Metal nanoparticles and biomaterials: The multipronged approach for potential diabetic wound therapy

2.1 Diabetic wounds

Wounds can be categorized into acute and chronic, depending on the duration of the healing process. If a wound fails to heal after 90 days, it is classified as chronic. Wound healing can be divided into four phases: hemostasis, inflammation, proliferation, and remodeling [13]. When an injury occurs, platelets in the blood will adhere to the injury site and release chemical signals to promote clotting, resulting in the activation of fibrin. Fibrin forms a glue-like material to bind platelets, preventing further bleeding, a process known as hemostasis. Inflammation involves phagocytosis and the release of platelet-derived growth factors (transforming growth factor-beta (TGF-β) and tumor necrosis factor-alpha (TNF-α)) and cytokines such as interleukin 1 alpha (IL-1 α). The release of growth factors and cytokines leads to proliferation, resulting in new tissue formation, angiogenesis, collagen deposition, and epithelization. Remodeling is the last phase of the wound healing process, during which collagen is realigned and excess cells are removed by apoptosis [14,15].

Diabetic wounds are a pathological condition caused by various factors, including mechanical changes in the conformation of the bony architecture of the foot, peripheral neuropathy, and atherosclerotic peripheral arterial disease, all of which occur with higher frequency and intensity in the diabetic population. The risk factors for diabetic ulcers include poorly fitted or poor-quality shoes, poor hygiene (not washing the feet regularly or thoroughly), improper trimming of toenails, alcohol consumption, ocular diseases associated with diabetes, heart disease, kidney disease, obesity, and tobacco use (inhibits blood circulation).

The delayed healing of diabetic wounds is caused by several factors. First, the increased blood sugar level stiffens arteries and narrows blood vessels to restrict the delivery of oxygen and nutrients essential for the natural healing process [6]. Second, patients with diabetes experience decreased or poor blood circulation as these patients are at risk for peripheral arterial disease (PAD), a condition restricting blood flow to the feet and legs. PAD usually affects those with chronic wounds, particularly diabetic foot ulcers. Third, peripheral neuropathy, also known as nerve damage, is caused by a lack of blood circulation. In the extremities, this condition leads to a reduced supply of oxygen and nutrients to tissues and nerves, damaging nerves around the area and reducing sensation to pain, temperature, and touch. Finally, diabetes also weakens the immune system, affecting the body’s ability to send white blood cells to fight bacteria, rendering the wounds more susceptible to infection. Therefore, diabetic wounds commonly develop into infections such as non-healing diabetic foot ulcers [6,16].

S. aureus and P. aeruginosa are the common bacteria infecting diabetic wounds and can form biofilms, a major contributor to the failure of antibiotic treatment [13]. S. aureus is the most common single isolate (76%) in diabetic wounds and foot ulcers, resulting in alterations in wound healing. Moreover, wound infections can result in more serious conditions, including bacteremia or sepsis, causing morbidity and mortality. Over the last 40 years, MRSA infections have become endemic in hospitals in the United States and worldwide. In 2002, the first clinical isolate of vancomycin-resistant S. aureus (VRSA) was found in diabetic foot ulcers [17]. Bacterial infections can lengthen the increased release of proinflammatory cytokines, such as IL-1 and TNF-α, and therefore extend the inflammatory phase. If this persists, the wound may enter a chronic state and fail to heal. Therefore, the optimal management of infections in diabetic wounds is crucial to reduce the rate of morbidity and mortality. In patients with diabetes, the common complication inducing morbidity and mortality is impaired skin wound healing [18] owing to decreased blood flow and nerve damage, both of which are more likely to occur in patients with high blood glucose levels [19].

Furthermore, wound healing is affected by local and systemic factors. Local factors such as oxygenation and infection influence the healing process directly. Oxygen is important in the metabolism of cells as it synthesizes energy and increases angiogenesis needed during the process of wound healing. In contrast, systemic factors concerning the disease state or overall health of patients affect the healing process, including factors such as age, stress levels, hormone production, diabetes, medications, and obesity [13].

3.1 Biomaterials

Biomaterial-based wound dressings are being developed for treating diabetic wounds, mainly to accommodate the need to reduce the inflammatory phase during the healing process. Biomaterials accelerate wound healing by maintaining moisture in the wound microenvironment, facilitating cell proliferation at the wound site [37]. Chitosan, hyaluronic acids (Has), poly(lactic-co-glycolic acid) (PLGA), collagen, and fibrin are several examples of biomaterials that have been developed for diabetic wound therapy to elicit healing through cell-material interactions and/or sustained delivery of drugs or active agents [2].

Integra Dermal Regeneration Template is a commercial biomaterial-based wound dressing initially designed by Yannas and Burke in 1980 [38]. Treatment with Integra involves a two-step procedure, where Integra is applied on the wound site and remains for at least 21–28 days before replacement with a skin graft [39]. Integra is an acellular bilayer matrix consisting of bovine tendon collagen type I and shark chondroitin-6-sulfate glycosaminoglycan, which bind to the silicone pseudo-epidermis. The bioscaffold is constructed to allow the integration of host fibroblasts into the dermal component, bovine collagen type I. When the cells populate the dermal layer of the bioscaffold, the dermal materials degrade as neodermis, leaving the pseudo-epidermal components at the wound site, preventing bacterial infection and acting as a water vapor barrier. In a clinical trial conducted in type I and type II diabetic patients with foot ulcers, the patients treated with Integra healed 5 weeks faster than the control group, with the average wound size reduction per week being 50% faster than the control group [40]. Despite the successful use of biomaterials as wound dressings, the main concern related to bioengineered products still exists, primarily related to a bacterial infection at the wound site. Hence, the addition of antimicrobial agents to biomaterial-based wound dressings such as metal nanoparticles is beneficial for improving their efficacy.

3.2 Metal nanoparticles and their synthesis

Nanoparticles are tiny materials ranging from 1 to 100 nm in size, which can be categorized into different classes based on their properties, shapes, or sizes. Nanoparticles can be divided into carbon-based, ceramic, metal, semiconductor, polymeric, and lipid-based nanoparticles [41]. Furthermore, they are unique owing to their optical and electronic conductivity, which greatly depends on the particle size and shape. They have achieved significant consideration for their potential applications in drug delivery systems and as antimicrobial agents. Nanoparticles, particularly metal nanoparticles, have been extensively evaluated as antimicrobial agents owing to their ability to combat multi-drug-resistant pathogens, including the inhibition of biofilm formation through several mechanisms, mainly attributed to the diversity of the intrinsic, as well as modified chemical composition properties. Moreover, the physicochemical properties of nanoparticles, including particle size and shape, chemical modification, and surface coating, greatly influence their antibacterial activity, rendering them adaptable as antimicrobial agents [42]. Most antibiotic resistance mechanisms are unrelated to nanoparticles as their activity is attributed to direct contact with the bacterial cell wall, without the need for cell penetration. Therefore, nanoparticles are less likely to promote bacterial resistance than antibiotics [43]. Metal nanoparticles such as AgNPs, AuNPs, and ZnO nanoparticles have been widely studied for their antimicrobial activities.

Nanoparticles can be synthesized via physical and chemical methods. Evaporation-condensation and laser ablation are the most common physical methods [44]. ZnO, as small as 30 nm, can be obtained by physical methods such as high-energy ball milling. The milling time is one parameter influencing nanoparticle size, shape, and antibacterial activity [45]. Despite the advantages of physical methods such as the absence of solvent contamination and uniform nanoparticles produced, factors such as high energy, time consumption, and heat generation remain limitations. Furthermore, physical methods may require a large space, such as the synthesis of AgNPs using a tube furnace at atmospheric pressure [44].

For the chemical synthesis of metal nanoparticles, common reducing agents include sodium citrate (a well-known method developed by Turkevich and French), sodium borohydride [46,47], ascorbate, Tollens reagent, N,N-dimethylformamide (DMF), and poly(ethylene glycol)-block copolymers [44]. Notably, chemical methods are the best methods to obtain a small size (<100 nm) and different nanoparticle shapes, by varying the molar concentration of the reactant, dispersant, and feed rate of the reactant. Furthermore, the selection of appropriate reducing agents is a crucial factor because the size, shape, and size distribution of particles are strongly dependent on the nature of the reducing agent [48]. AgNPs synthesized using a water-soluble polymer, poly(acrylic acid) (PAA), formed stable AgNPs. Polyacrylate anions (PA⁻) obtained from PAA with uncoordinated carboxylate groups bind with metallic cations (Ag⁺) to form an intermediate charged cluster and AgNPs [49]. Although markedly small sizes of AgNPs can be obtained using sodium citrate (42–58 nm), the nanoparticles tend to agglomerate owing to instability [50]. Similarly, extremely fine AuNPs (4–13 nm) could be obtained by reducing gold ions using trisodium citrate and sodium borohydride, but poor nanoparticle stability was reported [51]. A similar aggregation issue was reported with titanium nanoparticles synthesized using sodium borohydride [52]. Therefore, the selection of the reducing agent is important for the chemical synthesis of metal nanoparticles, in addition to specific catalysts and regulated reaction environments, including appropriate temperature, pressure, and pH.

Chemical reducing agents are generally expensive, limiting their application. Chemically synthesized metal nanoparticles may contribute to the harmful effects of chemical residues adhered to the nanoparticles. For example, the chemical synthesis of ZnO using hydrazine, a highly reactive alkali, and reducing agent requires protective equipment to avoid skin burns, severe damage to the respiratory tract, and death due to overexposure [53]. Therefore, synthesizing nanoparticles using green or biological methods remain preferable as the use of toxic chemicals can be avoided [54,55]. Green synthesis (biosynthesis) of metal nanoparticles uses natural sources as reducing agents, including microorganisms [56], fungi [57], plant extracts, and marine-derived compounds [58]. The biosynthesis of metal nanoparticles is shown in Figure 2.

Figure 2

Mechanism of green synthesis (biosynthesis) of metal nanoparticles. Metal ions such as Ag⁺ and Au³⁺ are reduced into metal nuclei/atoms (Ag⁰ and Au⁰) using extracts of natural sources such as plants and microorganisms. The metal nuclei/atoms are then accumulated or growing, and therefore, stabilizing/capping agents (e.g., chitosan) are added to produce stabilized metal nanoparticles with small particle sizes. Various shapes of metal nanoparticles can be produced by controlling parameters during the synthesis such as concentration of metal ions and reducing agents as well as pH and temperature.

Reducing agents from different sources present certain advantages and limitations. For example, microbes require more complex procedures (e.g., elaborate purification process), and bacterial adherence on the particle surfaces may increase the potential danger to the environment, as well as human health [59]. The plant-mediated synthesis of nanoparticles demonstrates more advantages when compared with other sources owing to its suitability for large-scale production and cost-effectiveness.

3.3 Applications of metal nanoparticles in diabetic wound therapy

The escalation of microbial resistance has resulted in extensive research on metal nanoparticles as potential antibacterial agents owing to their proven effectiveness in inhibiting and killing bacteria. Compared with eukaryotes, prokaryotes are known to be more susceptible to the toxic effects of silver. Higher silver concentrations are required to kill eukaryotes owing to the larger cell size, as well as the redundancy of structure and function within the eukaryotic cell [60]. For promoting wound healing, metal nanoparticles have been tested alone or in combination with other materials, as a strategy to enhance antibacterial activity. Similar to antibiotics, bacteria may develop resistance toward metal nanoparticles via several mechanisms: (1) reduction of Ag⁺ to its neutral oxidation state with lower toxicity and (2) active Ag⁺ efflux from the cell by P-type adenosine triphosphatases or chemiosmotic Ag⁺/H⁺ antiporters [60]. Rapidly evolving resistance toward citrate-coated AgNPs was reported for Escherichia coli [61], while Bacillus subtilis developed resistance after prolonged pre-exposure to metallic nanosilver [62]. These findings have prompted scientists to combine metal nanoparticles with other antimicrobial agents to overcome resistance and enhance their activity in killing microorganisms.

4.1 Relationship between physicochemical characteristics and biological properties of metal nanoparticles

Reportedly, several factors are known to affect the biological properties of nanoparticles, including their antibacterial activity. These factors include the type of synthesis, particle size, shape, and surface charge. All these factors play vital roles in determining the effectiveness of metal nanoparticles as antibacterial agents in wound healing applications. In addition to nanoparticle properties, their behavior in biological systems is further influenced by the shape, size, and surface charge of the nanostructure. Moreover, factors related to the synthesis of nanoparticles, such as type, process parameters, and metal salts, have been shown to affect the size, shape, and morphology of nanomaterials [86].

Physical and chemical synthesis are more labor-intensive and hazardous than the biological synthesis of metal nanoparticles. Biological synthesis or biosynthesis is more attractive as it offers advantages such as high yield, solubility, and stability [87]. Therefore, the type of synthesis can indirectly affect the antibacterial properties of metal nanoparticles [88]. Several studies have shown the higher antibacterial activity of biosynthesized AgNPs than the chemically synthesized AgNPs. AgNPs biosynthesized using a guava leaf extract demonstrated significantly higher antibacterial activity, as well as stability against E. coli, when compared with the chemically synthesized counterpart, possibly owing to the adsorption of biomolecules on the surface of biosynthesized AgNPs. Similar findings were reported with biosynthesized ZnO nanoparticles using C. halicacabum leaf extract at various concentrations (0.2, 0.4, and 0.6 mL); higher antibacterial activity was observed than that with chemically synthesized ZnO nanoparticles [89]. Additionally, different methods of synthesizing metal nanoparticles impacted the size of the produced particles. The particle size of ZnO nanoparticles biosynthesized using different concentrations of C. halicacabum (0.2, 0.4, and 0.6 mL) was in the range of 48–62 nm with low zeta potential values. The particles were relatively smaller than those synthesized chemically using zinc nitrate and potassium hydroxide with the size of 65 nm [89]. In contrast, smaller particle sizes were obtained with chemically synthesized AuNPs using sodium citrate than those biologically synthesized using L. rhinocerotis extracts, locally known as Tiger Milk Mushroom [90]. Diverse findings have demonstrated that several factors contribute to the physicochemical and biological properties of the final products, presenting a major challenge to the manufacture of metal nanoparticles, particularly for clinical use.

Monodisperse nanoparticles are an ideal characteristic for pharmaceutical and medical applications. Smaller sizes of metal nanoparticles are also desirable as they exhibit higher antibacterial activity than larger ones owing to their higher particle surface area, leading to better contact between particles and bacterial cells. Markedly small AgNPs (10–15 nm) were obtained by synthesizing them in an agar hydrogel matrix with the addition of fumaric acid. The hydrogel showed antibacterial effects against common bacterial strains causing wound infections, including E. coli, S. aureus, and P. aeruginosa [91]. However, smaller-sized particles can be more toxic, presenting another challenge for scientists to develop formulations or dressings selective to bacterial cells only, attempting to prevent adverse effects on human cells [88]. Additionally, particle shape plays an important role in the effectiveness of metal nanoparticles in killing bacteria or inhibiting bacterial growth. Metal nanoparticles with flower-shaped particles killed S. aureus more efficiently than star-shaped and spherical particles because of sharp edges presented on their surfaces, piercing and rupturing the bacterial membrane [64,92].

Chemical and/or biological coatings on the surface of metal nanoparticles could also be attributed to the cytotoxic effect. Biopolymers are commonly used to cap and stabilize metal nanoparticles. Biopolymers are polymeric biomolecules consisting of monomeric units covalently bonded to form larger molecules. The prefix “bio” refers to polymers that are synthesized from living organisms and are biodegradable [93]. For example, chitosan [94], gum arabic [95], and soluble starch [96] are used as reducing and stabilizing agents for the preparation of metal nanoparticles [97] to improve the stability and biocompatibility of metal nanoparticles [98].

4.2 Toxicity of metal nanoparticles

Despite the benefits of metal nanoparticles in wound healing, concerns regarding potential toxicity still arise, especially for long-term use. The toxicity of metal nanoparticles is influenced by numerous factors, including nanoparticle concentration, size, surface charge, surface coating, solubility, surface functionalization, distribution, mode of entry and action, growth media, exposure time, and cell type [85,88,99]. Silver dressings directly in contact with the damaged skin (a breached dermal barrier) are prone to systemic absorption, inducing toxicity [100].

In humans, a lethal dose of silver nitrate is approximately 10 g [101], and although different toxicity levels of AgNPs have been reported, the effects depend on the cell type as well as the particle size. AgNPs reportedly kill mammalian cells at concentrations as low as 2–5 µg/mL [102]. The cytotoxicity of AgNPs in human skin fibroblasts is significantly influenced by the size of nanoparticles and exposure time, with a smaller size and longer duration of exposure resulting in higher cytotoxicity [103]. Furthermore, the effect was dose-dependent, as a study showed that at the AgNP concentration range of 1–100 µM, the cytotoxicity was insignificant; at 200–300 µM, the effect gradually increased and became toxic at 1 and 2 µM after human dermal fibroblasts were exposed to AgNPs for 72 h [104]. The mechanism of AgNP cytotoxicity is still not fully understood. However, AgNP cytotoxicity is associated with the induction of mitochondrial dysfunction and the generation of ROS [105]. Smaller AgNPs generated more ROS than larger particles, killing more macrophages [106] and demonstrating the higher toxicity of smaller nanoparticles.

In general, AuNPs are less toxic than AgNPs. A study compared the cytotoxicity of AuNPs and AgNPs of the same particle size in human dermal fibroblasts. The cell proliferation rate with AuNPs was more than 90%, with no cytotoxicity observed. However, the cell proliferation rate decreased to 69% with AgNPS, with noted cytotoxicity [107]. Despite the lower toxicity of AuNPs, their cytotoxicity is dose-dependent, with lower concentrations (5 ppm) stimulating keratinocyte proliferation, but higher concentrations (>10 ppm) demonstrating toxic effects [107]. Furthermore, the particle size of AuNPs affected the secretion of proinflammatory cytokines and antibody production. Larger AuNPs (40 nm) stimulated high levels of proinflammatory cytokines, including IL-6, IL-12, and TNF-α, while smaller AuNPs (20 nm) failed to demonstrate any cytokine production. Furthermore, shape and surface charge may affect the cytotoxicity of metal nanoparticles. In vitro, rod-shaped AuNPs were found to be more toxic to culture cells when compared with spherical ones [108]. Conversely, the IC₅₀ of negatively charged AuNPs was >7.37 and 72 μmol/L for monkey fibroblasts (Cos-1) and red blood cells, respectively, while the IC₅₀ for positively charged AuNPs was 1 μmol/L for both cell lines, indicating the higher toxicity of positively charged AuNPs. The higher toxicity of positively charged AuNPs was associated with their ability to interact with the negatively charged cellular membrane. In another study, both charges of AuNPs were toxic to HaCaT (human keratinocytes) cell lines at a concentration of 10 μg/mL [85], demonstrating that the effect is multi-factorial. Therefore, cytotoxicity testing should be conducted for each type of metal nanoparticle, on all the cell types to be applied clinically. A rapid approach without the use of any toxic chemicals such as the application of microwave irradiation in biosynthesizing metal nanoparticles could be a good strategy to minimize cytotoxicity of the particles. Furthermore, the combination of metal nanoparticles with certain biomaterials can be strategically utilized to reduce cytotoxicity.