Inorganic nanomaterials do not contain either carbon or hydrogen atoms that are associated with biological matter. As an alternative, inorganic nanomaterials comprise metallic and non-metallic elemental compounds that have weak intermolecular interactions which form nanostructures with higher dimensionality. Among two classes of the inorganic nanomaterials, metallic inorganic nanomaterials (Ag, Au, Zn, Cu, Bi) have attracted significant attention over non-metallic nanomaterials (S, Si, B, Te and Se). This is particularly due to the inherent water insolubility of the non-metallic inorganic nanomaterials that restricted their use in antibacterial applications. Therefore, this review primarily focuses on the development of inorganic metallic nanomaterials in antibacterial applications. Other inorganic nanomaterials such as fluoride in oral treatment has also been researched for a long time. For instance, in the prevention of dental caries, the addition of fluoride has a significant antibacterial effect on *Streptococcus mutans*, *Lactobacillus acidophilus*, *E. faecalis*, *Actinomyces naeslundii,* and *Parvimonas micra* [163–166].

Inorganic metallic nanomaterials do not readily self-assemble into 1D nanowires, nanotubes, nanoribbons, and 2D nanowalls and nanofilms [167]. Therefore, complicated synthesis methodologies are required to promote the "growth" of inorganic metallic nanomaterials into nanostructures with higher dimensionality [168,169]. Zero-dimensional metal and metal oxide nanoparticles are the most popular candidates that can readily be synthesized for antibacterial applications, which are discussed in Sections 3.1 and 3.2 below.

#### *3.1. Metal Nanoparticles*

Metal nanoparticles are the most promising candidate in this class of materials with inherently strong antibacterial activities amongst the nanomaterials. A summary of the possible bactericidal effects of metal nanoparticles on different bacteria is shown in Table 3. Researchers reviewed a variety of metal nanoparticles including silver, gold, copper, zinc, and super-paramagnetic iron which demonstrated promising antibacterial effects, with silver nanoparticles (AgNPs) being the most effective against bacteria [169–172]. AgNPs exhibit bactericidal activity at concentrations well below their cytotoxicity and exhibit synergistic antibacterial efficacy with conventional antibiotics when used against MDR bacteria [173–176].

The antibacterial mechanisms of AgNPs are still poorly understood despite extensive studies [171,176]. Currently accepted antibacterial mechanisms include cell wall penetration and membrane damage, toxicity associated with metal ion release, and induction of oxidative stress [177–180]. AgNPs have already been used in various biomedical and antibacterial applications and products, including surface coatings on medical devices, topical treatments, wound dressings, dental fillings, personal care products with sanitizing effects, disinfectants, and detergents [180,181]. A recent study demonstrated the potential of AgNP-containing disinfectants as active ingredients for disinfecting surgical masks, effectively improving mask protection by inhibiting the growth of *E. coli*, *K. pneumoniae*,

and *S. aureus* [23]. Over the past few decades, the AgNP market has been growing steadily, with an estimated annual production of more than 500 tons of nanoparticles, which also reflects the widespread interest of AgNPs [182].

Despite the promising antibacterial effects and the wide use of metal nanoparticles in different applications, metal nanoparticles suffer from several drawbacks. The potential toxicity of metal nanoparticles affects the basic functioning of mammalian cells as metal nanoparticles or released metal ions via direct uptake from mammalian cells [24]. Colloidal metal nanoparticles tend to aggregate over time [25]. The aggregation along with increased particle size reduces their peculiar properties at the nanoscale, including their antibacterial activities. Bacteria have the ability to develop resistance to metal nanoparticles by using adhesive flagellin [183]. Phenotypic changes of adhesive flagellin production triggers the aggregation of metal nanoparticles, thereby reducing their antibacterial activity. Metal nanoparticles are potential environmental hazards and difficult to recover or deactivate in solid-waste incineration plants or wastewater treatment systems [184–186]. To overcome the above limitations, metal nanoparticles could be incorporated into other nanomaterials to form nanocomposites, which show a greater dispersion of metal nanoparticles, improved antibacterial activity, and reduced toxicity [187–189]. This will be further discussed in Section 4 below.

**Table 3.** Antibacterial activity of metal nanoparticles against different bacteria.

Abbreviations: NPs, nanoparticles; MDR, multidrug resistant; MRSA, methicillin-resistant *Staphylococcus aureus*.

#### *3.2. Metal Oxide Nanoparticles*

Metal oxide nanoparticles offer another alternative promising solution against MDR bacteria. A variety of metal oxide nanoparticles including titanium dioxide, zinc oxide, magnesium oxide, copper oxide, and aluminium oxide have been demonstrated to exhibit antibacterial effects [215], which will not be discussed in detail here. Zinc oxide (ZnO) nanoparticles are the most-established candidate of these metal oxide nanoparticles due to the following reasons: ZnO is one of the most important metal oxide nanoparticles with widespread applications [26] and worldwide production is up to 1 million tons per year [216]. ZnO can be easily biodegraded and absorbed in the body and has been listed as a Generally Recognized as Safe (GRAS) material by the FDA. ZnO nanoparticles have a higher biocompatibility and lower toxicity than other metal oxide nanoparticles [217,218]. The semiconductor properties of ZnO nanoparticles with a wide band-gap energy readily absorb ultraviolet (UV) light. This allows them to act as potential photosensitizing agents for various antibacterial applications [27]. ZnO nanoparticles exhibit multiple antibacterial mechanisms, as described by Figure 6. Current postulated mechanisms include photo-triggered production of ROS [219] and Zn2+ ions mediating a poisoning effect [220]. In a study by Azam et al., the potential of ZnO as an antibacterial agent was demonstrated against Gram-positive bacteria (*B. subtilis*, *S. aureus*) and Gram-negative bacteria (*P. aeruginosa*, *Campylobacter jejuni*, *E. coli*) [213]. In another study, ZnO nanoparticles were also shown to be significantly inhibitory against Gram-positive (*S. aureus*) and Gram-negative (*E. coli* and *P. aeruginosa*) strains [221].

**Figure 6.** Multiple antibacterial mechanisms that are associated with ZnO nanoparticles. (Adapted with permission from Ref. [222]. 2015, Sirelkhatim et al. More details on "Copyright and Licensing" are available via the following link: https://www.mdpi.com/ethics#10 (accessed on 10 October 2022)).

The aggregation of metal oxide nanoparticles over time remains a problem, which limits their use in vivo applications [223]. One approach to this problem is to disperse metal oxide nanoparticles into a polymer matrix, forming polymer–metal oxide nanocomposites. Polymer–metal oxide nanocomposites have been widely investigated and applied in the textile and polymer industries for various antibacterial applications, which will be discussed in the following section. [224].

#### **4. Nanocomposite/Nanohybrid Antibacterial Materials**

Nanocomposites or nanohybrids are a novel class of multiphase materials that exhibit a hierarchical structure, where one phase of the material has at least one dimension in the nanometer range [225]. They have attracted significant attention due to their unprecedented properties compared with their mono-constituent parts, largely attributed to strong reinforcing effects of additional materials. Currently, synthesizing a variety of nanocomposites with unprecedented physical properties and enhanced antibacterial activities is the main focus in the field over the last 10 years. Despite the large volume of studies on nanocomposites, the understanding of the structure–property–activity changes remains in its infancy [226]. It is important to understand the mechanisms behind the property's changes within nanocomposites in order to design materials with enhanced improvements in the desired properties for a specific purpose. Sections 4.1–4.5 discusses the development and potential applications of the nanocomposite antibacterial materials. Of note, graphene oxide is especially emphasized in the section below due to its versatility to form nanocomposites with organic and inorganic metallic nanomaterials, respectively.

#### *4.1. Polymer–Metal Nanocomposite Nanoparticles*

Polymer–metal composite nanoparticles are another promising solution to achieve a greater dispersion of metal nanoparticles and prevent metal nanoparticle aggregation. Polymer–metal composite nanoparticles comprise a metal nanoparticle core surrounded by a polymer shell with the alkyl tail arranged toward the surrounding environment. Polymer– metal composites are essentially insoluble in water and the colloidal stability of the polymer– metal composite nanoparticles in aqueous environments has a huge potential for in vivo antibacterial therapies [227]. The polymer macromolecular matrix acts as a reaction chamber for metal nanoparticle synthesis, a capping agent to prevent nanoparticle aggregation and as a scaffold for nanoparticle immobilization [228]. Moreover, synergies between the polymer and the metal nanoparticles confer the nanocomposite with unprecedented performance and improved antibacterial properties [229]. Tamayo et al. summarized the synthesis, properties, and recent applications of polymer composites with metal nanoparticles [230].

The incorporated metal nanoparticles are focused on the use of gold (AuNPs) and silver (AgNPs) due to their antimicrobial properties, catalytic activity, and conductivity properties enabling a wide range of applications.

#### 4.1.1. Development of Synthesis Approaches for Polymer–Metal Nanocomposites

Both in situ and ex situ approaches can be employed to synthesize polymer–metal composite nanoparticles and polymer-matrix metal nanocomposites. In the last decade, several studies have been conducted to develop and improve synthetic methods at higher efficiencies resulting in improved antimicrobial outcomes.

By using in situ methods, the precursor of the nanoparticle is required to be dispersed in a monomeric solution before polymerization. The metal ions can be reduced into the polymer matrix or simultaneous metal ion reduction and polymerization can occur [230]. In general, these in situ reduction methods need a relatively long time to produce nanocomposite films. Kazuhiko et al. developed a rapid and scalable synthetic method exploiting use of a mid-infrared laser, CO2 laser, at 10.6 μm without the use of reducing agents [231]. The polymer film (polyvinyl alcohol (PVA) or polyethylene glycol (PEG)) containing Ag ions were coated on a glass substrate and then the CO2 laser was used to heat the substrate. Subsequently, the thermal energy was absorbed by the polymer film, causing Ag ion reduction. Eventually, Ag-PVA or Ag-PEG nanocomposite films were formed in several seconds. This process is industrially scalable by increasing the power of the CO2 laser.

Ultrasound is a promising tool to be applied in for the in situ production of polymer– metal nanocomposites. Ultrasound radiation was employed as a homogenizing tool to fabricate composites with homogeneously dispersed metal nanoparticles [232]. The ultrasound was used to disperse organic liquids of polymerizing monomer (pyrrole) in the aqueous solution of the oxidizer (Ag+ or AuCl−). The aqueous solution was placed in an ultrasonic chamber and droplets of the organic solution were added continuously until achieving a volume ratio of 4:1. After polymerization, the nanocomposites could be obtained at the liquid–liquid interface [232]. Wan et al. used ultrasound as both an initiating and reducing agent in the nanocomposite preparation process, shown in Figure 7 [233]. Tertiary amine-containing polymeric nanoparticles were produced by ultrasound-initiated polymerization-induced self-assembly (sono-PISA), following which, the metal ions (Au and Pd) were reduced in situ by radicals generated via the sonolysis of water, forming polymer–metal composites.

**Figure 7.** Synthesis of tertiary amine-containing polymeric nanoparticles, and in situ formation of the Au and Pd nanocomposite by ultrasound. (Adapted with permission from Ref. [233]. 2021, Wan et al. More details on "Copyright and Licensing" are available via the following link: https: //www.mdpi.com/ethics#10 (accessed on 10 October 2022).)

In contrast, metal nanoparticles are synthesized before they are incorporated into the polymer via an ex situ method. The subsequent deposition of the nanoparticles into the polymer can exploit processes such as melt compounding or solution blending [230]. However, a significant drawback exists when using ex situ methods, which is the fact that the nanoparticles are not optimally distributed in the polymer.

4.1.2. Synergistic or Combined Antibacterial Effects When Using More Than Just a Metal Nanoparticle Agent

A combination of metal nanoparticles and cationic polymers also facilitates the enhanced antibacterial activity of a composite nanoparticle, possibly due to the synergism between the antibacterial mechanisms from two different nanomaterials [234–237]. Nanoparticle formation was also expected to increase the local density of cationic polymer, leading to stronger binding on the negatively charged bacterial membranes [234]. This polyvalent interaction between cationic polymers and bacterial membranes is followed by the synergistic antibacterial mechanisms, including bacterial membrane disruption, internalization of composite nanoparticles, inhibition of intracellular enzymatic activity, and eventual cell death [234]. Imidazole-capped chitosan–gold nanocomposites exhibited enhanced antimicrobial activity to eradicate staphylococcal biofilms in a rabbit wound infection model [238]. The antibacterial mechanism of the composite nanoparticles also involved the binding of cationic polymer to the bacterial surface, and the subsequent synergistic effects from the gold nanoparticles, imidazole, and the chitosan polymer to strongly eradicate the biofilm [238].

Polymer–metal composite nanoparticles could potentially solve the environmental hazards associated with many metal nanoparticles. Richter et al. postulated that a metallic core is not necessary for the antimicrobial action [187]. Instead of synthesizing the entire nanoparticle of metal, the composite nanoparticle can be produced by infusion with a minimum amount of silver ions to the biodegradable lignin core, followed by surface functionalization with a layer of cationic polyelectrolyte [187]. The resulting composite nanoparticles exhibited broad-spectrum bactericidal activity against *E. coli*, *P. aeruginosa,* and a quaternary-amine-resistant *Ralstonia*. This is attributed to the enhanced binding to bacterial membranes by the polyelectrolyte shell and the synergistic antibacterial activity between the silver ions and the polyelectrolyte. The required silver ions were ten times lesser than when using conventional silver nanoparticles. The gradual diffusion of silver ions from the silver-infused lignin core composite nanoparticles into water will rapidly lose their post-utilization activity and be biodegradable in the environment after disposal [187].

#### 4.1.3. On the Potential Clinical Use of Antibacterial Polymer-Matrix Metal Nanocomposites

Biocompatible and safe antibacterial materials are constantly sought to avoid inflammatory syndromes in patients. The formulation of polymer–metal composite nanoparticles generally improves their biocompatibility and reduces the toxicity associated with metal nanoparticles owing to protection by the polymeric shell. For instance, the viability of NIH3T3 cells was not affected at a dosage exceeding 20 times that of the minimum inhibitory concentration (MIC) of the polymer–silver composite nanoparticle, showing the low toxicity of these materials to mammalian cells [234]. Lu et al. further demonstrated that imidazole-capped chitosan–gold nanocomposites did not display hemolytic activity and significant toxicity towards L929 cell line [238]. Pryjmaková et al. modified the surface of polyethylene naphthalate (PEN) by a 248 nm KrF excimer laser and subsequently, Ag and Au nanowires were incorporated onto the modified PEN surface by vacuum evaporation [239]. The resulted nanocomposites displayed antibacterial effects against Gram-negative bacteria (*E. coli*) and Gram-positive bacteria (*S. epidermidis*) via a 24-hr incubation drop plate test and were suggested as a non-toxic material by a WST-1 cytotoxicity test. In addition to the complete eradication of the biofilm, accelerated wound healing by a composite nanoparticle was demonstrated in a rabbit model [238]. These studies

demonstrate the great potential of composite nanoparticles as novel antibacterial agents against bacterial infections.

#### *4.2. Polymer-Matrix Metal Oxide Nanocomposites*

Fine dispersions of metal oxide nanoparticles can be achieved by forming polymermatrix metal oxide nanocomposites in a manufacturing process similar to polymer-matrix metal nanocomposites. There is an increasing interest in using metal oxide nanoparticles to replace metal nanoparticles for synthesizing polymer-matrix metal oxide nanocomposites [240–243]. Metal oxide nanoparticles, especially zinc oxide nanoparticles, are more desirable than metal nanoparticles as nanofillers in forming polymer-matrix nanocomposites [244–247]. Zinc oxide nanoparticles feature new UV-absorption and photosensitizer characteristics, with higher biocompatibilities and lower toxicities than metal nanoparticles [218,248]. Furthermore, the low cost of production and high stability of zinc oxide nanoparticles present advantages over conventional metal nanoparticles, even in extreme synthesis conditions [249]. The advantages of metal oxide nanoparticles as nanofillers in forming polymer-matrix metal oxide nanocomposites has open a new avenue for research into novel bio-nanocomposites for use as antimicrobial surfaces in various antibacterial applications, which will be depicted in following sections.

Polymer-matrix metal nanocomposites exhibit several advantages. Polymer flexibility allows the final product to be fabricated into complex structures or forms for various antibacterial applications, of which the details are shown in Table 4. The polymeric matrix immobilizes metal nanoparticles and prevents their aggregation, thus extending the antibacterial activity of the metal nanoparticles [228]. Localized release of metal nanoparticles to the desired application site can also be achieved, reducing in vivo toxicity and the environmental hazards caused by undesirable release of metal nanoparticles [250]. Synergistic antibacterial activity between metal nanoparticles and polymers is obtained with inherent antibacterial activities [229]. The strong interfacial binding and intermolecular interactions between the well dispersed metal nanoparticles and the polymer matrix further enhance the mechanical properties of the polymer, including the tensile strength, Young's modulus, yield stress, and ductility [251].

**Table 4.** Versatility of polymer-matrix metal nanocomposites for various antibacterial applications.

4.2.1. Development of Synthesis Approaches for the Industrial Production of Polymer-Matrix Metal Nanocomposites

A prerequisite to the aforementioned advantages exhibited by polymer-matrix metal nanocomposites is the formation of homogenous dispersions of metal nanoparticles in the polymer matrix without metal nanoparticle aggregation [262]. The delicate synthesis conditions to meet this prerequisite is often time-consuming, laborious, and difficult to be industrialized [263]. Therefore, recent studies have focused on developing facile and convenient synthesis approaches, aiming to produce polymer-matrix metal nanocomposites on industrial scales [228,250,264]. Other recent review papers have summarized developments, so we will not discuss these in detail [240–242].

For instance, Tran et al., developed a simple one-pot synthesis method in producing polymer matrix silver nanocomposites [228]. The ionic liquid medium, butylmethylimmidazolium chloride, was utilized as the only reaction medium for dissolving the biopolymer keratin and cellulose, and reduction of a silver ion precursor in the polymeric matrix. The synthesized polymer-matrix silver nanocomposite was found to retain the enhanced mechanical strength by cellulose and controlled release of silver nanoparticles by keratin, with a homogenous dispersion of silver nanoparticles. At 0.48 mmol of silver content, the nanocomposite demonstrated good biocompatibility and excellent antibacterial activity against *E. coli*, *P. aeruginosa*, MRSA, and vancomycin-resistant *E. faecalis* (VRE). An in vitro release assay demonstrated that less than 0.02% of the silver nanoparticles were released from the nanocomposite even after 7 days of soaking in solution, indicating good immobilization of silver nanoparticles using this simple one-pot synthesis method [228].

A scalable approach was recently developed to produce a silver-nanoparticles-doped nanoclay–polylactic acid composite nanocomposite, which involved doped nanoclay with minimal alteration to the fabrication processes and industry standard equipment [264]. Loading the nanoclay can significantly reduce the affinity of the nanocomposites for bacterial adhesion. With this synthesis method, only a 0.1 wt % of silver loading content was required to have satisfactory antibacterial activity. *S. aureus* and *E. coli* numbers were reduced by 91.3% and 90.7% after 48 h of incubation. The material costs associated with the silver-loading content is dramatically reduced compared with other studies which utilize at least 1 wt % of silver nanoparticles in the polymer-matrix metal nanocomposite to achieve a 90% reduction in bacterial numbers [265,266]. This is a great advantage for industrial production, in which the high costs associated with higher loading amounts of metal nanoparticles is a considerable problem. In addition, 3D printing is a promising method to produce metal oxide nanocomposites, with the advantages of keeping the integrity and functionality of the materials and reduce waste from traditional manufacturing methods [267].

4.2.2. The Application of Polymer-Matrix Metal Oxide Nanocomposites as Self-Sterilizing Antimicrobial Surfaces in Healthcare Environments

There are some recent literature reviews on the application of polymer-matrix metal oxide nanocomposites as self-sterilizing antimicrobial surfaces in healthcare environments which will not be repeated here [268–270]. The antibacterial and photosensitizing activity of ZnO nanoparticles has been well exploited in the absence or presence of light irradiation [271]. With the exploitation of their light absorption characteristic, ROS are produced from ZnO nanoparticles to act on bacteria, leading to a self-cleaning or self-sterilizing effect of the polymer-matrix zinc oxide nanocomposites [272]. For instance, Sehmi et al. and Ozkan et al. have developed self-sterilizing surfaces that was coupled with light-activated photodynamic therapy in killing bacteria [272,273]. Both studies showed the polymer matrix zinc oxide nanocomposites demonstrated lethal photosensitization of *E. coli* and *S. aureus* under white light irradiation that has a similar light intensity to that in a clinical setting [272,273]. This could potentially lower the rates of healthcare-associated infections by eliminating bacterial transfer in healthcare environments.

#### 4.2.3. Wound Healing Applications of Polymer-Matrix Metal Oxide Nanocomposites

Polymer-matrix metal oxide nanocomposites are an attracting candidate in wound healing applications. Gobi et al. summarized the recent applications of nanocomposites in wound dressings [274]. A novel polymer-matrix metal oxide nanocomposite comprising a castor oil polymeric matrix reinforced with a chitosan-modified ZnO nanocomposite was recently developed [275]. This novel bio-nanocomposite showed enhanced mechanical properties, porosity, water absorption, hydrophilicity, water vapor transmission rate, and oxygen permeability [275]. These enhanced properties are important for wound healing, which provides porosity to absorb wound exudates and water, enables a moist wound healing environment, a cooling effect for pain alleviation, and gases to exchange for ventilation. ZnO nanofillers also enhanced the antibacterial activity and keratinocyte migration of a polymer-matrix zinc oxide nanocomposite, leading to stronger antibacterial activity that prevented the reoccurrence of a bacterial infection and promoted healing [276]. Recent research has reported a prepared nanocomposite consisting of a Lawsone-loaded o-carboxymethyl chitosan and ZnO which was evaluated against bacterial strains such as *Salmonella*, *S. aureus*, *P. aeruginosa*, and *E. coli* [277]. This prepared nanocomposite tended to prevent the evolution of these harmful bacteria compared to an o-carboxymethyl chitosan or nano-zinc oxide alone, further supporting this advantageous strategy of using a polymermatrix ZnO nanocomposite in wound dressings. Finally, a polymer-matrix ZnO nanocomposite demonstrated promising in vivo efficacy, biodegradability, cytocompatibility, and promoted cell attachment on the material [278,279]. Taken as a whole, polymer-matrix metal oxide nanocomposites could possibly satisfy all the required standards as wound materials, highlighting their huge potential in wound healing applications.

#### 4.2.4. Food Packaging Applications of Polymer-Matrix Metal Oxide Nanocomposites

For food packaging applications, the addition of ZnO nanoparticles as nanofillers to biodegradable polymeric materials greatly enhances the physiochemical properties and antibacterial activities of the resulting bio-nanocomposites to protect the environment [270,280,281]. ZnO nanoparticles create a barrier effect to hinder the diffusion of the decomposition products from the polymer matrix to the gas phase, which further improve polymer thermal stability and avoid thermal degradation under the wide polymer melt processing window [282]. In addition, ZnO nanoparticles act as a nucleating agent in raising the crystallinity level of the polymer matrix [283]. The combined effect of the increased crystallinity of the polymer along with the barrier effect of ZnO nanoparticles creates a highly tortuous path for the gases, water vapors, and organic compounds [284].

The barrier properties to gases, water vapors, and organic compounds subsequently improve the product quality and shelf life by blocking the diffusion of moisture and oxygen. Mechanical performance such as stiffness, glass transition temperature, tensile strength, and toughness is also enhanced because of the strong polymer matrix–ZnO nanofiller interactions [282,285]. The antibacterial and UV-absorption properties of ZnO nanoparticles inhibit the growth of food-borne pathogens and prevent the photo-oxidative degradation of food, respectively [286]. Taken together, polymer matrix–zinc oxide nanocomposites are promising materials to be used as cutlery, overwrap films, and containers in preventing growth of food-borne pathogens and achieving good quality packaged food with extended shelf-life.

#### *4.3. Graphene Oxide–Metal Nanocomposites*

As mentioned in Section 3, the shortcomings of metal nanoparticles limit their potential for medical applications. To overcome these issues, many nanocomposites composed of metal nanoparticles and graphene have been prepared experimentally and studied against various bacterial strains [287–292]. Some of the available literature reviewed the development of graphene–metal matrix nanocomposites which will not be repeated here [293–296]. Among them, a combination of graphene oxide and silver nanoparticles to form nanocomposites has attracted a lot of attention as antibacterial agents in antibacterial therapies, since Ag and its compounds have been used since the time of the ancient Egyptians. The antibacterial and antiviral properties of Ag, Ag ions, and Ag-based compounds have been thoroughly researched [202,297,298]. With the incorporation of GO as the supporting matrix, silver nanoparticles could be dispersed in aqueous solution while minimizing the aggregation problem that would otherwise greatly affect the antibacterial activity of the silver nanoparticles [299]. The large surface area and abundant functional groups on the basal plane of GO allows GO to interact with silver ions or silver nanoparticles through electrostatic interactions, charge–transfer interactions and physical absorption [300]. This allows GO–Ag nanocomposites to be synthesized through loading of pre-synthesized silver

nanoparticles into GO (ex situ approach) or via reduction of silver ions in a graphene matrix to form silver nanoparticles in situ [301–305].

#### 4.3.1. Development of Synthetic Approaches for Improving the In Vivo Performance of Graphene Oxide–Metal Nanocomposites

The synthesis process of GO/reduced GO (rGO)–Ag nanocomposites involves harsh conditions, as well as highly toxic reducing agents and organic solvents, which minimizes their use in biomedical applications, which is an unmet area of need that requires more exploration [306,307]. Despite the good dispersity of GO–Ag in aqueous solution, it has been discovered that GO aggregates irreversibly in physiological solutions over time [308]. This greatly affect its bioavailability, significantly weakening its antibacterial efficiency and long-term effectiveness. In an effort to solve the problem, the (polyethylene glycol) PEGylation of GO was carried out for long term antibacterial activity and stability of GO–Ag in physiological solution [309]. The PEGylated GO–Ag nanocomposite remained stable in a series of complex media over one month and resisted centrifugation (Figure 8). In contrast, non-PEGylated GO–Ag aggregated to varying degrees in the media after 1 h, and complete precipitation was observed after 1 week of equilibration. GO−PEG−Ag nanocomposites displayed remarkable long-term antibacterial activity after 1 week of storage in physiological saline, preserving >99% antibacterial activity against *S. aureus* and >95% antibacterial activity against *E. coli*. GO−PEG−Ag inhibited bacterial growth in nutrient rich Luria–Bertani (LB) broth for at least one week, and the repeated usage of GO−PEG−Ag up to three times did not reduce the antibacterial efficacy. In contrast, unmodified GO−Ag exhibited a >60% decline in antibacterial activity after 1 week of storage in physiological saline. This study provides a direct solution for the synthesis of homogenously dispersed and stable GO–Ag nanocomposites under physiological conditions. This result was also confirmed by a subsequent study, in which ternary hybrids of PEG-functionalized GO with Ag nanoparticles exhibited excellent bactericidal effects against *E. coli*, and it was found that those with smaller Ag nanoparticles (8 nm) showed better antibacterial activity than those with larger nanoparticles (50 nm) [310]. Furthermore, modification of GO with polyethyleneimine polymers dramatically enhanced the long-term antibacterial activity and stability of the GO–Ag nanocomposite [311]. In addition to this, Parandhaman and his colleagues recently designed GO–Ag nanocomposites functionalized with the natural antimicrobial peptide poly-L-lysine with remarkably improved stability and adhesion to *S. aureus* biofilms [312]. Notably, poly-L-lysine functionalization prevented the leaching of anions, thereby reducing the cytotoxicity of the graphene–silver nanocomposites. In order to obtain nanomaterials with long-term and stable antibacterial activity, a facile and green method has also been proposed to prepare AgNPs/polymer/GO composites with catalytic and antibacterial activities via the incorporation of furan-functional poly(styrene-*alt*-maleic anhydride) [313].

#### 4.3.2. Potential of Graphene Oxide–Metal Nanocomposites for In Vivo Therapies

In terms of biological activity, GO–Ag nanocomposites have been shown to demonstrate synergistic antibacterial activities against planktonic bacteria and biofilms, with low cytotoxicity and good biocompatibilities [314–317]. The enhanced antibacterial activity of GO–Ag nanocomposites is often ascribed to the synergistic activity of GO and Ag; however, the full antibacterial mechanism remains to be elucidated [299]. Malik et al. have also demonstrated that GO–Ag nanocomposites exhibit significantly enhanced growth inhibition of *E. coli*, *S. aureus*, and *P. aeruginosa* relative to silver nanoparticles alone [291]. Recent studies have accomplished the surface functionalization of GO and Ag nanoparticles by using lantana plant extract, and the results also affirmed the potential of GO–Ag nanocomposites as antibacterial agents against biological pollutants [290]. The negatively charged oxygen-containing groups of graphene oxide can absorb Ag ions through electron absorption, which can improve the confinement of Ag nanoparticle agglomeration and burst release, and synergistically enhance their antibacterial properties [292]. Intriguingly,

GO–Ag nanocomposites exhibit species-specific bactericidal mechanisms, with cell wall disruption being observed against *E. coli* and inhibition of cell division against *S. aureus* [318].

**Figure 8.** Long-term stability and antibacterial effectiveness of a PEGylated GO–Ag nanocomposite. (Adapted with permission from Ref. [309]. 2017, Zhao et al. More details on "Copyright and Licensing" are available via the following link: https://www.mdpi.com/ethics#10 (accessed on 10 October 2022).)

The promising physical properties of GO, along with its synergistic activity with Ag nanoparticles, hold great potential for a targeted nanocomposite system [319]. A photothermal nanocomposite was produced which was composed of hyaluronic-acid-coated Ag nanoparticles that were integrated with GO [319]. Hyaluronic-acid-coated Ag nanoparticles confer additional protection by preventing the release of metal ions to surrounding mammalian cells. Upon encountering bacteria that secrete hyaluronidase, such as *S. aureus*, hyaluronic acid is degraded, followed by interaction of the GO–Ag nanocomposite and bacteria to further enhance the antibacterial action. Together with the photocatalytic characteristic of GO, local photothermal therapy under light irradiation could be achieved to further enhance the antibacterial activity of the GO–Ag nanocomposite [291].

4.3.3. Potential of Graphene Oxide–Metal Nanocomposites to Reduce Membrane Biofouling Issues for Water Decontamination and Filtration

The intrinsic characteristics of GO, including its availability as single-atomic-thick sheets, high hydrophilicity, extraordinary electrical, thermal, mechanical, structural properties, and low systemic toxicity could potentially reduce membrane biofouling issues for water decontamination or filtration [320,321]. GO–Ag nanocomposites are also a promising membrane surface modifier that contributed to enhanced membrane hydrophilicity, wettability and permeability, and good water influx [322–325]. Surface modification of membranes using GO/Ag nanocomposites exhibited stronger antimicrobial activities than AgNP-modified membranes and GO-modified membranes, without significantly altering the membrane transport properties [326]. The feasibility of using GO–Ag nanocomposites in membrane regeneration for a long-term anti-biofouling effect was demonstrated by conducting, in situ, the Ag-formation procedure to regenerate AgNPs on GO–Ag-modified membranes [327]. This potentially solves the problem of weakening biofouling properties of the functionalized GO–Ag nanocomposite membranes over time due to constant leaching

of silver ions throughout the process. More complex membranes containing GO, Ag, and metal-organic frameworks (MOFs) in PES were prepared for water treatments, exploiting the synergistic effects of graphene oxide and silver to enhance the anti-biofouling properties of the membranes [328]. In conclusion, the metal oxide/graphene nanocomposites exhibit enhanced antibacterial properties under visible light irradiation and have great potential as photocatalysts in the field of water purification [321]. Nevertheless, more studies are required to examine the long-term usage, membrane reusability, and regeneration potential of functionalized GO–Ag nanocomposite membranes.

#### *4.4. Graphene Oxide–Polymer Nanocomposites*

Graphene oxide–polymer nanocomposites exhibit enhanced antibacterial activity, biocompatibility, hemocompatibility, hydrophilicity, and stability, compared with the polymeric based nanomaterials [329–331]. In addition, GO can greatly reinforce the mechanical properties of GO–polymer nanocomposites, including their breaking strength, Young's modulus, compressive strength, flexural strength, and tensile strength [332–334]. The reinforcing effect is usually explained via strong interactions and bonding between the homogenously dispersed GO and polymeric components [332,333].

The alignment of GO sheets on the polymer film has been suggested to greatly affect the antibacterial activity of the resulting graphene oxide-based polymer nanocomposite [335]. Lu et al. synthesized graphene oxide–polymer nanocomposites by aligning GO in planar, vertical, and random orientations with the aid of a magnetic field (Figure 9A). GO was then immobilized by cross-linking with the surrounding polymer matrix, followed by oxidative etching to expose GO on the surface (Figure 9B). The vertically aligned GO nanosheets on the polymer film exhibited enhanced antibacterial activity compared with the random and horizontal orientations. Mechanistic examinations revealed that direct, edge-mediated contact with bacteria was the major mechanism in causing a greater physical disruption of the bacteria membranes (Figure 9C) [335]. Subsequently, greater levels of intracellular electron donors, for instance, glutathione, would release into the external environment upon membrane disruption, favoring GO to induce antibacterial activity via an oxidative stress mechanism [335]. This study highlights the importance of GO alignment and provides direct implications for the designing of GO–polymer nanocomposite films with enhanced antibacterial activities.

The abundant functional groups present on GO–polymer nanocomposites provide various interactions with nanoparticles, creating GO–polymer-based metal nanocomposites with superior characteristics. For instance, GO–chitosan nanocomposites have been demonstrated to act as both nucleation sites for calcium phosphates mineralization and absorption sites for nanoparticles [336]. The resulting GO/chitosan nanocomposites comprise micro- and nanohierarchical porous structures that allow cell attachment and proliferation after biomineralization [336]. In addition, the immobilization of the AgNPs and growthfactor-encapsulated nanoparticles on the GO–chitosan nanocomposites greatly enhances the antibacterial activity and osteo-inductivity, respectively [336]. Taken together, GO– polymer nanocomposites have great potential for use as multifunctional nanocomposite materials in various antibacterial applications. This is due to the substantial property enhancements and their ability to interact with various metal or metal oxide nanoparticles. Díez-Pascual and Luceño-Sánchez described several antibacterial applications of GO–polymer nanocomposites [325].

4.4.1. Development of Synthetic Approaches for the Production and Use of Graphene Oxide–Polymer Nanocomposites

Various synthesis routes can be applied to prepare graphene oxide–polymer nanocomposite with covalent or non-covalent interactions [337]. Shahryari et al. summarized a range of synthesis routes in a thorough review [338]. Generally, three common methods are utilized: solution blending, melt blending, and in situ polymerization [338,339]. The solution blending approach is the most commonly used method to synthesize graphene

oxide–polymer nanocomposites in a dispersion form, due to its convenience and ease of implementation. With this method, facile synthesis is achieved by simply blending the polymer with GO in solution, followed by sonication, or magnetic stirring, or shear mixing to obtain homogenous dispersions of the nanocomposite products as depicted in Figure 10 [340,341]. In Rusakova et al.'s study, an ultrasonic bath was used to disperse GO in a solution of styrene or a polyester resin in styrene, adding toluene and benzoyl peroxide and then, the mixture was placed into a special mold for polymerizing the nanocomposite [342].

**Figure 9.** (**A**) Schematic illustration of the GO film with different orientations. From left to right: random, planar, and vertical orientations. (**B**) Schematic illustration of the film fabrication procedure. A magnetic field is applied to control the orientation of dispersed GO nanosheets, with the orientation preserved by photo-cross-linking the dispersing agents. (**C**) SEM micrographs pictured the intact *E. coli* morphology treated by no-GO film, retained *E. coli* morphological integrity treated by randomly aligned- or planar aligned-GO film and flattened and wrinkled *E. coli* morphologies after being treated by a vertically aligned-GO film. (Adapted with permission from Ref. [335]. 2017, Lu et al. More details on "Copyright and Licensing" are available via the following link: https://www.mdpi.com/ethics#10 (accessed on 10 October 2022).)

Melt blending is applied commonly in industry due to its low cost and scalability. Unlike solution blending, melt blending eliminates the use of any toxic solvents for the dispersion. Following melting the polymer at high temperature, the GO nanofillers are dispersed into a polymer matrix by mechanical shear forces. However, it is likely to induce particle aggregation by thermal heating and local mechanical stresses during melt blending. A study employed wet phase inversion to prepare a sponge-like structure of a polymer/GO/solvent mixture as an exfoliating agent. It was subsequently ground into powder form and mixed with a polymer using melt blending [343]. The hybrid method was exemplified with two polymers, polyamide 6 (PA6) and poly (ethylene-co-vinyl acetate) (EVA). The produced nanocomposites exhibited enhanced dispersions with improved mechanical and dynamic–mechanical properties, compared with the nanocomposites prepared via melt blending [343].

**Figure 10.** Formation of a well-dispersed graphene oxide–polymer nanocomposite with graphene being embedded in polymeric matrix.

In situ polymerization can lead to a more homogeneous dispersion of the graphene derivatives within the polymer matrix than the two methods mentioned above. In this technique, GO was initially mixed with the monomers, followed by polymerization. The polymerization can be initiated by heat or radiation. Microwave heating has been demonstrated to be a particularly efficient method to produce well-dispersed rGO polymer nanocomposites [344,345]. Hou et al. exploited microwave heating to simultaneously reduce GO and conducted a nitroxide-mediated polymerization of styrene, forming rGOpolystyrene nanocomposites [346].

The resulting nanocomposite could be further modified into various forms and shapes for different antibacterial applications [336,347,348].

4.4.2. Application of Graphene Oxide–Polymer-Based Metal Nanocomposites in Wound Healing

Graphene oxide–polymer-based metal nanocomposites have a potential emerging role in wound healing application as in situ-forming hydrogels. Yan et al. have synthesized a novel GO–polymer-based metal nanocomposite (PEP-Ag@GO) for a wound healing application [349]. PEP-Ag@GO comprises a poly(Nisopropylacrylamide166-co-nbutyl acrylate9)-poly (ethyleneglycol)-poly(N-isopropylacrylamide166-co-n-butyl acrylate9) copolymer, denoted as PEP and AgNPs decorated on reduced GO nanosheets, denoted as Ag@GO. An aqueous mixture of the PEP-Ag@GO could transit to a hydrogel immediately in situ upon contact with the skin area that has a higher temperature than the transition temperature of PEP-Ag@GO (30 ◦C). The in situ formation of the hydrogel allows the treatment of wound areas that are difficult to access and minimizes tissue damage that is associated with changing of the wound dressing material [350]. More importantly, the strong interactions and bonding that arises from the PEP-GO give rise to good stability of the composite network. Therefore, the PEP-Ag@GO hydrogel resisted a transition back to liquid form at lower temperatures, even at 5 ◦C. This is particularly advantageous as commonly synthesized thermo-responsive hydrogels would phase transition back to a liquid at lower temperatures [351]. In vitro and in vivo experiments have also demonstrated good biocompatibility and enhanced antibacterial activity of a PEP-Ag@GO against MRSA (Mu50), leading to a much faster wound healing rate of an MRSA-infected skin defect [349].

4.4.3. Application of Graphene Oxide–Polymer-Based Nanocomposites in Water Treatment

Membrane technologies have been widely applied in water treatments, but the growth of biofilms causes deterioration of water filtration membranes. Therefore, GO–polymerbased nanocomposites are promising materials for use in water treatments due to their antibacterial and antifouling properties. A recent study discussed newly developed GO- based nanocomposites in water treatments and identified limitations for future improvements [352]. Zeng et al. modified poly(vinylidene fluoride) (PVDF) membranes by covalently immobilizing graphene oxide quantum dots (GOQDs) to exert the antibiofouling and antibacterial properties and maintain excellent permeation properties of PVDF membranes [353]. It was found that the GOQDs–PVDF membrane inhibited the growth of *E. coli* and *S. aureus* more effectively than two-dimensional GO sheets. Cheng et al. evaluated the performances of GO-coated and GO-blended polysulfone ultrafiltration membranes and GO-coated membranes presented lower declines in water flux and higher flux recoveries than GO-blended membranes [354]. They showed strong antibacterial activity and biofouling resistance against *E. coli*. To enhance the antibacterial activity, metal nanoparticles can also be incorporated into GO–polymer-based nanocomposites. Mahmoudi et al. incorporated Ag-decorated GO nanoplates into polysulfone membranes, which demonstrated superior antibacterial properties and inhibited the formation of biofouling [355].

#### 4.4.4. Application of Graphene Oxide–Polymer-Based Nanocomposites in Food Packaging

Graphene oxide–polymer-based membranes are attractive candidates to be applied in food packaging due to the fact that their incorporation increases the permeability, selectivity, barrier, and antibacterial activities of packaging and prolongs the durability of the material. For example, GO was cross-linked with chitosan at 120 ◦C to form nanocomposite films which improved the tensile strength and thermal stability and exhibited antimicrobial properties against *E. coli* and *B. subtilis* [356]. As a result of these enhancements, the films are suitable for use in food packaging. Multiple reviews have been conducted on the development and applications of graphene oxide nanocomposites in food packaging [357–360]. However, the current state of GO–polymer composite membranes in food packaging applications are yet to be commercialized to the best of our knowledge.

#### *4.5. Lipid Polymer Hybrid Nanoparticles*

Lipid polymer hybrid nanoparticles (LPHNPs) have emerged as a potentially superior nanocomposite delivery system by combining the advantages and mitigating the limitations associated with liposomes and polymeric nanoparticles alone [361–366]. LPHNPs comprise a biodegradable polymeric core for drug encapsulation, an inner lipid layer surrounding the polymeric core, and an outer polymeric stealth layer (Figure 11). The polymeric core provides high structural integrity, mechanical stability, a narrow size distribution, and higher lipophilic drug loading capacities of the LPHNPs [367]. The inner lipid layer confers the biocompatibility and delays the polymeric degradation of LPHNPs by limiting inward water diffusion, contributing to the sustained release of the composite system [368]. Finally, the outer polymeric stealth layer provides steric stabilization of the nanoparticles, acting as a stealth coating that enhances the in vivo circulation time and protect the composite system from immune recognition [369].

**Figure 11.** Structure of a lipid–polymer hybrid nanoparticle that comprises a polymeric core, an inner lipid layer, and an outer lipid–polyethylene glycol (PEG) shell (polymeric stealth layer).

#### 4.5.1. Development of Lipid Polymer Hybrid Nanoparticles Using a Quality-by-Design Approach

LPHNPs are still in their early developmental stage for antibacterial applications despite the advances witnessed in cancer therapeutics [370]. The synthesis conditions of antibiotic loaded LPHNPs with desired particle characteristics is highly challenging due to the strong interplay between process variables. Therefore, a quality-by-design approach is often utilized to customize the LPHNPs in meeting the requirements or desired particle characteristics [371–375]. A quality-by-design approach is a statistical method that applies multiple factorial concepts and modelling to determine the interactions between two or more process variables and the desired and observed response conditions [374,376–378]. For instance, Dave et al. developed and optimized norfloxacin loaded LPHNPs for a topical drug delivery application [372]. They utilized a Box–Behnken design to determine the effect of process conditions including the concentration of soya lecithin (lipid) and the concentration of a polylactic acid (polymer) on the response conditions. This included parameters such as the entrapment efficiency, particle size, and cumulative drug release. It was found that the optimal norfloxacin-loaded LPHNPs have a high drug encapsulation efficiency, desired particle size with a narrow distribution range, and an improved drug release profile and stability [372]. In a study aimed at optimizing LPHNPs, Thanki et al. customized LPHNPs composed of lipidoid 5 (cationic lipid-like molecule) and poly(DLlactic-co-glycolic acid) (PLGA) for loading antisense oligonucleotide-mediated luciferase gene (Luc-ASO) transcripts and they achieved efficient cellular delivery by using a qualityby-design approach [374]. In their study, they determined the effect of process conditions including the lipidoid 5 content and the lipidoid 5: Luc-ASO ratio against the response conditions (intensity-weighted average hydrodynamic diameter, polydispersity index, zeta potential, Luc-ASO encapsulation efficiency, Luc-ASO loading, in vitro splice-correction efficiency, and in vitro cell viability) to achieve efficient cell delivery [374]. A recent study by Ma et al. involved efficient delivery of hydroxycamptothecin (HCPT) via PEGylated LPHNPs [375]. A quality-by-design strategy was used to optimize HCPT-loaded LPHNPs with desired properties, among them, the factors representing key process conditions were the lipid/polymer mass ratio, polymer concentration, medium chain triglyceride volume, water-solvent ratio, and poly(D,L-lactide-co-glycolide) molecular weight, and the response conditions are particle size, particle size distribution, and drug-loading capacity [375]. The experimental results showed that the optimal LPHNPs had greater controlled release behavior and good stability in plasma, and effectively increased the loading of HCPT [375].

#### 4.5.2. Potential of Lipid Polymer Hybrid Nanoparticles as Antibacterial Delivery Vehicles

Recently, LPHNPs have received great attention in antibacterial applications as efficient drug delivery systems due to their combined advantages of liposomes and polymer nanoparticles [361,363–366,379]. Cai et al. have investigated the application of LPHNPs to act as promising antibacterial delivery vehicles for biofilm eradication [379]. In this study, the lipid layer was designed to contain mixed lipids of phospholipids and rhamnolipids, which acted as anti-adhesive and disrupting agents against the biofilms. The inner polymer core comprises multidrug regimens including antibiotic amoxicillin to exert antibacterial activity and the amoxicillin potentiator pectin sulfate that prevent the re-adherence of *H. pylori*. As a result, a complete eradication of *H. pylori* biofilm with impaired antibacterial resistance was observed under in vitro conditions. The performance of vancomycin-loaded LPHNPs was enhanced via the synthesis using multiple lipid excipients, including glyceryl triplamitate, oleic acid, polymer excipients Eudragit RS100, chitosan, and sodium alginate [361]. Compared to LPHNPs using mono-constituents of the lipid and polymer, the LPHNPs with multiple co-excipients demonstrated higher drug loading capacities and enhanced antibacterial activity against both sensitive strains of *S. aureus* and MRSA [361]. In one recent experiment, Jaglal et al. designed a pH-responsive LPHNP system for codelivery of vancomycin and 18β-glycyrrhetinic acid, showing its ability to eliminate 75% of MRSA in less than 12 h with the advantage of sustained and rapid release of vancomycin

in acidic conditions [363]. LPHNPs consisting of a poly(lactic-co-glycolic acid) core and a dioleoyl-3-trimethylpropane lipid shell were developed for loading vancomycin and were shown to have prominent antibacterial effect against planktonic *S. aureus* cells [364]. In this study, enhanced interactions with bacterial cells and penetration into biofilms was due to the presence of lipid shells. These studies indicate that LPHNPs could be a useful strategy to deliver and enhance the antibacterial activity of the loaded drugs against planktonic cells and biofilms of diverse species. However, more studies are needed to accelerate the clinical translation of the LPHNPs in antibacterial application.

#### **5. Conclusion, Bottlenecks, and Future Perspective of Nanotechnologies Being Developed for Antibacterial Applications**

This review mainly focuses on the progress and development of the prospects of nanomaterials in antibacterial applications. As described, the use of nanomaterials to combat bacterial infections has great potential for human health and medical development. Over the past few decades, significant progress has been made in understanding the antibacterial activity and potential of different classes of nanomaterials as drug carriers, leading to the discovery of a number of particularly promising candidate nanoparticle systems. Moreover, Chieruzzi et al. discussed that nanomodification such as incorporating fluoroapatite nanobioceramics into traditional clinical treatment materials, such as dental restorative glass ionomer cement, can lead to significant changes in the mechanical properties of materials, which is a very noteworthy direction for the development of new nano antibacterial materials [380]. Given their vast therapeutic potential and wide range of antibacterial applications of these nanomaterials and nanocomposites, we anticipate that more studies with emphasis on aiding the clinical translation and subsequent clinical trials of these nanotechnology-associated products will increase rapidly in the next decade. Several challenges remain to be addressed. Despite the importance of these nanomaterials as therapeutics and use in the field of biomedicine, their current limitations on human health cannot be ignored. The high-efficiency properties of nanomaterials as antibacterial agents or drug delivery vehicles allow their diffusion in different bodily organs, and sometimes the accumulation of these nanomaterials in various cells and tissues may cause negative health effects. It should be noted that the establishment of the consensus of nanomaterials physiochemical properties leading to maximum antibacterial activity and minimum toxicity is of utmost importance. With the understanding of the structure–property–activity relationship, researchers are able to reduce off-target effects of nanomaterials and effectively deliver nanotherapeutics to a desired infected tissue. For example, the use of single nanoparticles such as metal particles have many drawbacks due to their inherent toxicity. Therefore, combining a variety of nanomaterials to develop a new type of nanocomposite or nanohybrid material to obtain enhanced antibacterial activity has become a major research trend. In addition, exploring the mechanisms behind the antibacterial and physical properties of nanomaterials and nanocomposites should not be neglected. The more we learn, the better we as a community can devise new strategies to combat antimicrobial resistance. More studies in assessing the dose calibration and identification of the appropriate routes of administration for a wide range of nanomaterials are also needed. This will greatly speed up the progress towards clinical trial progression and commercialization of nanotechnology-associated end products.

**Author Contributions:** Conceptualization, R.R., C.L. and S.L; writing—original draft preparation, R.R., C.L. and S.L.; writing—review and editing, Y.W., J.S., T.-W.L., B.W.M., H.-Y.H. and H.-H.S.; supervision, H.-H.S.; funding acquisition, H.-H.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the NHMRC Career Development Fellowship GNT1106798.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Abbreviations**

#### **References**

## *Article* **Mesoporous Silica Nanoparticles Enhance the Anticancer Efficacy of Platinum(IV)-Phenolate Conjugates in Breast Cancer Cell Lines**

**Ivana Predarska 1,2, Mohamad Saoud <sup>3</sup> , Dijana Draˇca 4, Ibrahim Morgan <sup>3</sup> , Teodora Komazec <sup>4</sup> , Thomas Eichhorn 2, Ekatarina Mihajlovi´c <sup>4</sup> , Duško Dunderovi´ ¯ c<sup>5</sup> , Sanja Mijatovi´c 4, Danijela Maksimovi´c-Ivani´c <sup>4</sup> , Evamarie Hey-Hawkins 1,\* and Goran N. Kaluderovi´ ¯ c 2,3,\***

**Abstract:** The main reasons for the limited clinical efficacy of the platinum(II)-based agent cisplatin include drug resistance and significant side effects. Due to their better stability, as well as the possibility to introduce biologically active ligands in their axial positions constructing multifunctional prodrugs, creating platinum(IV) complexes is a tempting strategy for addressing these limitations. Another strategy for developing chemotherapeutics with lower toxicity relies on the ability of nanoparticles to accumulate in greater quantities in tumor tissues through passive targeting. To combine the two approaches, three platinum(IV) conjugates based on a cisplatin scaffold containing in the axial positions derivatives of caffeic and ferulic acid were prepared and loaded into SBA-15 to produce the corresponding mesoporous silica nanoparticles (MSNs). The free platinum(IV) conjugates demonstrated higher or comparable activity with respect to cisplatin against different human breast cancer cell lines, while upon immobilization, superior antiproliferative activity with markedly increased cytotoxicity (more than 1000-fold lower IC50 values) compared to cisplatin was observed.  In vivo, in BALB/c mice, **1** and SBA-15|**1** inhibited the tumor growth while decreasing the necrotic area and lowering the mitotic rate.

**Keywords:** platinum(IV) conjugates; cisplatin; phenolic acid; nanoparticles; drug delivery; breast cancer

#### **1. Introduction**

Caffeic acid (CA) and ferulic acid (FA) belong to the most common phenolic acids, naturally occurring in fruits, grains, vegetables, spices, tea and coffee [1]. Apart from the key functions that these secondary plant metabolites exhibit on plant growth, development and defence, they are also found to have highly beneficial effects on humans. They are known for being potent natural antioxidants that are important for a variety of biological and pharmacological processes, such as cancer prevention, inflammation reduction, bacterial and viral infections prevention, blood clotting prevention, liver protection and many others [2–8]. The antioxidant properties of these phenolic acids are accredited to

**Citation:** Predarska, I.; Saoud, M.; Draˇca, D.; Morgan, I.; Komazec, T.; Eichhorn, T.; Mihajlovi´c, E.; Dunderovi´ ¯ c, D.; Mijatovi´c, S.; Maksimovi´c-Ivani´c, D.; et al. Mesoporous Silica Nanoparticles Enhance the Anticancer Efficacy of Platinum(IV)-Phenolate Conjugates in Breast Cancer Cell Lines. *Nanomaterials* **2022**, *12*, 3767. https:// doi.org/10.3390/nano12213767

Academic Editor: Cesare Malagù

Received: 29 September 2022 Accepted: 17 October 2022 Published: 26 October 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

their chemical structure which includes one (ferulic acid) or two (caffeic acid) free phenolic hydroxyl groups and a double bond in the aliphatic chain enabling the formation of a phenoxy radical stabilized through resonance. In addition to the free radical scavenging via hydrogen atom donation [9], these phenolic acids are also found to modulate enzymatic activity, affect signal transduction as well as activate transcription factors and gene expression. As an example, alkyl esters of caffeic and ferulic acid are found to demonstrate significant inhibition on the activity of cyclooxygenase enzymes (COX-1 and COX-2) [10–13] responsible for the formation of key proinflammatory mediators. Additionally, COX-2 is overexpressed in a variety of tumors [14], making it a desirable target for the creation of anticancer medications. Therefore, in the past decade, these phenolic acids have attracted extensive attention as potential chemopreventive and chemotherapeutic agents. In that sense, multiple groups have pointed to the ability of caffeic acid and its derivatives to inhibit the growth of colon cancer [15–18], express anti-hepatocellular carcinoma activity [19], suppress the cancer cell proliferation in the human HT-1080 fibrosarcoma cell line [20] as well as human cervical cancer cells (HeLa and Me-180) [21] and reduce not only the growth of breast cancer, but also the ability of the breast cancer MCF-7 cells to migrate [22,23] which is a crucial factor for cancer metastasis. Ferulic acid as well has been found to have tumor-suppression potential in colon and breast cancers [4,24]. Additionally, both caffeic and ferulic acid have been found to increase the therapeutic efficacy of known anticancer drugs or lower the drug-related side effects. In this regard, the combined therapy involving caffeic acid and paclitaxel has been shown to have synergistic anticancer activity on H1299 lung cancer cells leading to enhanced apoptosis in vitro and in vivo [25]. Furthermore, caffeic acid elevates the antitumor effect of metformin in HTB-34 human metastatic cervical carcinoma cells [26] and increases the susceptibility of gastric cancer cells to cisplatin and doxorubicin [27]. Synergistic effects are also found applying cisplatin and caffeic acid in combination resulting in apoptotic mode of cell death in human cervical cancer cells [28]. A very interesting finding has been made by Sirota et al. who have discovered that in ovarian carcinoma cell lines the timing of caffeic acid treatment determines whether they become cisplatin-sensitive or -resistant [29]. Their findings demonstrate that preincubation with caffeic acid before treatment with cisplatin resulted in acquired resistance to cisplatin and decreased DNA binding, whereas simultaneous treatment with cisplatin and caffeic acid can increase cytotoxicity of cisplatin and platinum binding to nuclear DNA [29].

Based on all these findings and bearing in mind the different pharmacokinetic profiles of phenolic acids and cisplatin, including rising uncertainties whether these agents can reach the tumors within the required time frame in vivo, we have designed conjugates that combine the two agents into a single platinum(IV) prodrug. In this way, together with the molecules of the platinum component, the molecules of the respective phenolic acid can also be delivered into the tumor cells. Upon intracellular activation of these prodrugs via 2-electron reduction and protonation, the two moieties can be released and together carry out their biological functions in a synergistic manner [30]. Another reason which makes this approach attractive is that the coordinatively saturated platinum(IV) complexes are more stable than platinum(II) complexes, such as cisplatin, thus preventing its premature activation occurring by hydrolysis of the labile platinum chlorido ligand bonds, which is associated with many of its drawbacks such as the severe side effects, fast detoxification and innate or acquired resistance [31,32].

In an effort to create chemotherapeutics with lower toxicity, we have also used an SBA-15 mesoporous silica as nanocarrier for the platinum(IV) conjugates, aiming to take advantage of the accumulation of nanoparticles in tumor tissues through the passive enhanced permeability and retention (EPR) effect [33]. An additional advantage of this strategy is the possibility to further protect the drug from premature interaction with biomolecules on the way to cancer cells as well as provide tailorable drug release. The mesoporous silica SBA-15 material was chosen as a drug delivery vehicle because it is nontoxic to cells [34] and it has advantageous properties, including high pore volume with tuneable nanoscale pores as well as large surface area [35–37]. The properties of the inorganic mesoporous silica materials that make them interesting for biomedical applications, especially as nanoplatforms for drug delivery, have been highlighted in several reviews in the recent years [38–44]. The use of SBA-15 as a cisplatin carrier in earlier studies has been associated with increased cytotoxicity in leukemic cells [45], a twenty-fold greater antiproliferative effect in HT-29 colon cancer cells [46] and activation of pathways that change the phenotype of cancer cells (i.e., melanoma B16 tumor cells differentiate into senescent cells) [47]. Formerly reported SBA-15 nanoparticles loaded with platinum(IV) conjugates have also demonstrated very high antiproliferative activity against different breast cancer cell lines, while maintaining the structural integrity and allowing for relatively slow release of the active compounds [48].

Thus, we report here the preparation of three platinum(IV) derivatives of cisplatin bearing phenolates as axial ligands and their immobilization in SBA-15 nanoparticles. In these conjugates, both axial positions are occupied with acetyl-protected caffeate (**1**) or ferulate (**2**), as well as ferulate (**3**) (Figure 1). It should be noted that a cisplatin-ferulate conjugate has previously been reported [49]. However, in our view, the spectra included in the supplementary information of the corresponding paper do not confirm or support the structure of the desired product [49]. The prepared platinum(IV) complexes and their respective nanomaterials demonstrated remarkable antiproliferative activity in four different human breast cancer cell lines with different characteristics. In addition, synthesized conjugates and appropriate nanomaterials showed notable anticancer potential against a mouse breast cancer cell line as well. The activity of conjugate **1** and its corresponding MSNs SBA-15|**1**, which proved to be the most potent compounds in vitro, was further assessed in vivo, in a BALB/c mouse model with breast tumor induced by inoculation of 4T1 cells. **1** and SBA-15|**1** demonstrated potential to inhibit the tumor growth while decreasing the necrotic area and lowering the mitotic rate.

**Figure 1.** Platinum(IV)-phenolate conjugates: cisplatin-acetyl-protected caffeate (**1**), cisplatin-acetylprotected ferulate (**2**) and cisplatin-ferulate (**3**).

#### **2. Materials and Methods**

Standard Schlenk methods and anhydrous, degassed solvents were used to conduct all reactions in a nitrogen atmosphere. Acetone was first refluxed with KMnO4, then distilled from drierite (anhydrous CaSO4), pyridine was distilled from CaH2; both solvents were stored over activated 3 and 4 Å molecular sieves, respectively; MBraun Solvent Purification System (MB SPS-800) was used to obtain dry toluene, dichloromethane (DCM) and dimethylformamide (DMF), which were afterwards kept over activated 4 Å molecular sieves. Chemicals (cisplatin (Carbolution, St. Ingbert, Germany), caffeic acid (Fluorochem, Dublin, Ireland), ferulic acid, oxalyl chloride, tetraethyl orthosilicate (TEOS), pluronic 123 (P123) and acetic anhydride (Sigma-Aldrich Chemie GmbH, Steinheim, Germany)) were used as supplied. The synthesis of oxoplatin, *cis*,*trans*,*cis*-[PtCl2(OH)2(NH3)2], was performed according to an already reported procedure [50]. In short, cisplatin was oxidized

with 30% (*w*/*w*) aqueous solution of hydrogen peroxide. Protection of the free phenolic hydroxy groups by acylation was done with acetic anhydride employing a literature-known procedure [51]. The acyl chloride derivative of acetyl-protected caffeic acid was prepared with oxalyl chloride and catalytic amounts of DMF in DCM [51], while for the preparation of the acyl chloride derivative of acetyl-protected ferulic acid, toluene was used as solvent.

A 400 MHz NMR spectrometer (BRUKER Avance III HD, Karlsruhe, Germany) was used for recording of 1H (400.13 MHz, tetramethylsilane (TMS) as an internal reference), 13C (100.63 MHz, internal reference TMS) and 195Pt (85.85 MHz, external reference Na2[PtCl6]), as well as two-dimensional 1H−1H COSY, 1H−13C HSQC, 1H−13C HMBC NMR spectra at 25 ◦C. The values of chemical shifts δ are given in parts per million (ppm). Electrospray ionization mass spectrometry was performed by using ESI-qTOF Impact II in positive mode (Bruker Daltonics GmbH, Bremen, Germany). Elemental analyses (C, H and N) were carried out with a microanalyser (VARIO EL, Heraeus Group, Hanau, Germany). VEGA3 (Tescan, Brno, Czech Republic) was employed for collection of scanning electron microscopy (SEM) images, as well as to conduct energy-dispersive X-ray spectroscopy (EDX) experiments (element detector EDAX Inc, Mahwah, NJ, USA). For nitrogen sorption measurements an Autosorb iQ/ASiQwin (Quantachrome Instruments, Anton Paar, QuantaTec Inc., Boynton Beach, FL, USA) was used. Small angle X-ray scattering (SAXS) measurements were performed on a D8 ADVANCE (Bruker, Karlsruhe, Germany) X-ray diffraction system. Inductively coupled plasma-optical emission spectrometry (ICP-OES) analysis was achieved on an Optima 7000 DV spectrometer (PerkinElmer, Waltham, MA, USA) using WinLab32 software.

#### *2.1. Synthesis of Platinum(IV) Conjugates* **1** *and* **2**

Pyridine (0.48 mL, 6.0 mmol, 10 eq.) was added to an oxoplatin (0.2 g, 0.6 mmol, 1 eq.) suspension in acetone (12 mL). After 10 min stirring, acyl chloride derivative, dissolved in acetone (12 mL), of either acetyl-protected caffeic acid or acetyl-protected ferulic acid (0.85 g or 0.76 g, respectively, each 3.0 mmol, 5 eq.) was added. The reaction mixture was refluxed at 75 ◦C (48 h). The precipitate formed was filtrated and thoroughly washed (water: 30 mL × 3; ethanol: 10 mL × 3; diethyl ether: 10 mL × 3) to eliminate the pyridinium salt. After drying in air, the pure products were collected.

**1**, pale yellow powder. Yield: 0.32 g (64%).

**1H NMR (DMSO-***d***6, ppm):** *δ* = 7.63 (d, <sup>4</sup>*J*HH = 2 Hz, 2H, C*H*aryl), 7.60 (dd, <sup>3</sup>*J*HH = 8 Hz, <sup>4</sup>*J*HH = 2 Hz, 2H, C*H*aryl), 7.38 (d, <sup>3</sup>*J*HH = 16 Hz, 2H, C*H*vinyl), 7.30 (d, <sup>3</sup>*J*HH = 8 Hz, 2H, C*H*aryl), 6.80–6.50 (br, 6H, N*H*3), 6.59 (d, <sup>3</sup>*J*HH = 16 Hz, 2H, C*H*vinyl), 2.29 (s, 12H, OCOC*H*3).

**13C{1H} NMR (DMSO-***d***6, ppm):** *δ* = 174.0 (qC, *C*OO), 168.7 (qC, *C*OO), 143.4 (qC, *C*aryl), 142.9 (qC, *C*aryl), 139.9 (CH, *C*vinyl), 133.9 (qC, *C*aryl), 126.7 (CH, *C*aryl), 124.6 (CH, *C*aryl), 123.3 (CH, *C*aryl), 122.8 (CH, Cvinyl), 20.9 (CH3, OCO*C*H3).

**195Pt{1H} NMR (DMSO-***d***6, ppm):** *δ* = 1213 (br s).

**HR-ESI-MS (positive mode, CH3OH):** *m*/*z* [2M + H]+: calc. for C52H57Cl4N4O24Pt2: 1653.139, found: 1653.135; *m*/*z* [M + H]+: calc. for C26H29Cl2N2O12Pt: 827.073, found: 827.070.

**Elemental analysis:** Found: C, 37.95; H, 3.2; N 3.5. Calc. for C26H28Cl2N2O12Pt: C, 37.8; H, 3.4; N, 3.4%.

**2**, yellow powder. Yield: 0.27 g (59%).

**1H NMR (DMSO-***d***6, ppm):** *δ* = 7.45 (d, <sup>4</sup>*J*HH = 2 Hz, 2H, C*H*aryl), 7.39 (d, <sup>3</sup>*J*HH = 16 Hz, 2H, C*H*vinyl), 7.21 (dd, <sup>3</sup>*J*HH = 8 Hz, <sup>4</sup>*J*HH = 2 Hz, 2H, C*H*aryl), 7.11 (d, <sup>3</sup>*J*HH = 8 Hz, 2H, C*H*aryl), 6.80–6.50 (br, 6H, N*H*3), 6.65 (d, <sup>3</sup>*J*HH = 16 Hz, 2H, C*H*vinyl), 3.83 (s, 6H, OC*H*3), 2.27 (s, 6H, OCOC*H*3).

**13C{1H} NMR (DMSO-***d***6, ppm):** *δ* = 174.2 (qC, *C*OO), 168.9 (qC, *C*OO), 151.5 (qC, *C*aryl)148.4 (qC, *C*aryl), 140.9 (qC, *C*vinyl), 133.9 (qC, *C*aryl), 123.6 (CH, *C*aryl), 121.9 (CH, *C*vinyl), 121.3 (CH, *C*aryl), 111.7 (CH, *C*aryl), 59.3 (CH3, O*C*H3), 20.8 (CH3, OCO*C*H3).

**195Pt{1H} NMR (DMSO-***d***6, ppm):** *δ* = 1212 (br s).

**HR-ESI-MS (positive mode, CH3OH):** *m*/*z* [2M + H]+: calc. for C48H57Cl4N4O20Pt2: 1541.159, found: 1541.141; *m*/*z* [M + H]+: calc. for C24H29Cl2N2O10Pt: 771.083, found: 771.086; *m*/*z* [M + Na]+: calc. for C24H28Cl2N2NaO10Pt: 793.065, found: 793.068.

**Elemental analysis:** Found: C, 37.6; H, 3.5; N, 3.9. Calc. for C24H28Cl2N2O10Pt: C, 37.4; H, 3.6; N, 3.65%.

#### *2.2. Deprotection of* **2** *to Obtain Platinum(IV) Conjugate* **3**

In a mixture of DCM, methanol and acetone (20 mL of each solvent) conjugate **2** (0.3 g, 0.39 mmol) was dissolved. Conc. HCl was added dropwise (10 mL) to the colorless solution, and a color change to light yellow was noticeable after a few minutes. To track the progress of the reaction TLC (ethyl acetate:*n*-hexane = 3:1) was used. After 3 h stirring at r.t., the starting material was no longer present. The volume of the solvents was reduced to ca. 15 mL, subsequently water (50 mL) was added. The reaction mixture was filtrated and the isolated product was washed (water: 10 mL × 3; ethanol: 10 mL × 3; diethyl ether: 10 mL × 3) and dried in air.

**3**, dark yellow powder. Yield: 0.25 g (93%).

**1H NMR (DMSO-***d***6, ppm):** *δ* = 9.45 (s, 2H, O*H*), 7.30 (d, <sup>3</sup>*J*HH = 16 Hz, 2H, C*H*vinyl), 7.23 (d, <sup>4</sup>*J*HH = 2 Hz, 2H, C*H*aryl), 7.02 (dd, <sup>3</sup>*J*HH = 8 Hz, <sup>4</sup>*J*HH = 2 Hz, 2H, C*H*aryl), 6.80–6.50 (br, 6H, N*H*3), 6.78 (d, <sup>3</sup>*J*HH = 8 Hz, 2H, C*H*aryl), 6.42 (d, <sup>3</sup>*J*HH = 16 Hz, 2H, C*H*vinyl), 3.82 (s, 6H, OC*H*3).

**13C{1H} NMR (DMSO-***d***6, ppm):** *δ* = 174.9 (qC, *C*OO), 149.1 (qC, *C*aryl), 148.4 (qC, *C*aryl), 142.1 (CH, *C*vinyl), 126.5 (qC, *C*vinyl), 122.7 (CH, *C*aryl), 118.4 (CH, *C*vinyl), 115.9 (CH, *C*aryl), 111.1 (CH, *C*aryl), 56.1 (CH3, O*C*H3).

**195Pt{1H} NMR (DMSO-***d***6, ppm):** *δ* = 1216 (br s).

**HR-ESI-MS (positive mode, CH3OH):** *m*/*z* [2M + H]+: calc. for C40H49Cl4N4O16Pt2: 1373.116, found: 1373.098; *m*/*z* [2M + Na]+: calc. for C40H48Cl4N4NaO16Pt2: 1395.098, found: 1395.068; *m*/*z* [2M + K]+: calc. for C40H48Cl4KN4O16Pt2: 1411.072, found: 1411.035; *m*/*z* [M + H]+: calc. for C20H25Cl2N2O8Pt: 687.062, found: 687.060.

**Elemental analysis:** Found: C, 35.1; H, 3.5; N, 4.2. Calc. for C20H24Cl2N2O8Pt: C, 35.0; H, 3.5; N, 4.1%.

#### *2.3. Solubility and Stability of Conjugates* **1***–***3**

For determination of their water solubility, compounds **1**–**3** (5 mg, each) were suspended in distilled water (5 mL) and stirred for 12 h. After that, the undissolved solid was separated by filtration and the amount of dissolved conjugate was evaluated after evaporation of the solvent from the filtrate.

To deduce the stability of **1**–**3** in DMSO, time-resolved (0, 3, 6, 12, 24, 48 and 72 h) 1H NMR spectroscopy was applied.

#### *2.4. Preparation of SBA-15*

The SBA-15 silica mesoporous nanoparticles were prepared according to a reported procedure [52]. Briefly, to a solution of P123 (48.4 g) in a mixture of HCl (2 M, 1400 mL) and H2O (360 mL) at 35 ◦C, TEOS (102 g) was added dropwise. The reaction mixture was vigorously stirred at 35 ◦C (20 h), then at 80 ◦C (24 h). The solid material was isolated by filtration and dried at 90 ◦C (14 h). Finally, the material was heated to 500 ◦C (1 ◦C min<sup>−</sup>1) and kept for 24 h at this temperature for calcification.

Yield: 30.2 g; BET surface: 517 m2 g<sup>−</sup>1; wall thickness: 3.66 nm; pore diameter: 4.74 nm; pore volume: 0.72 cm<sup>3</sup> g<sup>−</sup>1; lattice parameter: 8.4 nm; XRD (2*θ* in ◦, Miller indices): 1.0802 (100), 1.8032 (111), 2.0645 (200).

#### *2.5. Preparation of SBA-15 Material Loaded with Platinum(IV) Conjugates, SBA-15|***1***–SBA-15|***3**

The literature procedure was followed for loading the conjugates **1**–**3** in the MSN mesopores [53,54]. Namely, to the activated SBA-15 nanoparticles (vacuum, 150 ◦C, 16 h), a suspension of **1**–**3** in toluene was added and the reaction mixture was agitated at 80 ◦C

(48 h). Filtration was used to separate the solid material, which was then thoroughly washed using toluene and *n*-pentane. The MSNs loaded with drug (**1**–**3**) were obtained and dried at r.t.. Physical parameters of prepared MSNs are given in Table 1.

SBA-15|**1**: 165 mg of **1**, 200 mg of SBA-15; yield: 340 mg. SBA-15|**2**: 231 mg of **2**, 300 mg of SBA-15; yield: 498 mg. SBA-15|**3**: 103 mg of **3**, 150 mg of SBA-15; yield: 244 mg.

**MSNs** *<sup>S</sup>***BET Wall Thickness Pore Diameter Pore Volume Lattice Parameter XRD [m2 g**−**1] [nm] [nm] [cm3 g**−**1]** *a* **[nm] 2***θ* **[** ◦**]** *hkl* SBA-15 [48] 517 3.66 4.74 0.72 8.4 1.0802 100 1.8032 111 2.0645 200 SBA-15|1 245 3.34 4.75 0.34 8.1 1.1404 100 1.8543 111 2.1378 200 SBA-15|2 260 4.26 4.46 0.37 8.7 1.0311 100 1.7450 111 2.0075 200 SBA-15|3 292 5.03 3.68 0.37 8.7 1.0149 100 1.7603 111 2.0228 200

**Table 1.** Selected physical parameters of the MSNs.

#### *2.6. Drug Release Studies*

ICP-OES was used to track the release of the conjugates **1**–**3** from the MSNs in phosphate-buffered saline (PBS) depending on the amount of platinum in the liquid phase. Experiments were performed by suspending the drug-loaded MSN material (2 mg) in PBS (1 mL) and sampling at defined times (0.08, 0.5, 1, 3, 6, 12, 24 and 72 h). Quantification of platinum in the PBS was done by ICP-OES upon separation of the solid material by centrifugation. External platinum standard solutions, obtained from an ICP platinum standard reference solution (Pt 1000 μg mL−1, Specpure®, Alfa Aesar GmbH & Co KG, Germany), were used for the calibration of the ICP-OES device. The measurement was carried out on the 194Pt isotope on a previously chosen emission line of platinum of 265.9 nm. Using different mathematical models (zero/first order, Korsmeyer-Peppas and Higuchi), the drug release kinetics were analyzed.

In addition, SBA-15|**1** and SBA-15|**2** (2 mg each) were each suspended in PBS (1 mL). The PBS was exchanged with fresh PBS (1 mL) every hour for 10 h consecutively. Again, the platinum present in the liquid phase was quantified using ICP-OES.

#### *2.7.  The mouse breast cancer cell line 4T1 (derived from the mammary gland tissue of a mouse BALB/c strain), was a kind gift from Prof. Nebojša Arsenijevi´c from the Faculty of Medical Sciences, University of Kragujevac, Serbia. PBS, fetal calf serum (FCS) and basal cell culture media Roswell Park Memorial Institute (RPMI) were purchased from both Sigma-Aldrich (St. Louis, MO, USA) and Capricorn Scientific GmbH (Ebsdorfergrund, Germany). Trypsin/ethylenediaminetetraacetic acid (EDTA) and L-glutamine were bought from Capricorn Scientific GmbH (Ebsdorfergrund, Germany). Paraformaldehyde (PFA) was obtained from Serva (Heidelberg, Germany). DMSO, carboxyfluorescein diacetate succinimidyl ester (CFSE), propidium iodide (PI) and crystal violet (CV) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The penicillin/streptomycin solution was obtained from Biological Industries (Cromwell, CT, USA). Acridine orange (AO) was obtained from Labo-Moderna

(Paris, France). PAN Biotech GmbH (Aidenbach, Germany) supplied us with Endopan 3 with supplementing kits. ApoStat was purchased from R&D Systems (Minneapolis, MN, USA) while Annexin V-FITC (AnnV) was from Biolegend (San Diego, CA, USA). 4-Amino-5-methylamino-2 ,7 -difluorofluorescein diacetate (DAF-FM diacetate) and dihydrorhodamine 123 (DHR) were bought from Thermo Fisher Scientific (Waltham, MA, USA). The multi-well plates, culture flasks and other cell culture plastics were obtained from TPP (Trasadingen, Switzerland) and Greiner Bio-One GmbH (Frickenhausen, Germany).

#### *2.8.  RPMI-1640 medium containing 2 mM L-glutamine, heat-inactivated FCS (10%), and penicillin/streptomycin (1%) was used to sustain all cell lines. Prior to use or further subculturing, the cell lines were cultured in T-75 flasks at 37 ◦C in a humidified environment with 5% CO2. Before cell passaging and seeding, the adherent cells were washed with PBS and detached by using trypsin/EDTA (0.05% in PBS) [54].

#### *2.9. Cell Viability Studies*

Using the above-mentioned cell growth media, cells were seeded in 96-well plates in a density of 6000 cells per 100 μL per well for the in vitro investigations in human breast cancer cell lines. Following seeding, cells were given a 24-h period to adhere before being treated with platinum(IV) conjugates **1**–**3** and MSNs loaded with **1**–**3**, namely SBA-15|**1**, SBA-15|**2** and SBA-15|**3**. Stock solutions of platinum(IV) conjugates **1**–**3**, phenolic acids (ligand precursors) and cisplatin were prepared in DMSO. Serial dilution was afterwards done in standard growth media to reach the following concentrations: 10, 5, 1, 0.5, 0.1, 0.01 and 0.001 μM for the conjugates, 100, 50, 25, 12.5, 6.25, 3.125 and 1.6 μM for the phenolic acids, and 300, 100, 30, 10, 3, 1, and 0.1 μM for cisplatin. In a repeated experiment, HCC1937, MCF-7 and BT-474 cells were treated with various concentrations of the conjugates **1**–**3**, namely 50, 25, 12.5, 6.25, 3.125, 1.6, 0.8 and 0.4 μM. Stock suspensions of SBA-15 and drugloaded SBA-15 were prepared in PBS and serially diluted with standard growth media to achieve final concentrations of 50, 10, 5, 1, 0.5, 0.1, 0.01 and 0.001 μg mL−<sup>1</sup> for drug-loaded SBA-15, and 100, 50, 25, 12.5, 6.25, 3.125 and 1.6 μg mL−<sup>1</sup> for SBA-15 alone. Each 96-well plate contained a positive control using digitonin (100 μM). Three independent experiments were performed in quadruplicate.

For the in vitro studies in the mouse-derived breast cancer cell line, cells were seeded in a density of 2 × 103 cells per well (96-well plates) using the previously indicated cell growth media. Following seeding, cells were given a 24 h adhesion period before being treated with conjugate **1** and SBA-15|**1**. Using a DMSO stock solution of **1**, prepared at a concentration of 10 mM and retained at −20 ◦C for 3 days, a serial dilution was made in standard growth media to achieve concentrations of 25, 12.5, 6.3, 3.12, 1.56, 0.78 and 0.39 μM. A stock suspension of SBA-15|**1** was prepared in PBS and serially diluted in standard growth media until final concentrations of 50, 25, 12.5, 6.25, 3.12, 1.56 and 0.78 μg mL−<sup>1</sup> were reached. A DMSO solution of cisplatin at a concentration of 10 mM was prepared freshly before usage, and serial dilutions were made in culture medium to reach concentrations of 100, 50, 25, 12.5, 6.25, 3.12 and 1.56 μM. To determine the outcome of induced autophagy, cells were treated with an IC50 dose of conjugate **1** and SBA-15|**1** and concomitantly with 3-methyladenine (3-MA, 1 mM).

Cell viability was evaluated in each case after a 72-h incubation period. Colorimetric MTT- (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and CV (crystal violet)-based cell viability assays were used to examine the compounds' potential cytotoxicity. The MTT test was carried out in the following way: cells were rinsed with PBS before being incubated at 37 ◦C in a humid environment with 5% CO2 with an MTT standard solution (0.5 mg mL−<sup>1</sup> MTT in culture medium). After one hour, the MTT solution

was discarded and DMSO was used to dissolve the formazan that had formed. The absorbance of this formazan, along with the reference/background signal, could be measured at wavelengths of 540 nm and 670 nm. Measurements were taken utilizing SpectraMax M5 multi-well plate reader (MolecularDevices, San Jose, CA, USA). For the CV assay, cells were fixed with 4% PFA for 20 min at room temperature after being washed once with PBS. The PFA solution was then removed and the cells were left to dry for 10 min. Afterwards, cells were stained with 0.2% crystal violet solution for 20 min followed by removal of the staining solution, washing of the cells with water and letting them dry overnight at room temperature. Finally, upon addition of acetic acid (33% in aqua bidest.) to the stained cells, the absorbance was measured at reference wavelengths of 540 nm and 670 nm as described earlier [56]. A four-parametric logistic function was used to obtain the mean value of the cell viability, which is expressed as a percentage in comparison to untreated cells [57]. SigmaPlot 14.0 and Microsoft Excel 2013 were employed for data analyses and IC50 and MC50 calculation.

#### *2.10. Flow Cytometry*

Six-well plates were seeded with 4T1 cells (5 × 104 per well). After overnight adherence, cells were exposed to an IC50 dose of **1** and an MC50 dose of SBA-15|**1** and analyzed by flow cytometry. There were several staining procedures used: (1) CFSE for observing the influence on cellular proliferation, (2) AnnV/PI for identifying apoptotic cells, (3) Apo-Stat for caspase activity detection, (4) AO for autophagy detection, (5) DHR for reactive oxygen/nitrogen species (ROS/RNS) measurement, (6) DAF-FM for intracellular nitric oxide (NO) measurement, and (7) PI for cell cycle analysis. Results were acquired by CyFlow® Space Partec using the PartecFloMax® software v1.0.0 (Partec GmbH, Münster, Germany), with an exception for the results of cell cycle analysis which were obtained using BD FACSAria III and BD FACSDiva software v8.3 (BD Biosciences, Franklin Lakes, NJ, USA). Three separate replicates of the experiment were run. Depending on the particular staining agent, channels FL1 (green emission), FL2 (orange emission), and/or FL3 (dark red emission) were used for fluorescence detection.

For AnnV/PI, ApoStat, and AO staining, after 72 h treatment with the investigated agents, the cells were trypsinized and rinsed with PBS. Afterwards, cells staining according to the manufacturer's protocols was performed, with AnnV/PI in AnnV-binding buffer (15 min, room temperature), ApoStat in PBS 5% FBS (30 min, 37 ◦C) and AO 10 μM in PBS (15 min, 37 ◦C). Finally, upon washing, cells were resuspended in PBS (or in AnnV-binding buffer for AnnV/PI), and analyzed. For CFSE staining, pre-staining with PBS solution of CFSE (1 μM; 10 min, 37 ◦C) followed by washing and seeding of the cells was done, after which they were exposed to experimental agents over a period of 72 h. Before analysis, cells were once again washed, trypsinized and suspended in PBS. Similarly, pre-staining with 1 μM DHR for 20 min at 37 ◦C was first done also for DHR staining. For DAF-FM staining, the cells were first subjected to the experimental agents for 72 h, washed with PBS, and then stained with 5 μM DAF-FM diacetate in phenol red-free RPMI-1640 for 1 h at 37 ◦C. Thereafter, the dye was discarded and the cells were washed with PBS. Additional incubation of the cells in fresh RPMI-1640 not containing phenol red and serum, was done for 15 min to finish the reaction of de-esterification. Afterwards, cells were trypsinized, resuspended in PBS and analyzed. For analysis of cell distribution across cell cycle phases, after the treatments, 4T1 cells were fixed with 70% ethanol overnight at 4 ◦C, and then stained with PI (20 μg mL−1) in the presence of RNase (0.1 mg mL−1) during 45 min at 37 ◦C.

#### *2.11. In Vivo Studies*

Inbred female BALB/c mice (6–8 weeks old) from the Institute for Biological Research "Siniša Stankovi´c"—National Institute of Republic of Serbia (IBISS) were used for this study. Animals were kept in standard laboratory conditions, free of non-specific pathogens, with unlimited access to food and water. The handling of animals and the study protocol

adhered to national regulations established by the Law on Animal Welfare of the Republic of Serbia (Official Gazette of the Republic of Serbia No. 41/2009) and European Ethical Normative (Directive 2010/63/EU) on the protection of animals used for experimental and other scientific purposes. The national licensing committee at the Department of Animal Welfare, Veterinary Directorate, Ministry of Agriculture, Forestry and Water Management of Republic of Serbia granted approval for the experimental protocols (permission No. 323-07-07906/2022-05).

#### *2.12. Induction of Tumors and In Vivo Treatment*

BALB/c mice were orthotopically inoculated with 4T1 cells (2 × 104 cells in 50 <sup>μ</sup>L PBS) in the fat pad region of the fourth mammary gland region. On the fifth day following cell implantation when tumors were palpable, random assignment of the animals into different treatment groups was made. The administration regime consisted of i.p. application of the appropriate agent three times a week. Cisplatin was applied in a dose of 2 mg kg−<sup>1</sup> in 2% DMF/PBS, conjugate **1** at 5 and 10 mg kg−<sup>1</sup> in 2% DMSO/PBS, while SBA-15|**1** was applied in PBS at a dose of 17.5 and 35 mg kg−<sup>1</sup> for 25 days. Control mice were receiving 2% DMSO/PBS and 2% DMF/PBS as vehicle. Tumor growth was monitored every second day and on the 29th day after cell inoculation, mice were sacrificed. Tumors were extracted and measured in three dimensions. The tumor volume was calculated based on the following equation:

$$\mathbf{V} = 0.52 \times a \times b^2$$

where "*a*" is the longest and "*b*" the shortest diameter. On the day of sacrifice, urine was collected from the animals and different biochemical parameters were analyzed with Multistix 10 SG (Bayer, Leverkusen, Germany).

#### *2.13. Histopathological Examination*

Harvested tissues of sacrificed animals (tumor, liver and kidney) were macroscopically examined, measured and sectioned to 3 mm thick slices through the largest tissue plane. Tissues were processed in automatic tissue processor (Milestone SRL LOGOS ONE, Sorisole, BG–Italy). Embedding in paraffin blocks was done on embedding console (SAKURA Tissue-Tek TEC 5, Sakura Finetek, CA, USA). Sections 4 μm thick were cut from the tissue using microtome (LEICA RM 2245, (Leica Biosystems, Nussloch, Germany), and the slices were mounted on glass slides. The slides were then stained with hematoxilin eosin stain (H/E) in automated slide stainer MYREVA SS-30H (Especialidades Médicas MYR, S.L., Tarragona, Spain). After H/E staining, all glass slides were examined and microscopically analyzed under an Olympus BX43 microscope (OLYMPUS EUROPA HOLDING GMBH, Hamburg, Germany). Additionally, all slides were digitalized with a Leica Aperio AT2 slide scanner (Leica Biosystems, Nussloch GmbH, Germany) for analysis and documentation purposes. Morphometric analysis was done with a Leica Aperio ImageScope (version 12.4.6, Leica Biosystems, Nussloch GmbH, Germany).

#### *2.14. Statistical Analysis*

Differences between treatments were assessed using analysis of variance (ANOVA) accompanied with a Student-Newman-Keuls test. Results of in vivo experiments were evaluated by a Mann-Whitney test. Differences in histopathological changes between treatments were analyzed by a Oneway ANOVA test followed by a Tukey test. Statistical significance was considered if *p*-value was 0.05 or less. Statistical analyses were done using the program Statistica 10.

#### **3. Results and Discussion**

#### *3.1. Synthesis and Characterization of Platinum(IV) Conjugates* **1***–***3**

In this study, three cisplatin derivatives with acetyl-protected caffeate, acetyl-protected ferulate or free ferulate in the axial positions (complexes **1**, **2** and **3**, respectively, Figure 1) were prepared. Complexes **1** and **2** were synthesized by reacting oxoplatin (afforded by

cisplatin oxidation with hydrogen peroxide) [50] with an excess of the corresponding acyl chloride derivative of the acetyl-protected phenolic acid in the presence of pyridine as base. Acetyl-protected phenolic acids were used for the synthesis in order to avoid side reactions involving the hydroxyl groups present in their structure. Additionally, as reported by other authors, the reduction potential of the phenolic acids is directly linked to the existence as well as the number of hydroxyl groups [58,59]. Therefore, using caffeic and ferulic acid with free hydroxyl groups could induce reduction of the platinum(IV) in oxoplatin making the formation of the desired products impossible [60,61]. After complexes **1** and **2** were successfully obtained in yields of 64% and 59%, respectively, deprotection of **2** was done with conc. HCl to afford the cisplatin-ferulate conjugate **3**. The three obtained platinum(IV) conjugates were characterized by 1H, 13C and 195Pt NMR spectroscopy and mass spectrometry (ESI, Figures S1–S18) and their purity was further confirmed through elemental analysis. Deprotection of **1** was also attempted, employing different procedures and reagents, from conc. HCl to ones which have been reported to be mild and highly selective for deprotection of aromatic acetates such as guanidine·HCl [62], dibutyltin oxide [63] and ammonium acetate [64]. However, the desired cisplatin-caffeate conjugate could not be obtained; instead, degradation of the complex resulting in very complex 1H NMR spectra was observed in each deprotecting approach. Finally, we also attempted the deprotection using a commercially available enzyme Amano lipase A from *Aspergillus niger* (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) which has been reported to cause full *O*-deacetylation of different acetylated biomolecules under mild conditions [65]. Closely following the reaction by time-resolved 1H NMR spectroscopy (ESI, Figure S19), we noticed that as soon as the deacetylation of **1** begins, chemical shifts corresponding to free caffeate appear. This means that after deprotection, the free hydroxyl groups might reduce the platinum(IV) to platinum(II), leading to separation of the caffeate ligand from the cisplatin scaffold. This confirms the capacity of phenolic acids to induce metal reduction and the higher reduction potential of caffeic acid, which bears two hydroxyl groups in *ortho* position to each other, by comparison with ferulic acid, which has only one free hydroxyl group. As the deprotection of the acetyl protecting groups can occur under acidic conditions, it could be anticipated that the acidic tumor microenvironment would facilitate such a reaction in vivo. The free hydroxyl groups would then ease the reduction of the platinum(IV) to the cytotoxic platinum(II) and release the axial ligands, making both species available to act upon their respective targets.

The platinum(IV) conjugates are particularly poorly soluble in water (**1**–**3**: 0.09 g, 0.04 g and 0.56 g, respectively, in 1 L H2O) owing to the presence of lipophilic moieties. However, in polar aprotic solvents like DMF and DMSO, they are highly soluble.

As confirmed by time-resolved 1H NMR spectroscopy, the conjugates **1**–**3** are stable in DMSO solution over 72 h. During this time, no ligand exchange or complex degradation was observed (Figures S20–S22).

#### *3.2. Synthesis and Characterization of the Mesoporous Silica Materials*

For the preparation of SBA-15 nanoparticles, a synthesis described in the literature, with TEOS as a silica source and P123 serving as a structure-directing agent, was employed [52]. Calcination was performed to remove the organic template, resulting in rodshaped particles with consistent morphology as demonstrated by SEM imaging (Figure 2). Narrow size distribution of the particles ranging from 200–400 × 600–800 nm was observed.

Nitrogen physisorption experiments that resulted in a type IV isotherm with a hysteresis loop showing capillary condensation characteristic for highly organized mesoporous structures (Figure 3A) [66] were used to confirm the material's mesoporous nature. Carrying out these analyses also upon loading of the SBA-15 material with the platinum(IV) complexes **1**–**3** which afforded the three materials SBA-15|**1**, SBA-15|**2** and SBA-15|**3**, it was shown that the conjugates have not significantly influenced the SBA-15 structure, so the shape, morphology as well as the mesoporous nature of the materials are retained.

**Figure 2.** SEM images of MSNs: SBA-15, SBA-15|**1,** SBA-15|**2** and SBA-15|**3**.

**Figure 3.** (**A**) N2 adsorption-desorption isotherms and (**B**) SAXS patterns of MSNs: SBA-15, SBA-15|**1**–SBA-15|**3**.

As expected, after loading of the cisplatin derivatives, the high specific surface area, *S*BET (calculated based on the Brunauer–Emmett–Teller (BET) model), and pore volume, *V*p, which were corroborated for the SBA-15 material (517 m2 g−<sup>1</sup> and 0.72 cm3 g<sup>−</sup>1, respectively), were reduced to 245−292 m<sup>2</sup> <sup>g</sup>−<sup>1</sup> for the specific surface area and 0.34–0.37 cm<sup>3</sup> <sup>g</sup>−<sup>1</sup> for the pore volume. Additionally, there was a change of the pore diameter *D*<sup>p</sup> from 4.74 nm as determined for the pure SBA-15 to 3.68−4.75 nm for the loaded MSNs. On the contrary, the wall thickness *W*p, calculated by deducting the pore diameter from the lattice parameter *a*, increased upon immobilization of conjugates **2** and **3**, (3.66 nm 4.26 and 5.03 nm (SBA-15, SBA-15|**2** and SBA-15|**3**, respectively). The lattice parameter itself was attained by SAXS, and specifies the repetition rate of the hexagonal pores of the MSNs. Thus, the acquired data showing similar values for *a* for all pure and loaded MSNs are consistent with what is expected. Finally, as shown in Figure 3B, the SAXS patterns of pure SBA-15 and SBA-15|**1**–SBA-15|**3** exhibit diffraction peaks (see Table 1) typical for hexagonally ordered MSNs, highlighting the particles' unaltered structure once more. All corresponding values for *a* and 2*θ* are presented in Table 1.

The amount of immobilized platinum(IV) complexes **1**–**3** in the MSNs was calculated based on the amount of platinum detected with EDX analysis. Successful loading of all three compounds **1**–**3** with load contents of 7.51, 9.23 and 10.33 wt% Pt, respectively, was confirmed. The corresponding encapsulation efficiency was calculated to be 70.4%, 83.8% and 89.2%, for SBA-15|**1**–SBA-15|**3**, respectively (see ESI, Figure S23 and Tables S1 and S2).

Despite the successful production of the mesoporous material with the applied method, which resulted in MSNs with optimal drug loading, it is worth mentioning that significant advances have been made in the engineering of MSNs [67], including some greener approaches with less undesirable environmental impact [68], which should be considered in the future.

#### *3.3. Release of Platinum(IV) Conjugates from MSNs*

The in vitro release profiles of the platinum(IV) complexes **1**–**3** encapsulated in silica nanoparticles were determined in PBS (pH = 7.4) by assessing the platinum content in solution (ICP-OES) at predetermined time points (0.08, 0.5, 1, 3, 6, 12, 24, 48 and 72 h). As presented in Figure 4A, SBA-15|**1** and SBA-15|**2** exhibited very low release of only 18 and 5%, respectively, over 72 h. On the contrary, SBA-15|**3** released over 70% of its cargo over the same period of time. As these results are consistent with the solubility profiles of the three conjugates in water, it is probable that in this case drug release is driven by solubility of the drugs in the surrounding media. In the first hours, saturation of the solution is achieved and, therefore, prolonged contact with the fluid does not lead to increase the amount of platinum(IV) conjugate released. By exchanging the medium each hour in the time interval of up to 10 h, saturation of PBS with the drugs from nanomaterials SBA-15|**1** and SBA-15|**2** is avoided. As initially observed, the release is low, but continuous (Figure 4B). The obtained results indicate that the drug-loaded nanoparticles' structural integrity is maintained under the simulated physiological setting, preventing immediate leakage of the platinum(IV) complexes. In vivo, this could, in addition to the favorable EPR effect of MSNs, be very beneficial as it could imply minimal drug release during circulation, thus lowering the possibility of off-target interactions and side effects, and higher accumulation in the tumor, where the slow release could ensure prolonged drug exposure.

**Figure 4.** (**A**) Cumulative drug release (%) of the respective platinum(IV) conjugate from SBA-15|**1**, SBA-15|**2** and SBA-15|**3** over 72 h; (**B**) Cumulative drug release (%) of the respective platinum(IV) conjugate from SBA-15|**1** and SBA-15|**2** with exchange of the PBS solution every hour for 10 h.

Mathematical models *viz*. zero/first order, Korsmeyer–Peppas and Higuchi (see ESI, Figures S24–S26) were employed for a study of the kinetics of the drug release [69]. Data was quantitatively correlated with these models and the most suitable model was determined based on the degree of correlation R<sup>2</sup> (see ESI, Table S3). The results obtained

suggest that although the release of the cisplatin derivatives from the corresponding MSNs does not follow perfectly the principles of any of the investigated release kinetics (R2 < 0.9), diffusion-controlled drug release kinetics (Higuchi for SBA-15|**1** and Korsmeyer–Peppas for SBA-15|**2** and SBA-15|**3**) are the most fitting [70]. The value *n* obtained from the Korsmeyer–Peppas model (in all cases n < 0.45) further ascertained Fickian diffusion as prime release mechanism [71].

#### *3.4. Antiproliferative Activity*

The antiproliferative activity of the free platinum(IV) complexes **1**, **2** and **3** and the corresponding MSNs (SBA-15, SBA-15|**1**–SBA-15|**3**) was first determined against four human breast tumor cell lines, involving two triple-negative cell lines, namely MDA-MB-468 (human breast adenocarcinoma with COX-2 expression) and HCC1937 (human breast carcinoma with BRCA1 mutation), and the two ER- and PR-positive cell lines MCF-7 (with COX-1 expression) and BT-474 (both invasive ductal breast carcinoma). After 72 h of treatment with the examined compounds and MSNs, the cell viability was assessed using the two distinct assays, CV and MTT, and their activity was compared with that of clinically used cisplatin, as well as the phenolic acids used as ligands. IC50 and MC50 values are presented in Table 2. As expected, the ligands alone were found to be inactive (>100 μM) against all treated tumor cell lines. A similar observation was made for the SBA-15 material (>100 μg mL<sup>−</sup>1). On the contrary, the platinum(IV) conjugates **1–3** demonstrated antitumor activity in the range similar to (against MDA-MB-468 and HCC1937 cell lines) or higher (against MCF-7 and BT-474 cell lines) than cisplatin. Certain discrepancies were observed between the results acquired by MTT and CV assays against the MCF-7 cell line upon treatment with the platinum(IV) conjugates and against BT-474, HCC1937 and MDA-MB-468 cell lines upon treatment with cisplatin, implying that in these cases the mechanism through which cytotoxicity is achieved might be involving cell metabolism pathways [72–75].

**Table 2.** IC50 [μM] and MC50 [μg mL−1] values of cisplatin, **<sup>1</sup>**−**3**, free ligands, SBA-15, and SBA-15|**1**−SBA-15|**3** determined with CV and MTT assays (72 h).

(a) recalculated based on platinum content (EDX) and drug release (ICP-OES).

Immobilization of the platinum(IV) complexes **1**–**3** in MSNs led to a significant improvement of the antiproliferative activity of all three conjugates against all cell lines involved in the study. For comparative evaluation with the activity of cisplatin and the free platinum(IV) complexes, recalculation of the IC50 values for the drug-loaded MSNs (SBA-15|**1**, SBA-15|**2** and SBA-15|**3**) was made based on the obtained MC50 values considering the platinum content obtained by EDX, as well as the release of the drugs obtained by ICP-OES (detailed explanation in ESI). Submicromolar IC50 values which were at least two times and in some cases several thousand times lower than those for cisplatin were obtained. All drug-loaded MSNs showed the highest activity against the BT-474 cell line responsible for the development of invasive ductal breast cancer and known to be resistant

to the conventional treatment with tamoxifen [76,77]. Among the three drug-loaded MSNs, SBA-15|**1** was found to have the highest potency against this cell line with an IC50 value of only 0.1 nM.

Considering these very promising results, further investigations were necessary in an appropriate in vivo breast cancer model; thus, an orthotopic model of breast cancer by inoculation of 4T1 cells in BALB/c mice was selected. Therefore, the antiproliferative activity of the most active conjugate **1** and appropriate nanomaterial SBA-15|**1** was first assessed in vitro in this triple-negative mouse cell line. After 72 h of incubation, the viability of the cells was determined by measurement of total cell culture respiration or number of adherent cells using MTT and CV assays. The results summarized in Table 2 reveal that conjugate **1** strongly affected the cell viability of 4T1 cells similarly as cisplatin alone. Higher activity exceeding the one of cisplatin and **1** was observed upon drug-loading into SBA-15 particles, which is consistent with the results seen in the human breast cancer cell lines. Cell viability under **1**–**3**, SBA-15|**1**–SBA-15|**3** and cisplatin exposure is presented in the ESI, Figures S27–S31.

#### *3.5. Molecular Mechanism of Action*

Aiming to get an insight in the plausible mechanism of drug-induced cytotoxicity, flow cytometric evaluation of apoptotic cell death, cell cycle perturbation, rate of cell proliferation, caspase activation, and induction of autophagic response was employed upon treatment of the 4T1 cells with IC50 doses of **1** and cisplatin and an MC50 dose of SBA-15|**1**. The obtained results reveal that in all applied treatments, induction of massive apoptosis was in the background of diminished cell viability (Figure 5A). In concordance, significant accumulation of fragmented DNA in subG compartment of the cell cycle (Figure 5B), together with a strong caspase activation (Figure 5C) was found in cultures exposed to **1**, SBA-15|**1** and cisplatin. In parallel, surviving cell subpopulation lost the dividing potential (CFSE assay, Figure 5D). Since the process of autophagy is deeply involved in the cells' response to toxic stimuli, varying from protective to destructive, it is very important to define the eventual presence of autophagosomes in cultures exposed to the experimental therapeutics [78,79].

An intensified autophagic process (AO assay, Figure 5E) in all treated cultures was noticed. However, additional decrease in cell viability was observed also upon neutralization of autophagy through concomitant exposure of the treated cells to the specific inhibitor 3-MA (Figure 5F). Thus, the obtained data suggest that autophagy has a protective role, defending the cell from destructive stimuli.

In addition, treatment with conjugate **1** and SBA-15|**1** triggered increased production of nitric oxide (DAF-FM assay, Figure 6A), as well as ROS/RNS (DHR assay, Figure 6B).

Altogether, platinum(IV) conjugate **1**, free and loaded in SBA-15, clearly showed potential to downregulate cell growth through inhibition of proliferation and apoptotic cell induction which is even amplified with autophagy suppression. Behind the observed effect could be the enhanced production of reactive species which affect different cellular processes and subsequently the cell viability [80,81].

**Figure 5.** Effect of platinum(IV) conjugate **1** and corresponding MSNs SBA-15|**1** on 4T1 mouse breast cancer cells: (**A**) apoptosis (Annexin V-FITC/propidium iodide, (**B**) cell cycle—subG phase (\* *p* < 0.05 refers to untreated cells), (**C**) caspase activation (Apostat), (**D**) cell proliferation potential (CFSE), (**E**) induction of autophagy (AO staining), and (**F**) effect of 3-MA addition on viability of 4T1 cells (MTT assay) (\* *p* < 0.05 refers to culture treated with **1**, # *p* < 0.05 refers to culture treated with SBA-15|**1**).

#### *3.6. In Vivo Decrease of Breast Cancer Growth*

With the aim to check the effectiveness of conjugate **1** and SBA-15|**1** in vivo, an orthotopic model of breast cancer was used. Tumors were induced by inoculation of 4T1 cells in fat pad of the fourth breast in syngeneic BALB/c mice, and treatment started when tumors became palpable. Cisplatin was applied in a therapeutic dose [82], while **1** or SBA-15|**1** were applied in an equimolar dose with regards to cisplatin. Results revealed approximately 50% tumor volume reduction upon treatment with cisplatin and SBA-15|**1**, while conjugate **1** alone decreased the tumor volume by 20% (Figure 7A). As statistical significance was not reached after the application of the mentioned doses, the experiment was repeated with a dose of 10 mg kg−<sup>1</sup> for the free conjugate **1** and 35 mg kg−<sup>1</sup> for SBA-15|**1**. The obtained results revealed remarkable inhibition of tumor growth in all three experimental groups (Figure 7B). The experiment was concluded with the sacrifice of the animals and histopathological examination of their tumors, liver and kidney.

Average percentage of tumor's necrosis on tissue cross section was the largest in the control group (30%), while all treatments dramatically decreased necrotic area surface (conjugate **1** (14%), SBA-15|**1** (14%) and cisplatin (12%), Figure 7C). In addition, mitotic index, represented as mitotic figure count per one square millimeter, again was the highest in the control group, while rate of mitosis was diminished in tumor tissue sections of all treated groups (Figure 7B,D). Having in mind that tumors with large necrotic areas are recognized as highly aggressive and associated with poor prognosis [83], reduced necrotic zones together with lower mitotic rate upon the treatments indicates decreased neoplastic potential of the tumor tissue and underlines the advantage of the applied treatments.

In liver, findings characteristic for 4T1 metastatic breast cancer model with presence of extramedullary hematopoiesis, mainly represented by cells of myeloid lineage and in lesser amount by megakaryocytes, were observed [84,85]. Hematopoietic cells were present around and within the portal tracts, as well as in the sinusoid of the liver. The highest level of hematopoiesis was found in the control group, while upon treatment, in all treated groups it was almost absent (Figure 8). This fact led to the conclusion that all three treatments were effective in a way that inhibits hematopoiesis which was conditioned with the amount of tumor's cell burden.

No relevant signs of hepatotoxicity were found in any of the groups. There was no inflammation nor necrosis in this experimental setting (Figure 8). Some degree of dilatation of small blood vessels (mainly venules) mostly in the SBA-15|**1** group, without knowledge of functional derangement, was noted.

In kidney tissue, there were no significant signs of nephrotoxicity in all groups. Normal histomorphology was dominantly registered (Figure 8). Concordantly, all parameters (blood, bilirubin, pH, ketone, protein, specific gravity, leukocyte, urobilinogen and nitrite) tested in urine of animals which were exposed to treatment, showed no difference to the control group of untreated animals (ESI, Table S4). In the group treated with cisplatin, however, protein casts were found in the tubules, and dilatation of small periglomerular blood vessels was observed. This indicates that despite the fact that previous findings suggested the absence of toxicity, cisplatin has a nephrotoxic input [86] which was not observed for **1** and SBA-15|**1**.

**Figure 7.** In vivo effect of platinum(IV) conjugate **1** and the corresponding MSNs SBA-15|**1** on 4T1-inoculated breast tumors: (**A**) tumors' growth suppression, (**B**) tumor necrotic areas, (**C**) mitotic index, and (**D**) mitotic figures (marked with yellow arrows, H/E stain, 400× magnification).

**Figure 8.** In vivo effect of platinum(IV) conjugate **1** and the corresponding MSNs SBA-15|**1** on 4T1 inoculated breast tumors—histopathological evaluation: tissues in columns 1, 2, 3, and 4 represent control, cisplatin, **1** and SBA-15|**1** groups, respectively. Tissues in rows **A** and **B** are tumor tissues (8× and 40× magnification, respectively) with red lines delineating necrotic areas and green lines denoting largest diameter of tumor. Row **C** is liver tissue with red arrows pointing to hematopoietic cells (40× magnification). Row **D** is kidney tissue (200× magnification). In picture **2D**, black arrows point to protein cast in kidney tubules and red arrows point to dilated blood vessels. Red arrows in **3D** and **4D** point to areas of inflammatory infiltrate.

#### **4. Conclusions**

In summary, we report on three cisplatin derivatives bearing acetyl-protected caffeate (**1**), acetyl-protected ferulate (**2**) or ferulate (**3**) as axial ligands. Upon loading of these platinum(IV) complexes into mesoporous silica SBA-15 material, the MSNs SBA-15|**1**, SBA-15|**2** and SBA-15|**3** were prepared. Under simulated physiological settings, the MSNs maintained their structural integrity, enabling a moderate (for SBA-15|**3**) to extremely slow release (for SBA-15|**1** and SBA-15|**2**) of the appropriate platinum(IV) complex. This delayed release is particularly advantageous in vivo because it results in low drug release during circulation and longer drug exposure upon accumulation in the tumor.

The free platinum(IV) conjugates **1**–**3** demonstrated higher or similar antiproliferative activity with respect to cisplatin in four human breast cancer cell lines (BT-474, MCF-7, MDA-MB-468 and HCC1937). Great anticancer potential was, however, observed for all three complexes upon their loading into the nanostructured SBA-15. Thus, SBA-15|**1**, SBA-15|**2** and SBA-15|**3** demonstrated significantly higher cytotoxicity as compared to cisplatin with nanomolar IC50 values in all four cell lines.

For the most potent complex **1** and the corresponding SBA-15|**1**, mechanistic investigations made in the mouse breast cancer cell line 4T1 indicated the potential of drug and nanomaterial to downregulate cell growth through inhibition of proliferation and apoptotic cell induction. Drug toxicity can be further amplified via autophagy suppression. All observed effects could be due to the enhanced production of NO and ROS/RNS, which affect different cellular processes and, consequently, the cell viability. Furthermore, **1** and SBA-15|**1**, caused in vivo inhibition of tumor growth in BALB/c mice, decreasing the necrotic area and lowering the mitotic rate. Importantly, nephrotoxicity present in mice treated with cisplatin, was not observed when **1** or SBA-15|**1** were used.

**Supplementary Materials:** The following supporting information can be downloaded at https://www. mdpi.com/article/10.3390/nano12213767/s1. Figure S1. 1H NMR spectrum of **1** in DMSO-d6; Figure S2. 13C{1H} NMR spectrum of **1** in DMSO-*d*6; Figure S3. 195Pt{1H} NMR spectrum of **1** in DMSO-*d*6; Figure S4. HR-ESI-MS (positive mode, CH3OH) of **1**, *m*/*z* [2M+H]+; Figure S5. HR-ESI-MS (positive mode, CH3OH) of **1**, *m*/*z* [M+H]+; Figure S6. 1H NMR spectrum of **2** in DMSO-*d*6; Figure S7. 13C{1H} NMR spectrum of **2** in DMSO-*d*6; Figure S8. 195Pt{1H} NMR spectrum of **2** in DMSO-*d*6; Figure S9. HR-ESI-MS (positive mode, CH3OH) of **2**, *m*/*z* [2M+H]+; Figure S10. HR-ESI-MS (positive mode, CH3OH) of **2**, *m*/*z* [M+H]+; Figure S11. HR-ESI-MS (positive mode, CH3OH) of **2**, *m*/*z* [M+Na]+; Figure S12. 1H NMR spectrum of **3** in DMSO-*d*6; Figure S13. 13C{1H} NMR spectrum of **3** in DMSO-*d*6; Figure S14. 195Pt{1H} NMR spectrum of **3** in DMSO-*d*6; Figure S15. HR-ESI-MS (positive mode, CH3OH) of **3**, *m*/*z* [M+H]+; Figure S16. HR-ESI-MS (positive mode, CH3OH) of **3**, m/z [2M+H]+; Figure S17. HR-ESI-MS (positive mode, CH3OH) of **3**, *m*/*z* [2M+Na]+; Figure S18. HR-ESI-MS (positive mode, CH3OH) of **3**, *m*/*z* [2M+K]+; Figure S19. Attempted *O*-deacetylation of **1** with enzyme Amano lipase A from *Aspergillus niger*; time-resolved 1H NMR spectra in DMSO-*d*6, aromatic region section; Figure S20. Stability of **1** in DMSO-*d*<sup>6</sup> over 72 h; time-resolved 1H NMR spectra; Figure S21. Stability of **2** in DMSO-*d*<sup>6</sup> over 72 h; time-resolved 1H NMR spectra; Figure S22 Stability of **3** in DMSO-*d*<sup>6</sup> over 72 h; time-resolved 1H NMR spectra; Figure S23. Spatial distribution of silicon and platinum in SBA 15|**1**, SBA 15|**2** and SBA 15|**3** determined through EDX mapping; Figure S24. Zero order, first order, Higuchi and Korsmeyer-Peppas kinetic release of **1** from SBA-15|**1**; Figure S25. Zero order, first order, Higuchi and Korsmeyer-Peppas kinetic release of **2** from SBA-15|**2**; Figure S26. Zero order, first order, Higuchi and Korsmeyer-Peppas kinetic release of **3** from SBA-15|**3**; Figure S27. Cell viability of cisplatin, **1**, **2** and **3** determined by CV and MTT assays in MDA-MB-468 and HCC1937 human breast cancer cell lines; Figure S28. Cell viability of cisplatin, **1**, **2** and **3** determined by CV and MTT assays in MCF-7 and BT-474 human breast cancer cell lines; Figure S29. Cell viability of SBA-15|**1**, SBA-15|**2** and SBA-15|**3** determined by CV and MTT assays in MDA-MB-468 and HCC1937 human breast cancer cell lines; Figure S30. Cell viability of SBA-15|**1**, SBA-15|**2** and SBA-15|**3** determined by CV and MTT assays in MCF-7 and BT-474 human breast cancer cell lines; Figure S31. Cell viability of cisplatin, **1** and SBA-15|**1** determined by CV and MTT assays in mouse-derived 4T1 breast cancer cell line; Table S1. Relative weight % of silicon and platinum at six points in SBA-15|**1**, SBA-15|**2** and SBA-15|**3** determined by EDX analysis; Table S2. Platinum load (wt%) and encapsulation efficiency (%) in SBA-15|**1**, SBA-15|**2** and SBA-15|**3**; Table S3. Constants and coefficient of determinations (R2) for each model; Table S4. Urine parameters of untreated animals (control) and animals exposed to treatment with cisplatin, **1** and SBA-15|**1**.

**Author Contributions:** I.P., S.M., D.M.-I., E.H.-H. and G.N.K. conceptualized the project. I.P., M.S., I.M., T.E., T.K., E.M., D.D. (Dijana Draˇca) and D.D. (Duško Dunderovi´ ¯ c) did investigation and formal analysis of data. I.P., S.M. and D.M.-I. did data curation. I.P., D.D. (Duško Dunderovi´ ¯ c), S.M. and D.M.-I. wrote the original draft. S.M., D.M.-I., E.H.-H. and G.N.K. supervised the work and reviewed and edited the original draft. S.M., E.H.-H. and G.N.K. acquired the funding and administrated the project. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was financially supported through the program FEM POWER (funded by the operational program ESF Saxony-Anhalt WISSENSCHAFT Chancengleichheit with funds from the European Union; doctoral grant for I.P.), the Graduate School "Building with Molecules and Nanoobjects (BuildMoNa)", the Research Academy Leipzig and the Ministry of Education, Science and Technological Development of the Republic of Serbia, grant number 451-03-68/2022-14/200007.

**Institutional Review Board Statement:** The animal study protocol was approved by the national licensing committee of Department of Animal Welfare, Veterinary Directorate, Ministry of Agriculture, Forestry and Water Management of Republic of Serbia (permission No. 323 07 07906/2022 05).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The authors thank Prof. Dr. Valentin Cepus for the ICP-OES measurements and Prof. Dr. Stefan Ebbinghaus for the SAXS measurements.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

## *Article* **Silver Nanoparticles Functionalized Nanosilica Grown over Graphene Oxide for Enhancing Antibacterial Effect**

**Qui Quach and Tarek M. Abdel-Fattah \***

Applied Research Center at Thomas Jefferson National Accelerator Facility, Department of Molecular Biology and Chemistry, Christopher Newport University, Newport News, VA 23606, USA

**\*** Correspondence<EMAIL_ADDRESS>
**Abstract:** The continuous growth of multidrug-resistant bacteria due to the overuse of antibiotics and antibacterial agents poses a threat to human health. Silver nanoparticles, silica-based materials, and graphene-based materials have become potential antibacterial candidates. In this study, we developed an effective method of enhancing the antibacterial property of graphene oxide (GO) by growing nanosilica (NS) of approximately 50 nm on the graphene oxide (GO) surface. The structures and compositions of the materials were characterized through powdered X-ray diffraction (P-XRD), transmission electron microscopy (TEM), scanning electron microscopy coupled with energy dispersive X-ray spectroscopy (SEM-EDS), ultraviolet–visible spectroscopy (UV–VIS), dynamic light scattering (DLS), Raman spectroscopy (RM), Fourier-transform infrared spectroscopy (FTIR), Brunauer–Emmet–Teller (BET) surface area, and pore size determination. The silver nanoparticles (AgNPs) with an average diameter of 26 nm were functionalized on the nanosilica (NS) surface. The composite contained approximately 3% of silver nanoparticles. The silver nanoparticles on nanosilica supported over graphene oxide (GO/NS/AgNPs) exhibited a 7-log reduction of *Escherichia coli* and a 5.2-log reduction of *Bacillus subtilis* within one hour of exposure. Both GO/NS and GO/NS/AgNPs exhibited substantial antimicrobial effects against *E. coli* and *B. subtilis*

**Keywords:** silver nanoparticles; nanosilica; graphene oxide composites; antibacterial effect; *Escherichia coli*; bacillus subtilis

#### **1. Introduction**

Multidrug-resistant bacteria have become a global health threat in the 21st century [1]. Antibiotic resistance has caused common infections to become more difficult to treat and has led to expensive healthcare costs [1]. In order to reduce the dependency on antibiotic treatments which are ineffective against resistant bacterial strains, various studies have been conducted to develop novel materials to treat and prevent the growth of these pathogens [2–5]. Among those materials, silver nanoparticles appear to have the most potential and be the most attractive solution due to their antibacterial properties [6,7]. However, some studies have shown that bacteria can develop resistance to silver nanoparticles after repeated exposure [8,9]. The resistant strains produced adhesive flagellum that caused the nanoparticles to aggregate, resulting in a reduction in the surface area and antibacterial effect [8,9].

In previously reported studies, support templates or capping agents were often used to improve the size, shape, and stability of nanoparticles [10–14]. It is important to control the surface properties, morphology, and functionality for bio-medical applications [15–17]. Among the materials, silica-based and graphene materials are excellent support templates for enhancing antimicrobial activity [18,19]. These materials are well-known in medicine and biotechnology [19–21]. In a study from Liu et al., silver nanoparticles were loaded on to the graphene oxide and a large silicate template, but the synthesis process required heating which caused the graphene oxide to reduce [22]. Previous research reported that graphene

**Citation:** Quach, Q.; Abdel-Fattah, T.M. Silver Nanoparticles Functionalized Nanosilica Grown over Graphene Oxide for Enhancing Antibacterial Effect. *Nanomaterials* **2022**, *12*, 3341. https://doi.org/ 10.3390/nano12193341

Academic Editors: Goran Kaluderovi´ ¯ c and Nebojsa Pantelic

Received: 18 August 2022 Accepted: 20 September 2022 Published: 25 September 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

oxide had a higher antibacterial ability than that of the reduced form [23]. Additionally, the large mesoporous silica template was less effective than small-sized silica nanoparticles in improving the antimicrobial property of the composite [24]. Based on different review studies, the combination of graphene oxide, nanosilica, and silver nanoparticles was neither synthesized nor tested for antibacterial activity [25,26]. Furthermore, the method which we applied in this study to grow nanosilica on graphene oxide was different from other reported methods [27–29]. In a Yang et al. study, the SiO2 was functionalized with an amine group and then loaded on to the graphene oxide; the pH of the synthesis process was required to be 7 for the coupling process to be successful [27]. Another study by Kou and Gao grew the silica nanoparticles on the graphene oxide with the assistance of ammonia, but the resulting product had to be stored in alcohol [28]. In our study, our product was formed in a basic condition and was stored without using any solvent.