**1. Introduction**

The conservation of stones in historical buildings around the world is receiving a growing amount of interest due to the importance of preserving historical memory for new generations. Different authors have highlighted the importance of stone conservation using varied scientific approaches due to the complexity and diversity of problems that require solutions. Stone conservation is an area where chemistry, physics, material science, biology, history, architecture, archeology, restoration, geology, etc. find a common point of confluence [1–4].

In an overview of research on stone conservation, the process referred to as consolidation is considered to be an active conservation process "where stone is severely weakened by decay, and some form of consolidation may be necessary to restore some strength. Ideally, one might hope to make the stone at least as strong as it was originally, so it might resist further decay" [5]. The same overview points out that "some of the causes of stone decay are sudden and rapid in their effect. Those toward the latter part of the following list are slow and more insidious: earthquake, fire, flood, terrorism, vandalism, neglect,

tourism, previous treatments, wind, rain, frost, temperature fluctuations, chemical attack, salt growth, pollution, biodeterioration, intrinsic factors, and so on" [5].

Stone consolidants have been widely used to protect historical monuments. Commercial products containing alkoxysilanes, such as tetraethoxysilane (TEOS), are commonly applied as consolidants for decayed natural stones. They are applied as low viscosity monomers or dimers in solutions that may include water, ethanol, or other organic solvents (generally MEK and acetone). An organometallic catalyst such as di-n-butyltin dilaurate (DBTL) is commonly used to increase the rate of polycondensation. Hypothetically, the fluid deeply infiltrates the porous network of the stone and, through a sol–gel process, forms a silica gel that works as a new cement for the matrix. This is done to re-establish the cohesion between loosened material grains as well as to restore the original mechanical resistance in the decayed material. The use of alkoxysilane-based products as stone consolidants to conserve decayed quartz-bearing rocks, such as sandstone, or siliceous natural materials in general, has been a common practice for several decades [6], although the first patent was approved much earlier in 1924 [7].

However, in the case of calcareous stones, their non-compatibility with alkoxysilanes has been established, so several approaches have been investigated [8–10].

In a recent review regarding the application of alkoxysilanes in the field of stone conservation, it is pointed out that conventional alkoxysilane-based consolidants have several drawbacks that hinder their successful application in carbonate stones. In terms of addressing these issues, the modification of alkoxysilanes has resulted in some improvements. The cracking tendency of alkoxysilanes has been solved by the introduction of elastic segments, surfactant templates, and nanoparticle loading, in addition to other elements. Nevertheless, there is still much room for improvement. The complexity of sol–gel chemistry and the conceptual incompatibility between alkoxysilane-based consolidants and carbonate minerals do not allow for successful prediction of the consolidation behavior. The proposed consolidants often render the treated stone hydrophobic, which is interesting if the objective is to obtain both a hydrophobic and consolidation treatment, although the risk of incompatibility exists. Despite the drawbacks, the study of new alkoxysilanebased consolidants that can provide multifunctional (consolidant, self-cleaning, or biocide) properties is still being conducted. The development of such properties again makes alkoxysilanes a feasible alternative for consolidating carbonate stones in important stone heritage if their compatibility and durability are effective. Of course, any consolidation action should not hinder or limit future interventions. In addition, due to the complexity of the interaction between the alkoxysilanes and the stone, a more detailed study of the mechanism is needed [11].

A recent investigation addresses the impact and importance of the carbonate medium in the sol–gel processes of stone consolidation based on alkoxysilanes and the possible detrimental effects in practical applications, highlighting the need for the design and development of new alkoxysilane-based consolidants to consider this effect [12].

The interest of our research group in the use of alkoxysilanes in the conservation of historical building stone has led to investigations on many topics, such as chemical, physical, geological, and mineralogical characterization of historical building stones; considering decay and biodecay evaluation; and the synthesis and application of consolidant and hydrophobic formulations based on alkoxysilanes in siliceous stones, with the aim of solving reported problems in the performance of some of the commercial formulations through the development of hybrid systems based on TEOS/SiO2St/PDMS-OH. Most of our work in the field has focused on siliceous stones [13–21].

Tetraethoxysilane (TEOS) and alkylalkoxysilanes are widely used as precursors in consolidant and hydrophobic formulations. Due to their lack of solubility in water, the formulation must be applied in organic solvents (VOCS), promoting a clear disadvantage in terms of "green chemistry" in this field. In the search for "greener" silicon derivatives with potential use as consolidants, the use of tetrakis(2-hydroxyethyl)silane (THEOS) and tris(2-hydroxyethyl)methyl silane (MeTHEOS) as precursors is suggested because of their remarkable water solubility. Initial studies of the THEOS precursor were conducted by Mehrotra and Narain [22] and, subsequently, introduced by Hoffmann and his group through the transesterification reaction of TEOS and ethylene glycol. The complete characterization of properties achieved by Hoffman demonstrated that THEOS possesses high solubility and stability in water and, as a result, the use of typical organic solvents is suppressed [23].

On the other hand, in 2004, Shchipunov, Tatyana, and Karpenko reported the compatibility of THEOS with different natural polysaccharides, including chitosan. As observed by the authors, polysaccharides worked as accelerators, catalysts, and templates for the silica generated in situ by the sol–gel process; modification of the synthesis conditions led to different properties and structures suitable for obtaining monolithic hybrid materials. For the THEOS–chitosan system, a transparent hybrid material was obtained, and no phase separation or syneresis was detected [24–27].

An important aspect of the hybrid THEOS–chitosan was described when it was shown that it accelerated, catalyzed, and served as a template for silica generated in situ by sol–gel, thus manipulating its synthesis as well as the properties and structure of the produced monolithic hybrid materials [24]. The potential application in drug encapsulation is an example of a very important and actual field of investigation where these biocompatible hybrid materials can have an impact [28]. The hydrolysis of THEOS produces silicic acid. As a result of the complete compatibility with chitosan, the presence of sol–gel in solution results in gelation of the non-gellable chitosan as well as other polysaccharides. For example, the gelation by mineralization of carbohydrate macromolecules strengthened them and provided cross-linking [25]. The gelation time required for the sol–gel transition and the dynamic rheological properties of the resultant gel matrix could be modulated by the amount of added THEOS. The hybrid material has found application in electrochemical biosensors [27,29,30] and is able to obtain stable and intact thin films or monolithic hybrid gels [31].

On the other hand, it is important to consider that the hybrid has the presence of primary amines and hydroxyl groups in the chitosan structure, and such functionalities have a key effect on the biopolymer solubility. Additionally, they can act as reactive sites for the covalent interaction via condensation in the chitosan–siloxane network. In this direction, we have recently reported a detailed study to elucidate the covalent interaction between reactive silanols (from the complete hydrolysis of THEOS and MeTHEOS) and chitosan. The results sugges<sup>t</sup> that the condensation site forming the silyl–ether bond is located at C6 of the chitosan structure [32].

The characteristics developed by the hybrid system THEOS–chitosan enable us to sugges<sup>t</sup> a new application in the area of conservation of historical stone buildings that is based on the preparation of hybrid consolidant and hydrophobic formulations that can be applied in the conservation of stones with a siliceous and calcareous composition; THEOS–chitosan can be used in the consolidant formulation and MeTHEOS in the hydrophobic formulation. Bearing in mind that a grea<sup>t</sup> number of important historical monuments in different parts of the world have siliceous or calcareous stones as building materials, some advantages can be considered in the introduction of glycol alkoxysilane–chitosan in the field of stone conservation, i.e., that the "green" formulations based on water-soluble silanes do not need organic solvents for their application, these hybrid formulations can potentially be used as an alternative to solve the problem of compatibility between alkoxysilanes and calcareous stone, and the synergy originating from the alkoxysilane–chitosan interaction in terms of the film formation capacity of chitosan, as well as the antimicrobial activity, suggests the possibility to avoid the biodegradation of stones by many organisms. It is important to consider that the material aptitude to biological colonization by certain organisms is called the bioreceptivity and is dependent on different environmental factors, such as the pH, water availability, climate exposure, mineral composition, porosity, permeability, and nutrient sources [5,33].

The antimicrobial activity of chitosan has been widely studied; for example, the antibiofilm properties of chitosan-coated surfaces, where chitosan offers a flexible, biocompatible platform for designing coatings to protect surfaces from infection [34]. The state of the art of antimicrobial chitosan and chitosan derivatives and the effects of structural modifications on the activity and toxicity have been reviewed toward improving the understanding of the bioactivity and to develop more useful chitosan conjugates [35]. On the other hand, it has been pointed out that chitosan and its derivatives can be called environmental purification functional materials as they can effectively control the growth and reproduction of hazardous bacteria and also control toxic pollutants [36]. The antibacterial activity of chitosan extracted from a pen shell against both Gram-positive and Gram-negative bacteria was recently reported [37]. Furthermore, chitosan and its derivatives have been studied due to their antimicrobial properties in the context of preventing and treating denture stomatitis, which can be caused by fungi [38].

#### **2. Results and Discussion**

The results and discussion section considers two main aspects: synthesis and characterization of the formulations THEOS–chitosan and MeTHEOS–chitosan and their application in siliceous and calcareous building materials. Films of the silane–chitosan hybrids were obtained and characterized using different methods. Such characterization was useful for revealing the film behavior inside or on the stones as a result of the consolidation or hydrophobic treatment.

The precursors of the hybrid formulations—THEOS and MeTHEOS—were obtained according to the most reported and used method (the transesterification reaction of TEOS or MeTEOS with ethylene glycol) [22,23]. At the reaction conditions used (140 ◦C and 15 h of reaction time), high yields are obtained (83% to 92%). Nevertheless, the morphology of the product obtained from different syntheses under the same conditions is diverse (liquid, translucid viscous, or gel), without altering the water solubility. The analysis of the transesterification reaction products (THEOS and MeTHEOS) was conducted by 29Si-NMR in solution (DMSO-d6), in respect of the TEOS and MeTEOS spectra as the reference (singlet at −82.5 ppm and −43 ppm, respectively). As demonstrated in previous studies and by recent observations, THEOS does not exist as a single molecule, as can be seen from the diverse morphology that the isolated product exhibits, and various silicon species appear in the Q unit region ( −81.7, −82.3, −83.6, and −88.6 ppm) for THEOS; for MeTHEOS ( −41.7, −44.1, −49.8, −52.3, and −58.2 ppm), different peaks appear in the T unit region [32,39,40].

#### *2.1. Synthesis and Characterization of Silane–Chitosan Hybrid Films*

A very wide range of tests of THEOS–chitosan and MeTHEOS–chitosan solutions were prepared, using different proportions of the reagents, in order to find out the most appropriate formulations to be applied in stone treatment. The selection of formulations to be used was carried out through observation of the film characteristics obtained in terms of the flexibility, homogeneity, transparency, and resistance to syneresis, where the excellent capability of chitosan as a film formulation was a key aspect in terms of the concentration of chitosan used. No phase separation was observed. The extensive testing revealed that a selection of formulations with similar physical characteristics were obtained, offering the possibility to apply them in experiments with different goals (see Materials and Methods). For example, the selected formulation for the films characterized by Fourier-Transform Infrared Spectroscopy-Attenuated Total Reflectance (FTIR-ATR), Scanning Electron Microscopy-Energy Dispersed X-ray Spectroscopy (SEM)–EDX, thermal stability, and solid-state Nuclear Magnetic Resonance (NMR) analysis was based on 10 mL of an aqueous solution of chitosan (0.5% in acetic acid at 1% with 72% deacetylation) and 0.5 g of THEOS. Some variations in the formulation composition were used in several analyses, such as hardness and contact angle determinations, in order to study the effect of the silane–chitosan ratio (formulations referred to as 1, 2, and 3 in Materials and Methods).
