparticles. HD is an inherited autosomal dominant disease caused by repeated trinucleotide sequence CAG that encodes for huntingtin protein [67]. Various laboratory findings suggest that oxidative stress is a major factor in Huntington's disease's pathogenesis. But due to poor knowledge of particular oxidative biomarkers, no antioxidants have effectively prevented neurodegeneration

*Emerging Selenium Nanoparticles for CNS Intervention DOI: http://dx.doi.org/10.5772/intechopen.109418*

in Huntington's disease [68]. Studies have found Se to play a protective role in Huntington's disease, such as sodium selenite has the potential to lower mutant huntingtin aggregation and oxidized glutathione levels in the brain of HD mice model [34]. Recent studies have found a low level of Se in the brain of HD patients. Attenuating neuronal dysfunction can be achieved by maintaining the level of Se in the brain. In a preclinical study of HD models, Selenium nanoparticles have been found to prevent neuronal loss and improve behavioral dysfunction. Molecular testing has shown that selenium nanoparticles also prevent the damage produced by oxidative stress and attenuate the aggregation of huntingtin proteins. Thus Selenium nanoparticles are an effective therapy for Huntington's disease [50].

### **3.4 Amyotrophic lateral sclerosis**

ALS is a chronic, deadly and irreparable NDs which involves the degradation of motor neurons in the motor cortex, brainstem, and spinal cord, which results in paralysis and death due to respiratory failure [69]. Because of the absence of efficient treatment, the majority of patients pass away within 3–5 years of assessment. Several cases of ALS are sporadic, while 15% of the cases are familial [70]. Genetic defects in SOD1 were found to be the first causative mutation involved in the pathology of ALS [71]. Apart from this, more than 50 genes were reported to be involved in ALS. The most common ones include mutations in chromosome 9 open reading frame 72 (*C9orf72*), TAR DNA-Binding (*TARDBP*), and fused in sarcoma (*FUS*) [72]. Recently in vitro studies have shown organo-selenium compound to potentially protect the neuronal damage and thus can be used as an alternative therapy in ALS [73].

### **3.5 Epilepsy**

Selenium nanoparticles can also be used in other CNS disorders such as epilepsy. SeNP also has potential anticonvulsant activity due to its extensive antioxidant, antiinflammatory, and neuromodulatory effect. Administration of SeNP decreases the duration of tonic, myoclonic and generalized seizures and can be used as an effective therapy in epilepsy [35]. SeNP can effectively cross the BBB and thus can be used to enhance the delivery of anti-cancer drugs in the brain, such as in the case of glioma in humans [74]. Drug delivery to cross the BBB is a complex process, and it requires a nanosized particle so that the drug can reach the brain. In this regard, SeNPs plays a vital role in the management of NDs (**Table 1**).

### **4. Mechanism of SeNPs in neurodegenerative disease**

Selenium, a crucial trace element in both man and livestock, is essential in managing the biological stability of the brain and possesses neuroprotective properties. Various selenoproteins were also found to be involved in controlling NDs [80]. SeNPs significance in NDs has been widely reported in recent years (**Figure 4**), considering that neurons are highly vulnerable to damage from oxidative stress-related injury for variety of reasons, including excessive oxygen utilization (about 25% of the total body utilization). There is a substantial quantity of polyunsaturated fatty acids and low amount of antioxidant enzymes [81]. Natural antioxidants are frequently utilized to treat neurological illnesses since oxidative stress is one of the primary contributors to their etiology. Yet, they are ineffective [80], and as a result, using antioxidants



*Selenium nanoparticles showing promising results in neurodegenerative disease.*

*Emerging Selenium Nanoparticles for CNS Intervention DOI: http://dx.doi.org/10.5772/intechopen.109418*

### **Figure 4.**

*Schematic representation of the mechanism of SeNPs in neurodegenerative disease.*

in the form of nanoparticles is growing in popularity. Se's capacity to pass the BBB and suppress Aβ aggregation are two of its key effects in AD [82]. Se was found to have a beneficial impact on the activity of H2O2 absorption, the generation of intracellular ROS, and the aggregation of Aβ, which is found in the investigation of Se-containing clioquinol derivatives during the oxidation of Aβ caused by Cu2+ [83]. It is well recognized that the formation of improperly folded proteins in the brain and their aggregation is one of the primary causes of neurodegenerative disorders. Since Aβ may acquire several formats in AD, amyloid plaques are recognized due to improper protein folding and aggregation in the brain. According to studies, metal ions like Cu2+, Zn2+, and Fe2+ can bind to Aβ and co-localize with amyloid plaques in exceptionally high concentrations [84]. As a result, the use of metal chelators, like clioquinol (CQ ), for AD treatment is of great interest. However, most chelators can also bind to other metal-containing proteins, which is undesirable and might disturb normal physiological functioning in the body [85].

SeNP has been shown to attach to Aβ, influence metal ions, and change their surfaces, such as ligands, charges, or reactivity [86]. As a result, it was demonstrated that l-Cys-modified SeNP (Cys-SeNP) could prevent Aβ40 fibril formation caused by Zn2+ [87]. A multifunctional therapy for AD treatment has also been developed using chondroitin selenium sulfate (CS@Se) nanoparticles [63]. Chondroitin sulfate is a sulfated glycosaminoglycan (GAG) that binds to the protein core to generate the proteoglycan chondroitin sulfate (CSPG). The primary constituent of Perineuronal networks is

CSPG [88]. Aβ1–42 induced cytotoxicity in SH-SY5Y cells (human neuroblastoma) was prevented by CS@Se, which demonstrates its ability to prevent amyloid-β aggregation efficiently. Additionally, CS@Se lowered the hyperphosphorylation of tau (Ser396/ Ser404), reduced the levels of ROS and MDA, and enhanced the levels of GSH-Px [63].

In HD, a hereditary neurodegenerative condition that results in the loss of brain cells and is linked to motor, cognitive, and behavioral impairments in adult patients, recent investigations have shown that Se has a protective function. Variable CAG trinucleotide repeats, found in the transcript that codes for the HTT, are a feature of this autosomal dominant disorder. The DNA repair system and mitochondrial malfunctions can be harmed by cumulative oxidative stress, which is thought to play a significant role in HD and other NDs. It has been demonstrated that sodium selenite can lessen mutant huntingtin aggregation and oxidize glutathione levels in HD mouse brains [34]. There is no viable treatment to stop HD from progressing or cure it. SeNP has been demonstrated to protect *C. elegans* from oxidative stress, ameliorate behavioral dysfunction, and prevent neuronal death at doses below 2 μM [50]. Nanoparticle therapy reduced the quantity of ROS, demonstrating their antioxidant properties, and stopped mutant HTT from aggregating in vivo.

After AD, PD is the second progressive neurological illness. The pathophysiology of PD is still unknown, although studies have revealed that oxidative stress, which causes neuronal death and apoptosis, is a key pathogenic component of PD [89]. An established neurotoxin called MPTP is used as a model for PD research. It has been demonstrated that MPTP can cause PD by boosting oxidative stress, which causes dopamine neurons to degenerate and causes neurobehavioral problems. By raising SOD and GSH-PX activity and lowering MDA levels, glycine-SeNP had an anti-oxidative effect on neurons. As a result, glycine-SeNP has the potential to treat Parkinson's disease [65]. Behavioral, molecular, and neurochemical alterations are the hallmarks of the persistent neurological condition known as epilepsy. Epilepsy is a neurological condition that affects between 0.5% and 1% of the world's population and is characterized by repeated, spontaneous seizures. Numerous conditions, such as cerebrovascular diseases, trauma, cancer, oxygen deprivation, infections, and genetic problems in brain development, can contribute to the development of seizures [90]. SeNP is a promising epilepsy treatment because of its excellent BBB-crossing capacity and few side effects. The malfunction of the mitochondria and endoplasmic reticulum causes oxidative stress, which increases the formation of free radicals and depletes neuronal antioxidant molecules. Oxidative stress is linked to both neuronal hyperexcitability and epileptogenesis [91]. The structure of selenoproteins and selenoenzymes include Se, which can inhibit ROS and, consequently, the onset of oxidative damage. Additionally, SeNP help to restore the levels of the neurotransmitters ACh, NE, DA, 5-HT, and GABA in brain tissue, which helps to restore neuronal connections and reduce apoptosis [35].

### **5. Diagnostic applications of SeNPs in CNS disorders**

One of the main obstacles in treating and diagnosing CNS diseases is the inability of therapeutics to cross the BBB. A more comprehensive and accurate nanoparticle design is required to deliver therapeutic and diagnostic compounds to the CNS95 effectively. The evaluation and management of neurological conditions such as AD, PD, HD, head injuries, brain tumors, and epilepsy remain difficult tasks at this time. Numerous prospective medications have been studied to treat various neurological illnesses, but their efficacy is still constrained due to various difficulties [18].

For biomedical applications such as medication administration, bioimaging, and biosensing in CNS illnesses, a variety of inorganic NPs provide considerable efficiencies [92]. SeNPs offer a wide range of applications, including assessing and treating health-related problems that would otherwise be impossible to identify or address. Khalid et al. have examined the intrinsic fluorescence of SeNPs and their diagnostic potential. They discovered SeNPs' inherent fluorescence and its usefulness for nanoscale monitoring of cellular mechanisms. SeNPs' photoluminescence spectrum ranges from the visible to the near-infrared, making it useful for neuroblastoma cell tracking and their in vitro imaging. SeNPs have also been investigated as a peroxide biosensor. For accurate H2O2 sensing, Wang et al. produced semiconductor monoclinic SeNPs and they can be used to diagnose and assess the state of oxidative stress [93]. To produce an H2O2 sensor, plant-based rod-shaped SeNPs were created utilizing lemon fruit extract as a reducer and capping agent. Hydrogen peroxide sensing is a crucial component since it initiates a variety of cellular processes [94]. As selenium levels are said to be low in patients, SeNPs could be utilized to diagnose AD and HD.

Further research can be done on diagnostic techniques for detecting GPx or selenium levels. By focusing on many physiological pathways that control the metabolic status, inflammatory responses, oxidative defense system, and apoptosis, SeNPs functionalized with monoclonal antibodies (OX26) may be capable of defending against ischemic stroke [95]. The nano-based technique has significance for multiple sclerosis diagnosis and its involvement in treatments. The very sensitive DNA-carrier gold NPs-based coding technique can identify biomarkers in CSF or damaged brain tissue. This diagnostic test may be very helpful in diagnosing MS because radio imaging is the standard gold method for MS assessment (**Figure 5** and **Table 2**) [18].

**Figure 5.** *Mechanism of selenium nanoparticles in the diagnosis of neurodegenerative disorders.*

*Emerging Selenium Nanoparticles for CNS Intervention DOI: http://dx.doi.org/10.5772/intechopen.109418*


### **Table 2.**

*List of some selenium nanoparticles in the diagnosis of CNS diseases.*

### **6. Future perspective**

Nanotechnology-driven formulation techniques have enormous potential in the twenty-first century's drug research and discovery. Several nanotechnology-based preparations are available and can be easily accessible from the market, and this fact is not hidden from everyone. Se nanoparticles, an important trace element needed as a co-factor for various enzymes, have become an important tool in diagnostics and therapeutics for treating various illnesses, including neurodegenerative disorders. Indeed, the bulk of reported research has a significant SeNPs-based justification [98]. The development of nanotechnology has increased the number of possible therapeutic approaches to halt the course of AD. Oral/gastric barriers and the BBB are conventional neurotherapeutic obstacles that are effectively overcome by the proper design and production of NPs, improving the physicochemical characteristics of drugs in biological systems. However, the field of AD nano-therapeutics still has several limitations. There have been many in vitro experiments demonstrating the capability of SeNPs and its effectiveness, but there have been few in vivo trials. Therefore, future studies of these SeNPs may show systemic efficiency or toxicity in biological systems over the long run that can be contrasted to *in vitro* methods. Therefore, in the near future, potential, affordable AD treatments may result from evaluating the safety and efficacy of appropriate SeNPs in human clinical trials [99]. Recent research findings suggest that SeNPs can provide promising results in for the treating of HD

through diets. In the future, an in-depth knowledge is required to know the mechanism of nano-Se in preventing the HD and their connection between physicochemical features and therapeutic potential will be beneficial for treating HD disease.

Furthermore, compared to other selenium species, the rational design of nanoSe may enhance dosage tolerance for HD therapy in the future [50]. It is abundantly evident that the current situation demands immediate and effective treatment for neuroprotection, neurorestorative, and neuroregeneration. Clinical translation in neurodegenerative disorders has become more challenging due to the lack of appropriate biomarkers, delayed diagnosis, incomplete understanding of molecular pathogeneses, lack of useful disease models, insufficient clinical protocol, and the generally asymptomatic nature of the disease. The deficiency of proper animal models that accurately reflect some crucial characteristics of ND in humans and a shortage of samples of patients are the two key limitations to therapeutic advancement. Therefore, the successful development of effective human disease-modifying medicines can be achieved through representative animal models.

Furthermore, inadequate knowledge of the molecular rationalization of aging and its biological impact and clinical consequences of neurodegenerative disorder contributes to delayed progress in translation. Success in therapeutic trials may result from shifting the focus from the primary pathogenic proteins to a plethora of diseaserelated proteins. In the future, using human CNS organoids to model neurological diseases is a realistic choice.3D brain organoids with the appropriate physicochemical signaling cues can be used to simulate patient-specific tissue patterns. Although organoids greatly improve the deep understanding of the development of brain and neurodegenerative illnesses, there are still several gaps in the field, including vascularization and non-neuronal cells. The targeting of particular brain cells in various NDs, such as in PD dopaminergic neurons are mainly targeted and this must be questioned when nanoformulation are prepared.

Additionally, it is important to consider adjusting pharmacokinetics and pharmacodynamics characteristics before administering NP [100]. Nanomedicine-based delivery systems raise concerns about their potential for toxicity, including the possibility of inflammation of neurons, excitotoxicity, mitochondrial and DNA damage, and some allergic reactions. Therefore, thoroughly researching the biocompatibility as well as biodegradability of nanodrugs is important [101]. The main function of the SeNPs in pharmacological defense against different types of inflammatory as well as oxidative stress-mediated situations is already discussed. However, nothing is known about how the SeNPs influence the pharmacokinetics and pharmacodynamics properties of selenoproteins. Most of the available research was not well structured and did not include comparisons to other Se sources. Future research should focus on understanding how selenoproteins contribute to the reported pharmacological effect and include relevant sources of Se [98].

### **7. Conclusion**

Leading contributors to the world's disease burden are CNS illnesses, which encompass a wide range of brain diseases with both short- and long-term disabilities. Because of the shift in lifestyle and the swift, ongoing environmental degradation, CNS disorders like AD, PD, stroke, brain tumors, and neuroinflammation are distressingly damaging to humanity. With their complicated anatomy, specific microenvironment, and specificity to any foreign material such as drugs, BBB and BCSFB are the primary physiological barriers that pose a significant bottleneck for the effective

### *Emerging Selenium Nanoparticles for CNS Intervention DOI: http://dx.doi.org/10.5772/intechopen.109418*

therapy of CNS disorders and brain tumors. This presents the greatest hurdle to CNS drug discovery. SeNPs may help with the current issue of the lack of multifaceted drugs for various CNS disorders, which may contribute to various distinct biological mechanisms. SeNPs have led to cutting-edge nanoscale targeting approaches among the different treatment approaches. They are at the forefront of a new paradigm that could administer active agents with intriguing dynamics to treat these disorders. SeNPs offer superior medicinal qualities to selenium salts and lesser toxicity, despite their narrow therapeutic window. The current demand for effective nano-based treatments is concentrated on neuroprotection and restoration, which would greatly benefit from other nano-based strategies and advancements in the anatomy, pathology, and physiology of neuronal cells. Several nanoscale treatments (SeNPs) were found to treat neurological illnesses in AD, PD, and stroke models, including the suppression of Aβ oligomerization, reduction of ROS, and enhancement of functioning neural networks (**Figure 5**). SeNPs have allowed it to administer chemotherapy and antisense gene therapy in malignant brain tumors with pinpoint accuracy. This has led to a striking reduction in disease development in both *in vitro* and *in vivo* research.

SeNPs could completely alter how we address CNS-targeted therapeutics because of their competency to be nanoengineered so that the drug or carrier can encounter the BBB, diffuse inside the brain, and target specific cells or signaling systems for therapeutic delivery. This opens up new paths in the intervention of neurological diseases and has an extremely great prospect. It is very conceivable that SeNPs will alter how CNS disorders are treated. Shortly, the real objective of drastically increasing survival rates will be accomplished.

### **Abbreviations**


**95**


### **Author details**

Jonaid Ahmad Malik1 , Jeba AjgarAnsari<sup>2</sup> , Sakeel Ahmed3 , Archana Rani4 , Shabana Yasmeen Ansari<sup>5</sup> and Sirajudheen Anwar<sup>6</sup> \*

1 Department of Biomedical Engineering, Indian Institute of Technology Ropar, Rupnagar, Punjab, India

2 Department of Pharmaceutics, Government College of Pharmacy, Aurangabad, Dr. Babasaheb Ambedkar Marathwada University, Maharashtra, India

3 Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research, Ahmedabad, India

4 Department of Pharmaceutical sciences, Guru Nanak Dev University, Amritsar, India

5 Pharmaceutical Unit, Department of Electronics, Chemistry and Industrial Engineering, University of Messina, Messina, Italy

6 Department of Pharmacology and Toxicology, College of Pharmacy, University of Hail, Hail, Saudi Arabia

\*Address all correspondence to: si.anwar@uoh.edu.sa

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Emerging Selenium Nanoparticles for CNS Intervention DOI: http://dx.doi.org/10.5772/intechopen.109418*

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### **Chapter 5**

## Antibacterial Strategies: Photodynamic and Photothermal Treatments Based on Carbon-Based Materials

*David Giancarlo García Vélez, Karina Janneri Lagos Álvarez and María Paulina Romero Obando*

### **Abstract**

The problem of bacterial resistance is based on the abuse of antibiotics such as trimethoprim, fluoroquinolones, chloramphenicol, and some carbapenems. For this reason, conventional treatments to treat diseases caused by bacteria have become ineffective. Therefore, developing new therapies with multifunctional materials to combat bacteria is mandatory. In this context, photodynamic treatment (PDT) and photothermal treatment (PTT) have been proposed to combat bacteria. These lightstimulated treatments are minimally invasive and have a low incidence of side effects. In addition, they are simple, fast, and profitable. The antibacterial effect of PDT, PTT, or synchronic PDT/PTT arises from the generation of reactive oxygen species (ROS) and heat caused by a photoactivated specific photosensitizer (PS) and photothermal agents (PTAs), respectively. The effectiveness of photoinduced treatment depends, among other parameters, on the nature and concentration of the PS/PTAs, light dose, and irradiation wavelength. PS/PTAs based on carbon-based materials (CBMs), such as graphene oxide, reduced graphene oxide, carbon dots, and carbon nanotubes as antibacterial agents, will be discussed in this chapter. These CBMs have emerged as excellent antibacterial alternatives due to their excellent physicochemical properties, biocompatibility, low toxicity in the dark, specificity, and excellent response to light. Moreover, several composites and hybrids employing polymers, metal oxides, and metals have been tested to enhance the antibacterial activity of the CBMs.

**Keywords:** photodynamic therapy, photothermal therapy, carbon-based materials, photosensitizers, photothermal agents

### **1. Introduction**

Food and water for human consumption, medical equipment, lung walls, upper respiratory tract, and external wounds, that accommodate a small number of bacteria, can generate a potential health risk due to their high adaptability and bacterial proliferation [1]. Infections caused by bacterial pathogens have claimed many human and

animal lives, mainly when the development of antibacterial treatments is deficient, for example, the plague pandemic coined as the "black death" in medieval Europe caused by the bacterium *Yersinia pestis*, cholera (*Vibrio cholera*), and tuberculosis (*Mycobacterium tuberculosis*), among others. Likewise, viral pathogens such as HIV and COVID-19 can suppress the immune system, leading to enhanced conditions for coinfection with bacterial pathogens [2, 3]. The shortage of drinking water and medical procedures exposed to bacterial pathogens in the air, or contaminated medical instruments, have become problems of great interest to the world because they are sources that produce bacterial infections that can lead to the death of people, mainly in developing countries [3, 4]. Bacterial infections significantly affect the health of people with cancer, diabetes, and HIV and transplant patients, a high-risk population. Likewise, they considerably affect the wound healing mechanism, reaching the amputation of affected regions or limbs [5, 6].

Antibiotics emerged in the previous era (1940–1980, the "Golden Age" of antibiotics) as an effective treatment for bacterial infectious diseases caused mainly by *Streptococcus pneumoniae* and *Staphylococcus aureus* [3, 6]. Undoubtedly, the general administration of antibiotics revolutionized the treatment of infections caused by pathogenic bacteria, saving countless lives, and they are still considered of great importance in modern bacterial therapies [1, 6]. Antibiotics are a subgroup of antimicrobial agents classified according to their effect, mechanisms of action, and spectrum. Antibiotics are designed to inhibit the growth and multiplication of susceptible bacterial cells selectively, interfering with the synthesis of the bacterial cell wall, protein synthesis, and nucleic acid synthesis, or affecting metabolic pathways [7, 8]. If antibiotic inhibits cell growth and multiplication, they are bacteriostatic, while when they cause internal mechanisms that lead to cell death, they are known as bactericides. However, they present specific mechanisms of action depending on the type of cell membrane of gram-positive or gram-negative bacteria. Likewise, they are broad spectrum when they can eliminate bacteria of both types [8]. Bacteria show the affinity of forming colonies in any solid or liquid substrate (catheters, prosthetics, human body parts, heart valves, and teeth), and proliferation in the presence of nutrients to release exopolysaccharides gives rise to biofilms [3, 6].

Biofilms are associated with the physiological states of bacteria and can be monostrains or multistrains, and there may be synergy or antagonism between the different strains [9, 10]. These biofilms are extremely difficult to eradicate because of the extracellular matrix (exopolysaccharides) that prevents the diffusion of antibiotics in the structure of the biofilm, as well as prevents the free entry and exit of nutrients and waste from bacteria. This situation leads to metabolic reduction (a subcritical condition that activates bacterial survival mechanisms). Therefore, most antibiotics become deficient because they were designed for exponential growth conditions [4, 6, 11].

Biofilms of multidrug-resistant (MR) bacteria are considered a source of infection that generates a high risk of affecting and causing death to people at any stage of life. For this reason, these biofilms are urgent public health problems in the world, charging 10 million human lives per year and costing 100 billion dollars by 2050 in the world economy. Thus, this strengthens the challenge to innovate current antibacterial treatments since the exchange processes of the genetic expression of bacterial pathogens are linked to the food chain, water sources, clinical care, and the environment in general, modifying the virulence of bacterial pathogens [3, 4, 6, 12], as shown in **Figure 1a**.

Searching for methods or treatments to control or eliminate resistant bacteria is not new, but there are limitations to their use in *in vivo* applications, such as selectivity *Antibacterial Strategies: Photodynamic and Photothermal Treatments Based on Carbon-Based… DOI: http://dx.doi.org/10.5772/intechopen.109780*

### **Figure 1.**

*(a) Exchange cycle of the genetic mutation of bacteria. (b) ROS and photothermal effect generation mechanisms for cell death in PDT and PTT. (c) Optical window of melamine, water, hemoglobin, and collagen, depending on the absorption coefficient [13]. Copyright 2022 MDPI. (d) Internalization mechanisms of the PS and PTAs in target cells.*

and activation control [1]. Nanotechnology has presented successful solutions to this problem, such as metal nanoparticles (NPs) [9], metal oxide nanoparticles [10], carbon-based materials (CBM) [11], and nanocomposites [12], as antibacterial agents. In the CBM group, there are single-walled carbon nanotubes (SWCNTs), multiwalled carbon nanotubes (MWCNTs), graphene (G), graphene oxide (GO), reduced graphene oxide (r-GO), carbon dots (CDs), and fullerenes [4, 6]. These nanotechnology solutions have spread into applications such as water treatment [14–17], antimicrobial

textiles [18], antimicrobial food packaging [19, 20], antibacterial coatings for medical instrumentation and equipment [21], bacterial distinguishment [22], regeneration of living tissues [12, 23], photocatalytic disinfection [1, 3, 12, 24], light-induced acidification [1, 25], photodynamic therapy (PDT) to antibacterial applications or antimicrobial PDT (APDT) [26–29], and photothermal treatment (PTT) or photothermal bacterial lysis (PTBL) [1, 30–33].

On the other hand, thermotherapy is a widely applied technique in medical treatments, mainly in the oncology area [34, 35]. It is based on using heat (conduction, convection, or conversion) to tissues (local, regional, or general) to induce damage to its cellular structure, causing death in target cells [3]. It also promotes an increase in blood flow that facilitates the supply of proteins, nutrients, and oxygen at the injury site. The rise of 1°C in the tissue temperature induces improvement between 10 and 15% of the local tissue metabolism [36]. Thermotherapy comprises two categories, that is, hyperthermia and thermal ablation, depending on the range of temperatures in the treatments. Hyperthermia encompasses a temperature range between 41 and 45°C, while thermal ablation encompasses temperatures above 46°C [37–39]. Thermotherapy supplies heat through different energy sources, for example, radio frequency, microwaves, high-intensity ultrasound, light (visible, near-infrared [NIR], and ultraviolet [UV]), and magnetic fields [40], and its name depends on the energy source.

PTT and PDT are antibacterial techniques that are derived from thermotherapy by using a light source (visible, NIR, and UV) to provide heat and reactive oxygen species (ROS) agents, and they differ mainly in the range of temperature and duration of treatment (PTT: > 46°C, 4–6 min; PDT: 41–45°C, 15–60 min) [13, 41], as well as by the mechanisms of action. PPT is based on the use of photothermal agents (PTAs) that produce heat in the presence of electromagnetic radiation, causing the rupture of cell membranes, protein denaturation, and irreversible cell destruction [5, 42]. If metal nanoparticles (NPs) or metal oxide NPs are used as PTAs, an effect known as "localized surface plasmonic resonance" (LSPR) [43] is produced, which allows the temperature of the nanoparticles to increase. When using CBM as PTAs, heating mechanisms are achieved through nonradiative relaxation pathways (internal conversion) (see **Figure 1b**). For this reason, CBM with high absorption and low fluorescence quantum yield will present higher photothermal conversion efficiency [44].

Antibacterial PDT employs three critical components for its application: photosensitizers (PSs), electromagnetic radiation (typically NIR region), and molecular oxygen (O2). The PS absorbs light and donates electrons or energy interchange with surrounding O2, promoting the formation of ROS, inducing irreversible damage to the cell membrane leading to the cell apoptosis or necrosis of the target cell [37]. Two different mechanisms achieve ROS generation as an agent of action in PDT. The first type of ROS is formed by the transfer of electrons between the PS and O2 or substrate, generating oxygen radicals such as superoxide anion ( •<sup>−</sup> O ), hydroxyl radical ( • *HO* ), and hydroperoxyl radical ( • *HOO* ). This is done by transitioning PS molecules from a ground state (S0) to a singlet excited state (S1,2) and the excited triplet state, as shown in **Figure 1b**. In excited triplet states, these PS molecules exchange electrons with a target cell (O2 mainly), producing free radicals that cause oxidative stress and cell death. The second type of mechanism to generate ROS consists of energy transfer between the PS in an excited triplet state and O2, giving rise to singlet oxygen (1 O2), which is more reactive and interacts more with proteins, lipids, and nucleic acids of target cells, in a perimeter of around 20 nm, producing cell apoptosis or necrosis [45, 46].

PPT and PDT are typically used under near-infrared (NIR, 700–950 nm) laser irradiation due to the optical window that this region presents (see **Figure 1c**), in

### *Antibacterial Strategies: Photodynamic and Photothermal Treatments Based on Carbon-Based… DOI: http://dx.doi.org/10.5772/intechopen.109780*

which absorption by hemoglobin (HbO), melanin, and water is reduced, increasing in this way the availability of photons to interact with the PS or PTAs and greater penetration in tissues [13, 42, 47–49]. The treatment effectiveness improves under an incubation time when the PS and PTAs are internalized into the cells by different mechanisms [45] (see **Figure 1d**). The selectivity of this treatment can improve by tuning the PS and PTAs with agents related to the target cells to be treated [41, 50]. Several CBMs, such as CDs, SWCNTs, and MWCNTs, have been used in PDT and PTT applications as PS and PTAs due to their low toxicity, biocompatibility, tunable fluorescence properties, easy functionalization, and antimicrobial activity, which are ideal for *in vivo* applications [5, 7].

### **2. Graphene oxide and reduced-graphene oxide**

Graphene, which is also called the "wonder material," constitutes a revolutionary discovery of the twenty-first century. This CBM has a two-dimensional planar structure like sheets of sp2 -hybridized carbon atoms packed into a hexagonal arrangement [48]. Graphene comprises isolated layers of graphite. Graphene possesses unique and fascinating properties such as large surface area, resistance, impermeability, hardness, lightweight, flexibility, and conductivity, which have encouraged its application in diverse and multidisciplinary fields [49]. Therefore, graphene has been employed in medicine, electronics, aerospace, energy, nanotechnology, and so on [50].

Graphene has been used in anticancer therapy, drug delivery, tissue engineering, and biomedical imaging. Nevertheless, pristine graphene is hydrophobic and relatively expensive to prepare. Thus, two alternatives of graphene derivatives have been proposed due to their better water affinity and the ability for mass production: graphene oxide (GO) and reduced-graphene oxide (r-GO) [51, 52]. GO is obtained by an oxidation procedure of graphite [53], generally carrying out the following routes of synthesis: Brodie method, Staudenmaier method, Tour method, Hofmann method, and Hummers method and its modification [54, 55]. On the other hand, r-GO is prepared by a reduction process of GO, commonly by chemical or thermal procedures [56]. The presence of carbon and oxygen functional groups, such as alkoxy, carboxylic, epoxy, hydroxyl, and carbonyl in the basal planes, and peripheries of these graphene derivatives, promotes a better hydrophilic character and solubility than graphene (see **Figure 2a**) and facilitates biointeractions with molecules like nucleic acids and proteins [58, 59]. Furthermore, these linked molecules determine the oxidative level of GO or r-GO [57, 60].

Regarding the antibacterial action of GO and r-GO, referring to the inhibition of growth and microorganism destruction, it is not only attributed to the photoinduced mechanisms like PDT by the generation of ROS or PTT by the generation of heat, but it is also a consequence of their two-dimensional structures. These CBMs physically kill the bacteria by direct contact with the sharp edge layers (thicknesses of 0.8–1.2 Å) of GO or r-GO and scrape the membrane, causing the rupture of the intracellular matrix and, consequently, the microbe's death [52]. The lateral size of GO and r-GO sheets influences the antibacterial effect. It covers the bacterial pathogen due to the electrostatic interaction between the functional groups of GO, r-GO (basal plane), and the bacterial membrane, thus inhibiting their nutrient absorption and proliferation mechanisms, leading to the death of the bacteria. **Figure 2b** shows AFM images of *S. Aureus* and *Escherichia coli* bacteria free of GO and with GO, showing that GO sheets superficially cover (folds formation) the bacteria; monolayer GO sheets

### **Figure 2.**

*(a) Structure of GO and r-GO. Adapted with permission [57]. Copyright 2019 Dove Medical. (b) Atomic force microscopy (AFM) images of S. aureus and E. coli with and without GO treatment [31]. Copyright 2020 Frontiers.*

with an area > 0.4 μm2 have higher antibacterial activity than GO sheets with an area < 0.2 μm<sup>2</sup> (nanographene oxide [NGO]) [61]. It is important to note that GO and r-GO have intrinsic antibacterial properties; additionally, they attack bacteria by two extra mechanisms: oxidative stress and cell entrapment [62].

### **2.1 Graphene oxide and reduced-graphene oxide in APDT**

The antibacterial capacity of GO has been successfully tested against *S. aureus*, *E. coli*, *Klebsiella pneumoniae*, *Pseudomonas aeruginosa*, *Streptococcus mutans*, *Porphyromonas gingivalis*, *Fusobacterium nucleatum*, and *Pseudomonas putida*, among others [63]. It has been demonstrated that GO only produces singlet oxygen (1 O2, energy transfer) under irradiation [64]; hence, it marginally contributes to the biocide capacity compared to the simultaneous generation of electron–hole pairs. In this context, GO with a lateral size of a few micrometers and a thickness of 1 nm reduced the percentage of bacterial survival to ~24.9% in *E. Coli* under irradiation-simulated sunlight exposure at 380 mW⋅cm−2, causing bacterial death mainly by the generation of <sup>1</sup> O2, to a lesser extent by ROS and intrinsic mechanisms of GO, but with a negligible photothermal effect [65]. The light exposure might reduce GO into r-GO (primarily by electron transfer), forming carbon-centered free radicals, which increase its antibacterial activity. Thus, r-GO performs better as a biocide than GO [63]. Within light-stimulated processes, GO and r-GO are considered ideal materials for the diagnosis and treatment by PDT and PTT because they can be absorbed in the first (650–950 nm) and second (1000–1350 nm) biological windows, where a sufficient tissue penetration of light is attained [31].

To improve the antibacterial capacity of these graphene derivatives, some researchers have proposed its usage along conventional PSs, such as indocyanine green, methylene blue, and toluidine blue [66]. Thus, several works have proved that these composites promoted higher ROS production and an enhanced antibacterial effect compared with the single components. For example, one work used a GO-based composite with indocyanine green to combat *Enterococcus faecalis* (an anaerobic

*Antibacterial Strategies: Photodynamic and Photothermal Treatments Based on Carbon-Based… DOI: http://dx.doi.org/10.5772/intechopen.109780*

gram-positive coccus bacterium). It demonstrated that GO upgraded the photodynamic action of indocyanine green, being 1.3 times more effective in the antibiofilm activity [67]. Likewise, nanoparticles of metals such as Ag have been employed to prepare composites showing excellent results in bacteria elimination [68, 69]. One work used GO along Ag nanoparticles (AgNPs) within *in vivo* subcutaneous tests and proved that after 20 min of irradiation with visible light (600 nm), an antibacterial efficacy of 96 and 99% for *E. coli* and *S. aureus* is obtained, respectively [70].

The NGO (~ 21.3 nm) functionalized with DNA-aptamer (short sequences of artificial DNA) is selective with *P. gingivalis*, reducing their viability in the order of 4.33 Log10 CFU under 980-nm irradiation (1 W for 1 min) and concentration of 1/2 MIC (minimum bacteriostatic concentration, 62.5 nM—obtained without irradiation), as seen in **Figure 3a**. The DNA-aptamer-NGO presents an intrinsic antibacterial activity and increases under irradiation. Their action mechanism reduces bacterial metabolic activity, as observed in **Figure 3b**, which leads to a higher rate of bacterial apoptosis as a function of concentration and irradiation (see **Figure 3c**) [71]. In APDT with GO, there is a dependence between the light dose and the loss of bacterial viability, for example, for the bacteria *E. coli*, its loss of viability is aggravated for 40–60 J⋅cm−2 at higher concentrations. However, a higher light dose is required for lower concentration, which also suggests the dependence on GO concentration (see **Figure 3d**). This behavior is similar in *S. aureus* with GO and NGO. That is, there is a threshold of light dose and concentrations of the PS, where the photons are available to excite GO and generate 1 O2 and the ROS is optimal for causing bacterial death and avoiding affecting healthy tissues.

### **Figure 3.**

*(a) Bacterial viability of P. gingivalis with DNA-aptamer-NGO in APDT [71]. (b) Metabolic activity of P. gingivalis with DNA-aptamer-NGO in APDT [71]. (c) The apoptosis rate of P. gingivalis with DNA-aptamer-NGO in APDT [71]. Copyright 2022 Springer Nature. (d) Loss of viability in E. coli in APDT with GO as a function of light dose at 630 nm [31]. Copyright 2020 Frontiers.*

### **2.2 Graphene oxide and reduced-graphene oxide in antibacterial PTT**

The photothermal effect of GO and NGO in antibacterial PTT is a function of its concentration and size. In NGO, its smaller lateral size allows it to keep its temperature below 60°C (in aqueous solutions). In contrast, the temperature of GO having larger lateral size reaches above 60°C under the same conditions (630 nm, 65.5 mW⋅cm−2). Likewise, the heating efficiency is higher in GO (~1.45) than in NGO (~1.3) for a light dose of 60 J⋅cm−2 (see **Figure 4a**) [31]. This suggests that a larger surface has greater availability of photons for internal conversion. Therefore, GO and NGO are potential PTAs in antibacterial PTT. However, its selectivity can be improved by incorporating functionalizing agents that positively charge GO for better attraction to bacteria. The amino groups (NH2) and polyethylene glycol (PEG) provide a positive charge to GO. Likewise, they can soften the sharp edges of GO, improving its cytotoxicity and biocompatibility but reducing its antimicrobial activity. Even so, nanocomposites, such as GO-PEG-NH2, exhibit excellent antibacterial activity in PTT, as seen in **Figure 4b**. These GO nanocomposites inhibit susceptible bacteria such as *E. coli* and *S. aureus* at 50 μm⋅mL−1 under 808-nm irradiation and 1.5 W⋅cm−2 for 5 min. These nanocomposites partially damage the membrane in the two bacterial strains due to their intrinsic antimicrobial activity. When irradiated, the destruction of the bacterial membrane and a subsequent union of the sample bacteria are produced, as indicated in **Figure 4c** [72].

Furthermore, synergistic mechanisms have been achieved since GO and r-GO also present photothermal effects. One work used amino-functionalized GO and determined that it was easily targeted by electrostatic attraction into gram-negative and gram-positive bacteria surfaces. After the irradiation of 159 mW cm−2, it was

### **Figure 4.**

*(a) Thermal study of NGO and GO in aqueous solution for different irradiation times and concentrations [31]. Copyright 2020 Frontiers. (b) Bacterial survival rate of S. aureus and E. coli in antibacterial PTT using GO-PEG-NH2 as PTAs [72]. (c) SEM images of the damage produced by GO-PEG-NH2 in E. coli and S. aureus bacteria in antibacterial PTT [72]. Copyright 2020 MDPI.*

proved that the temperature increased to 80°C using a concentration of 0.25 mg mL−1 [73]. Using GO and r-GO as PTAs and PS promises improved antimicrobial activity because bacteria are killed by oxidative stress (APDT) and photothermal effect (PTT), producing synergy between both therapies [74].

### **3. Carbon dots**

Carbon dots (CDs) are considered nanospheres of diameter between 1 and 10 nm [54, 56] with carbonaceous nuclei that present sp2 and sp3 domains in crystalline and amorphous structures [52, 60, 75]. CDs are divided into the following four groups: carbon quantum dots (CQDs), graphene quantum dots (GQDs), carbon nanodots (CNDs), and carbon polymer dots (CPDs) (CQDs and GQDs exhibit quantum confinement) [60, 61, 65]. The electrons (lone pairs) of sp2 domains absorb light (visible, NIR, and UV) [1, 57], passing from one energy level (π) to a higher one π → π\*, surpassing the forbidden band. Likewise, the presence of functional groups in its structure (typically: -COOH, -OH, and -NH2) [76–78] allow surface trap states (n) that reduce the bandgap, allowing electrons to absorb light, to reach a higher energy level n → π\*. In this way, the CDs generate photoluminescence (PL) by different mechanisms: a photon emission due to the π-conjugated domains of the nucleus (CQDs and GQDs) and photon emission by their surface trap states and by the state of the molecule [76]. In recent years, CDs have gained significant attention for antibacterial applications [76] due to excellent photoluminescence properties, low toxicity, ease of surface functionalization, chemical stability, dispersion in aqueous media, and low cost [23, 61]. Studies of the antimicrobial activity with *S. aureus* and *E. coli* show that CDs have more permeability toward the bacterial cell membrane than traditional antibiotics [4, 62] due to their nanometric size, and it can be improved by reducing the size of CDs [79, 80]. As shown in **Figure 5a**, more significant numbers of small CDs cross the cell membrane than the larger ones.

A highlighting factor of antimicrobial activity is the surface charge of CDs. It must be positive to generate electrostatic attraction between CDs and teichoic and lipoteichoic acids in the gram-positive bacteria cell membrane, likewise with lipopolysaccharides (LPS) in the gram-negative bacteria cell membrane [81]. The surface charge of CDs is modified by their functionalization with suitable molecules, antibiotics, such as biguanide [80], levofloxacin [47], lysine, and folic acid [82], or antimicrobial nanoagents such as AgNPs [83], which increase the antibacterial activity. CDs (4.5–7 nm in size) passivated with amino, carbonyl, and hydroxyl functional groups can diffuse through the *S. aureus* and *E. coli* bacterial membranes without affecting them and continue until CDs disrupt the double helix of naked bacterial DNA, inhibiting bacterial proliferation [81] or activation of other bacteria-killing mechanism observed in **Figure 5b**. If CDs are functionalized with antibiotics, such as levofloxacin hydrochloride (which inhibits bacterial topoisomerase IV and DNA gyrase), the mechanisms to kill bacteria become more potent than the antibiotic action alone [84–86]. In such a way, the CDs induce ROS generation to damage the bacterial cell membrane partially, and the internalization of levofloxacin hydrochloride is easier, causing cytoplasmic leakage and early death of bacteria [47]. A superior feature of CDs is the low probability of causing bacterial resistance due to their excellent biodegradation (short time for resistant response, no efflux pump) [4, 65], and no known enzyme is capable of inhibiting the ROS as • *HO* and <sup>1</sup> O2 [5].

### **Figure 5.**

*(a) Diffusion of CDs of various sizes in the bacterial wall until reaching the DNA. (b) Mechanisms of bacterial death by generating ROS in the presence of CDs.*

### **3.1 Carbon dots in APDT**

The features of CDs follow the requirements of the new generation of PSs in APDT. CDs achieve inhibition of even MR bacteria such as multidrug-resistant *S. aureus* (MRSA) or multidrug-resistant *Acinetobacter baumannii* (MRAB) at relatively low concentrations (32–64 μg⋅mL−1) of CDs (~ 3 nm, obtained from 2,4-dihydroxybenzoic acid and 6-bromo-2-naphthol by the solvothermal method), irradiated with red light (590 nm) by a 30-mW⋅cm−2 source for 15 min [87, 88]. Likewise, susceptible bacteria, such as *E. coli*, are inhibited at 50 μg⋅mL−1 of Cl-GQDs (~3–5 nm, obtained from

### *Antibacterial Strategies: Photodynamic and Photothermal Treatments Based on Carbon-Based… DOI: http://dx.doi.org/10.5772/intechopen.109780*

sucralose by the electrochemical method), irradiated with sunlight simulated in a 100 mW⋅cm−2 source for 2 hours [89]. However, the negligible cytotoxicity of these PSs makes them even more attractive for *in vivo* applications. The surface charge of CDs is a significant aspect of CDs-bacterial membrane coupling and must guarantee a positive charge. This surface charge depends on the treatment conditions, for example, pH and dispersion medium; *in vitro* or *in vivo* applications modify surface estate. Usually, the surface charge of CDs gives the Z potential [89]. The surface charge is related to the number of functional groups in CDs as PL emissive centers. With a negative or low charge, the antimicrobial activity decreases due to the low or null electrostatic attraction of the CDs-bacterial membrane and the few surface heteroatoms that promote the formation of ROS [5]. The irradiation times, environmental conditions, irradiation source, and estimation of the light dose administered are crucial in the correct development of APDT. However, in various studies, the incubation time is not reported [64, 77, 78, 90–93]. The incubation time refers to the process of internalization or endocytosis of CDs toward the membrane bacteria (see **Figure 1d**). In this sense, Liu

### **Figure 6.**

*(a) Cell viability by CFU number in Bacillus subtilis cells for samples with different concentrations and quantum yield (QY) of PL [94]. Copyright 2017 Royal Society of Chemistry. (b) SEM and TEM images of E. coli bacteria before and after APDT [5]. Copyright 2020 MDPI. (c) TEM image of E. faecium before and after APDT [95]. (d) Inhibition (orange arrows) of E. faecalis bacteria by increasing the irradiation dose in APDT [95]. Copyright 2020 Royal Society of Chemistry.*

et al. [87] extended the antimicrobial activity of red carbon dots (R-CDs) against MRAB and MRSA by increasing the incubation time from 0 to 45 min, achieving bacterial survival rates of 97.7% at 0 min and 3.3% in MRAB and 7.5% in MRSA at 45 min, because CDs can effectively induce the formation of ROS once inside the bacteria.

An exciting aspect of CDs as PSs in APDT is the dependence between the antibacterial activity and its QY; **Figure 6a** shows this effect. The QY refers to the transformation of absorbed and emitted photons in the structure of the CDs. However, it is common to use the quinine sulfate standard that provides adequate information for the interchange of results [96]. **Figure 6a** indicates a relationship between the irradiation time (hours) and the bacterial viability (CFU mL−1) in APDT, as well as a dependence between the bacterial activity (CFU mL−1) and the concentration of CDs (μg⋅mL−1) in the treatment. Bacterial viability reduces as more CDs have free electron pairs that promote π → π\* or n → π\* transitions, which, in turn, induce more ROS, thus evidencing the ROS production mechanisms shown in **Figure 1a**, which is the characteristic of APDT. The doping and functionalization of CDs is an essential aspect of QY and significantly affects the antimicrobial activity in APDT and can induce new bacterial death mechanisms, as is the case of bromine-doped carbon nanodots (Br-CNDs). The Br-CNDs in a change of pH (basic-acid-basic) and darkness conditions induce reactive nitrogen species that generate dark toxicity [97].

SEM/TEM observations are a helpful tool to elucidate bacterial damage and are typically acquired before and after APDT. Together with staining assays and confocal laser scanning microscopy imaging techniques, it is possible to propose mechanisms of cell death [23]. **Figure 6b** shows the damage caused to the *E. coli* bacterial membrane before and after APDT. A change in morphology is evidenced by TEM and bacterial lysis by SEM. Similarly, TEM images in **Figure 6c** reveal damage caused to the cytosol of *Enterococcus faecium* bacteria without affecting the cell membrane after APDT [89]. The dose of light used in APDT is a parameter of significant consideration. **Figure 6d** shows that by increasing the power of the irradiation source (0, 11, and 17 W) in APDT, the antibacterial effect increases until viability is inhibited by the *E. faecium* bacteria [89].

### **3.2 Carbon dots in antibacterial PTT**

In antibacterial PTT, the CDs cause initial damage to the bacterial membrane due to the absorption of photons and their internal conversion that increases their temperature, as indicated in **Figure 1b** and **5b**. CDs bind to the bacterial membrane mainly by electrostatic interactions and transfer heat to bacteria [98]. In this way, the bacterium becomes vulnerable to heat and allows the incoming of CDs that increase cell damage by inducing the ROS. Therefore, it is common for PTT to synergize with APDT to improve the bacteria-killing mechanism. The initial damage caused by the CDs is increased by incorporating an antibiotic or antibacterial agent, such as quaternary ammonium, which increases the damage to the bacterial membrane, allowing a more significant action than the action of the CDs alone [99]. The photothermal effect is effective in gram-positive and gram-negative bacteria due to the heat the CDs provide, affecting their different structures of peptidoglycan, phospholipids, and LPS. Therefore, CDs in antibacterial PTT present properties like those of broad-spectrum antibiotics. However, the antibacterial effect without irradiation may reduce effectiveness in gram-positive bacteria due to multiple layers of peptidoglycan. It is proved using nanohybrids of GQDs-AgNPs at a concentration of 2 μg⋅mL−1 and bacterial strains of *S. aureus* (gram-positive) and *E. coli* (gram-negative). Nevertheless, the

*Antibacterial Strategies: Photodynamic and Photothermal Treatments Based on Carbon-Based… DOI: http://dx.doi.org/10.5772/intechopen.109780*

total inhibition of bacterial strains is achieved by irradiation with red light (808 nm) from a 2-W⋅cm−2 source for 10 min and the same concentration of GQDs-AgNPs [83].

A relevant aspect of antibacterial PTT is the photothermal performance of CDs (conversion of photons into heat). In dispersion in a liquid medium, they act as heat-emitting sources, and depending on the medium to distribute this thermal energy efficiently, it is possible to reach high temperatures that cause damage to healthy tissue. The temperature reached in *in vivo* and *in vitro* applications is a function of photothermal performance, the concentration of CDs, irradiation time, the dispersion medium, and the dose of light supplied. **Figure 7a** shows the temperature dependence on the dispersion of CDs (doped with Fe), concentration, and irradiation time. The main parameter for temperature control is the concentration of CDs. However, in *in vivo* applications, temperature measurements are usually real time to avoid unwanted tissue damage. This procedure also depends on the depth of the treated infection [83]. CDs doped with Fe or Ag nanoparticles (AgNPs) can acquire a behavior like an enzyme peroxidase (POD) [83] interacting with H2O2, increasing its antimicrobial activity (99.85% inhibition *E. coli*) and promoting healing (see **Figure 7b** and **c**).

Antibacterial PTT with CDs allows bone infection treatment through hybrid nanomaterials such as chitosan (CS)-nanohydroxyapatite (nHA) scaffolds doped with CDs (CS-nHA-CDs). CS-nHA-CD scaffolds help as a base material for the new bone tissue with antibacterial features. These antibacterial scaffolds achieve an inhibition (*in vivo*) of up to 97 and 99% for *E. coli* and *S. aureus* bacteria, respectively, under red light irradiation (808 nm, 1 W⋅cm−2) for 10 min. Healthy tissues reduce the dose of light reaching antibacterial scaffolds. Therefore, the temperature control and thermal performance of these materials are essential [23].

### **Figure 7.**

*(a) Thermal study of CDs doped with Fe for different concentrations as a function of irradiation time. (b) The antibacterial activity of Fe-CDs for a strain of E. coli, with an irradiation of 2 W⋅cm−2 at 808 nm, (I) control, (II) Fe-CDs, (III) H2O2, (IV) Fe-CDs + H2O2, (V) NIR, (VI) Fe-CDs + NIR, (VII) H2O2 + NIR, (VIII) Fe-CDs + H2O2 + NIR. (c) Scheme of antibacterial PTT in vivo, with wound healing effect. All the images were obtained from [30]. Copyright 2021 Elsevier.*

### **4. Carbon nanotubes**

CNTs comprise sheets of graphene rolled in the form of a tube, and transverse dimensions are in the nanometric range, but the length is over the nanometric scale. Therefore, CNTs are one-dimensional material. The number of graphene sheets forming CNTs allows the classification of this material into multiwalled carbon nanotubes (MWCNTs) with a diameter of ~10–100 nm and single-wall carbon nanotubes with a diameter of ~0.4–2 nm [100]. CNTs have crystalline sp2 domains with graphene as a precursor. Their physicochemical structure makes them hydrophobic and cytotoxic. CDs are usually functionalized with noncovalent bonds to improve their biocompatibility and solubility in aqueous media [6]. The CNTs present a spontaneous interaction with bacteria and a strong absorbance in the NIR. For this reason, CNTs are suitable photothermal antibacterial agents in PTT [1]. CNTs are not eligible for APDT because they energetically reduce or inhibit singlet oxygen 1 O2 generation (SOG) and have QY below ~1 [101]. However, an appropriate functionalization or formation of a nanocomposite can modify their properties, allowing CNTs to perform as PSs. CNTs act like needles in the bacterial cell membrane, inducing damage according to the surface resistance of each bacterial strain. CNTs also act like a nanochannel once located in the bacterial membrane, the needle effect is more evident in SWCNTs due to reduced diameters, and the channel-like effect stands out in MWCNTs [100].

CNTs can be vertically directionally grown, producing a nanoforest of CNTs suitable to inhibit bacterial biofilms, causing immobilization due to their needle-like effect. The nanoforest of CNTs is a nanostructure like insects' wings (biomimetics) with excellent antimicrobial activity by their tower-like nanostructures. **Figure 8a** shows the antibacterial activity of the nanoforest of MWCNTs in a strain of *Klebsiella oxytoca*.

SWCNTs coupled with surfactants in antibacterial PTT show the inhibition of bacterial strains such as *E. soli* and *E. faecium* with more significant antimicrobial activity in *E. faecium*. The *E. faecium* bacterium is more susceptible to surfactants, allowing a better penetration of the CNTs in the cell membrane [103].

### **4.1 Carbon nanotubes in APDT and antibacterial PTT**

CNTs' functionalizing agents, such as menthol-zinc phthalocyanine (ZnMintPc), zinc monocarboxyphenoxyphthalocyanine (ZnMCPPc), spermine, protoporphyrin IX, or nanocomposites of CNTs with a matrix such as GO and poly (N-vinyl caprolactam-co-poly (ethylene glycol diacrylate)) poly (VCL-co-PEGDA) polymer, significantly improve SOG in antibacterial PDT, generating oxidative damage or alterations in bacterial DNA [12, 27]. The photothermal effect (photons to heat) in MWCNTs is produced by internal conversion, just like GO, because they share the same carbon structure in a hexagonal honeycomb arrangement (see **Figure 8a**). However, magnetic nanoparticles (MNPs) that generate heat by the LSPR effect increase a slightly lower photothermal effect. Thus, MWCNTs (cyan curves) convert photons to heat more efficiently than MNPs (blue curves).

MWCNTs embedded in VCL/PEGDA (hydrogel) and ZnMintPc as PSs form a nanocomposite VCL/PEGDA-MWCNT-ZnMintPc with excellent antibacterial activity (see **Figure 8c**, C1). The complete inhibition of *E. coli* bacteria ascribes to the photothermal effects of MWCNTs (irradiated with a red light at 360 nm in a 65.5-mW⋅cm−2 source) and the generation of ROS by the PS (ZnMintPc). The action mechanism of this nanocomposite consists of cell membrane damage by direct contact (see **Figure 8d**) and oxidative damage. The gram-positive bacteria (*S. aureus*) *Antibacterial Strategies: Photodynamic and Photothermal Treatments Based on Carbon-Based… DOI: http://dx.doi.org/10.5772/intechopen.109780*

### **Figure 8.**

*(a) SEM observations of morphology and K. oxytoca bacteria in a glass substrate and vertical MWCNTs' forest [102]. (b) Evaluation of the photothermal effect of MWCNTs and other molecules of interest as a function of time [12]. (c) Extended plate count method, CFU, of a bacterial strain E. coli under NIR irradiation (630 nm, 65.5 mW⋅cm−2) [12]. Copyright 2016 Royal Society of Chemistry. (d) STEM observations in bacterial strains S. aureus and E. coli before and after APDT-PTT with different nanocompounds (C1, C2) [12]. Copyright 2022 MDPI.*

did not completely inhibit. However, the nanocomposite bacteria coupling mechanism is similar. This nanocomposite exhibits synergistic antimicrobial properties in APDT and antibacterial PTT, identical to a nanocomposite with GO and MNPs (C2 in **Figure 8c**) toward *E. coli* bacteria under the same conditions. The potential application of MWCNTs as PTAs and PS (within antibacterial PTT and APDT, respectively), has the advantage that they present an additional mechanism of coupling-bacterial death by its needle-like effect.

### **5. Perspectives**

Carbon-based materials (CBMs), such as graphene oxide, reduced-graphene oxide, carbon dots, and carbon nanotubes, have promising possibilities as photosensitizers and photothermal agents within photodynamic and photothermal treatments to combat bacteria. These materials have been used as platforms and components to develop complex composites. Thus, to encourage their practical application in medicine, it is necessary to standardize large-scale production by maintaining high quality, reproducible, and uniform morphology and size of these CBMs.

Besides, exploring their killing or inhibition action against other microorganisms, like viruses and fungi, has become a topic of interest. Hence, it is important to continue the research on the toxicity of these materials in human health and the environment.

Antibacterial photodynamic and photothermal therapies have been extensively investigated in susceptible and multidrug-resistant (MR) monostrain bacteria and MR monostrain biofilms. However, MR dual-strain biofilms can proliferate

synergistically under specific conditions studied in recent years. The wide variety of bacterial pathogens and their potential coupling in biofilms sustained new research to understand and combat this warning to health. In addition, the possibility of continuing to find new multistrain bacteria biofilms acting synergistically in hospital substrates and the obsolete antibiotics proves the seriousness of this bacterial risk. Therefore, it is essential to evaluate, propose, and develop proper culture conditions, as well as the new era of antibacterial agents, including CBMs, for their promising antibacterial activity in thermotherapy treatments.

### **6. Conclusion**

This chapter summarizes the recent progress of carbon-based materials (CBMs) as a novel alternative to combat bacteria. The excessive use of antibiotics triggered bacterial resistance that caused severe diseases, even becoming a health risk. Therefore, developing new treatments has become mandatory to overcome this public problem. In this context, several authors have proposed CBMs as antibacterial agents, mainly focusing on their applications within light-assisted treatments as photodynamic and photothermal therapies since they are rapid, affordable, and minimally invasive and have less side effects. The main CBM employed to achieve this aim comprises graphene oxide, reduced-graphene oxide, carbon dots, and carbon nanotubes; nevertheless, the preparation of hybrids and composites has also been proposed to improve their antibacterial effect. Metal nanoparticles, biopolymers, metal oxide nanoparticles, and so on have been employed. We discussed some of the mechanisms whereby bacteria are inhibited or killed. Several works reported in the literature have achieved the complete elimination of bacteria. The most studied species are *E. coli* and *S. aureus*. Hence, this chapter evidences that CBM could be used as a benchmark antimicrobial agent.

### **Acknowledgements**

The authors would like to thank Escuela Politécnica Nacional.

### **Conflict of interest**

The authors declare no conflict of interest.

*Antibacterial Strategies: Photodynamic and Photothermal Treatments Based on Carbon-Based… DOI: http://dx.doi.org/10.5772/intechopen.109780*

### **Author details**

David Giancarlo García Vélez, Karina Janneri Lagos Álvarez and María Paulina Romero Obando\* Materials Department, National Polytechnic School, Quito, Ecuador

\*Address all correspondence to: maria.romerom@epn.edu.ec

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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