From Synthetic Route of Silica Nanoparticles to Theranostic Applications

Abstract

The advancements in nanotechnology have quickly developed a new subject with vast applications of nanostructured materials in medicine and pharmaceuticals. The enormous surface-to-volume ratio, ease of surface modification, outstanding biocompatibility, and, in the case of mesoporous nanoparticles, the tunable pore size make the silica nanoparticles (SNPs) a promising candidate for nano-based medical applications. The preparation of SNPs and their contemporary usage as drug carriers, contrast agents for imaging, carrier of photosensitizers (PS) in photodynamic, as well as photothermal treatments are intensely discussed in this review. Furthermore, the potential harmful responses of silica nanoparticles are reviewed using data obtained from in vitro and in vivo experiments conducted by several studies. Moreover, we showcase the engineering of SNPs for the theranostic applications that can address several intrinsic limitations of conventional therapeutics and diagnostics. In the end, a personal perspective was outlined to state SNPs’ current status and future directions, focusing on SNPs’ significant potentiality and opportunities.

1. Introduction

Nanomedicine is defined as the use of nanotechnology in medical science, which provides nanoscale materials for the diagnosis, treatment, and screening of diseases [1,2,3]. The use of nanosized drug moieties can achieve targeted delivery with increased bioavailability and therapeutic efficacy [4,5]. Various nanomedicine-based drug delivery systems have been utilized in the development and targeted drug delivery systems of different biomolecules [6,7,8,9,10,11]. It is clear that biomedical applications of a large number of nanomaterials, such as silica nanoparticles (SNPs), graphene, gold nanoparticles, superparamagnetic iron oxide nanoparticles (SPIONs), and other metal and metal oxide nanoparticles, are receiving more attention [12,13,14,15,16]. Among all other nanomaterials, the use of silica nanoparticles in medicine has been increased due to their excellent properties [12]. Silica nanoparticles are usually spherical amorphous materials, and they can be fabricated in a wide range of sizes from 1 to 100 nm [17,18]. Their straightforward synthesis techniques distinguish SNPs for reliable delivery mechanisms and the chemistry of their surfaces can be adjusted to suit required purposes.

In the past few decades, silica nanoparticles have become a key component of biological imaging and the delivery of drugs and genetic material, due to their chemical and physical stability, well-defined hydrophilic surfaces, and their ability to protect drugs from active immune responses [19,20]. SNPs are available in many forms, including core-shell silica nanoparticles, nonporous SNPs, hollow mesoporous silica nanoparticles (HMSN), and mesoporous silica nanoparticles (MSN) [21]. There are several physicochemical parameters associated with SNPs, such as size, shape, porosity, and surface area, that play a crucial role in the delivery of the payloads to the target site and their subsequent elimination from the body [22,23]. The size of the silica nanoparticles is one of the most important factors that determine their uptake by cells, as well as their biocompatibility, and it is controlled by varying the reaction parameters [24,25]. The shape of silica nanoparticles has also been found to be a key player in modulating their physiological behavior and activity [26,27]. A wide variety of therapeutic and diagnostic applications use nanospheres and nanorods with different aspect ratios as SNPs [28]. Nanoparticle uptake and biodistribution are primarily influenced by size and shape, but payload delivery was more affected by porosity.

Silica nanoparticles have received more interest in medicinal applications than silicon, popularly known as silicon quantum dots, because of their wide range of commercial approaches in pharmaceuticals, pigments, humidity sensors, and medical insulators [29,30]. The structure of mesoporous silica nanoparticles (MSNs) is highly porous and can load many drugs and other desired agents. Fluorescence molecules loaded on the MSNs are kept safe from severe external environments, which improve the stability of the fluorophore [31]. Light focusing is of higher resolution, which can overcome the limitations of biomedical imaging and other monitoring applications. The biggest safety concerns regarding SNPs are their toxic behaviors leading to immunotoxicity, genotoxicity, and cytotoxicity, which affect their size, hemolysis, aggregation, and bioaccumulation [32].

This review discusses the preparation of SNPs and their applications as drug carriers, contrast agents for imaging, and carriers of photosensitizers (PS) in photodynamic as well as photothermal treatments. In addition, the potential harmful responses of SNPs and the present scenario in medicines in terms of clinical trials are reviewed using data obtained from in vitro and in vivo experiments conducted by several studies.

2. Different Methods to Prepare SNPs

2.1. Microemulsion Method

The microemulsion is a synthetic “nonreactor” made of two incompatible liquids: isotropic, surfactant stable, and thermodynamically stable [33]. The standard composition consists of organic solvent such as cyclohexane, surfactants such as alkylphenol ethoxylate, water, and silica alkoxides such as tetraethylorthosilicates or tetramethylorthosilicates. In aqueous phase, reactants often produce a microemulsion, which is subsequently combined with another reactant to generate nanoparticles (NPs). The NPs produced by this technology have a narrow particle shape. Varying particle sizes and shapes are achieved with the alteration in the properties of the microemulsion system (Figure 1). The microemulsion process may be used to synthesize SNPs with a size less than 100 nm. The approach could be improved if the organic compounds created in the synthesis procedure were removed, resulting in higher SNP purity [34].

2.2. Gas-Phase Method

The raw material used in the gas-phase process involves silicon tetrachloride, which is hydrolyzed with oxygen and hydrogen at a high temperature to produce pyrogenic silica. Heat is then applied to the droplets to form the required NPs with less density and a distinct highly porous structure. The gas-phase method provides advantages of high dispersion, increased purity, and low number of hydroxyl groups on the surface [35]. The associated drawbacks are typically the cost of precursors, energy consumed during the process, sophisticated technology, and special instruments. Because of their great strengthening qualities, the nanoparticles synthesized by the gas-phase technique are primarily employed to improve the rubber characteristics [12,36].

2.3. Stöber’s Method

In 1968, Stöber, Fink, and Bohn proposed the Stöber technique, one of the sol-gel procedures [37,38]. They are widely utilized in the production of silica nanoparticles (size ranging from 5 to 2000 nm) because they can generate monodispersed particles, even without any template. A mixture of tetraethyl orthosilicate (TEOS) with alcohol, ammonia, and water can produce a large percentage of silicic acids (Figure 2). Si–O–Si condensate can be generated either by dealcoholization or dehydration of silicic acid [39]. The main SNPs immediately combine to form primary silica particles once the concentration approaches supersaturation, and then develop into more stable silica particles. SNPs are formed when soluble silicic acid condensates continuously react and have a more substantial influence on the development on their surface. The Stöber’s technique can be used to make solid and mesoporous SNPs [40]. SNPs have a great degree of homogeneity and purity, as well as a clear crystal structure. On the other hand, the precursors necessary in the synthesis process are time-consuming, expensive, and pollute the environment. As a result, additional in-depth research with the development of the Stöber’s technique will necessitate more cost-effective raw materials and more efficient production.

$$\mathrm{Si}-\mathrm{OR}+{\mathrm{H}}_2\mathrm{O}\stackrel{\mathrm{Hydrolysis}}{\to }\mathrm{Si}-\mathrm{OH}+\mathrm{ROH}(\mathrm{R}=\mathrm{alkyl}\mathrm{group})$$

$$\mathrm{Si}-\mathrm{OH}+\mathrm{OH}-\mathrm{Si} \stackrel{\mathrm{Polycondensation}}{\to } \mathrm{Si}-\mathrm{O}-\mathrm{Si}+{\mathrm{H}}_2\mathrm{O}$$

$$\mathrm{Si}-\mathrm{OH}+\mathrm{RO}-\mathrm{Si} \stackrel{\mathrm{Polycondensation}}{\to } \mathrm{Si}-\mathrm{O}-\mathrm{Si}+\mathrm{ROH}$$

$$\mathrm{Si}{\left(\mathrm{OR}\right)}_4+2{\mathrm{H}}_2\mathrm{O}\stackrel{{\mathrm{H}}^{+}{\mathrm{or}\mathrm{OH}}^{-}}{\to }{\mathrm{SiO}}_2+4\mathrm{ROH}$$

2.4. Precipitation Method

Refluxing silica is one precipitation technique that includes combining multiple chemicals in the solution and then mixing the precursor (sodium silicate, Na₂SiO₃) to the priorly prepared solution to produce the precipitate [41]. SNPs are made by drying or calcining the precipitate. To obtain excellent SNP dispersion in the silicate solution, it is necessary to ensure the solution contains monodispersed organic compounds. The use of specific solvents in the precipitation method results in excellent silica particles. Compared to the sol-gel approach, the precipitation method is a simple and efficient way to prepare silica nanoparticles but, sometimes, impurities and aggregation can quickly occur. Highly pure and amorphous silica was more acceptable in many applications. Methods for the extraction of silica from rice husk ash (RHA) were also developed [42].

2.5. Green Synthesis

Green and sustainable routes are used for the synthesis of nanoparticles with desired physicochemical properties [43]. SNP produced by green routes is useful in medicine, cosmetics, pharmaceuticals, and many other fields. In green synthesis, eco-friendly reagents replace harmful chemicals to use the synthesized nanoparticles for biomedical applications. Several natural precursors are used as silica sources to react with sustainable biological bases. Many clays, agricultural wastes, industrial wastes, and some common plants are prime sources of silica with different forms for synthesizing SNPs [44]. The aqueous extract of different parts of plants, fruit pulps, and microbial extracts are used as an alkaline catalyst for the green synthesis of SNPs.

Table 1. Comparison of different synthetic routes in terms of advantages and disadvantages.

Sl.No.	Methods	Advantages	Disadvantages
1.	Microemulsion	Thermodynamically stable Exhibit low viscosity compared to emulsions Do not require any energy contribution	Limited solubility for compounds that possess high melting points Requires large amount of surfactants
2.	Gas-phase	Can form binary and ternary NPs Wide variety of substrates can be used Uniform size distribution	Low production rate Temperature-sensitive
3.	Stöber’s	Simple, cost-effective, and efficient Functionalization with thick coating can be conducted to avoid corrosion High possibility of Sinteration at low temperature	Prolonged processing time Organic solvents usage may lead to cytotoxicity Toxic byproducts
4.	Precipitation	Simple and effective Homogenous yield	Requires further purification Less reproducible
5.	Green Synthesis	Cost-effective Nontoxic and eco-friendly High reproducibility	Size distribution Requires more processing time Low yield

shows the advantages and disadvantages of different synthetic routes of SNPs synthesis.

3. Mesoporous SNPs

Mesoporous SNPs (MSNs) are porous, spherical nanomaterials with unique features that include easy surface functionalization, mesoporosity, and high biocompatibility [45]. MSNs provide the potential ability to load a large number of drugs onto the nanocarrier due to their unique structural features. Nucleic acids, such as DNA and RNA, are frequently bound on the exterior surface of MSNs due to their tiny pore diameters. They exhibit greater drug encapsulation capacity and stimulus-responsive drug-releasing profile when it comes to drug delivery systems [46,47]. MSNs are very accessible to multifunctionalize, and their magnetic, fluorescent, and photothermal characteristics enable nanotheranostics to perform simultaneous bioimaging and drug administration. Moreover, the release of the drug of MSNs can be regulated through a nanovalve structure that responds to internal and external stimuli. The electrostatic interaction between MSNs and negatively charged RNA is facilitated by the positive surface charge and, also, allows MSNs to escape from endosomes through the proton sponge effect [48].

4. Core-Shell SNPs

A research group has developed a method for synthesizing core-shell SNPs that are now widely utilized. They used poly-L-lysine to cover polystyrene templates before performing a polycondensation with tetramethyl orthosilicate in hydrolyzed form [49]. The thickness of the hollow nanoshells is about 6–10 nm after calcination, and the diameter of the prepared nanoparticles was reduced by 10–20 percent. Metals might be used in the core for templating, resulting in core-shell SNPs. The magnetite–silica core-shell nanoparticles were synthesized by oxidation of silicon dust [50]. The above technique would be used to generate core-shell NPs for biological applications. Some researchers created a novel polymeric covering for gold–core SNPs. By mixing varying ratios of two polymers, β-cyclodextrin (β-CD) and poly-2-ethyl-2-oxazoline, biological characteristics of the silica-coated gold nanoparticles were enhanced, and novel cancer therapy concepts were generated [12].

5. Biomedical Applications

5.1. Drug Delivery System

Due to the advancement of nanotechnology, materials generated at the nanoscale level have received growing attention in disciplines such as drug delivery [3,51,52,53]. Because of its unique qualities, such as a vast surface-to-volume ratio, controlled particle size, pore volume, and high biocompatibility, porous silica nanoparticles (NPs) have been studied extensively among all known nanomaterials [54,55,56,57]. Mesoporous silica nanoparticles (MSNs) are good prospects for drug delivery and biological applications, with pore sizes ranging from 2 nm to 50 nm. MSNs are synthesized in the presence of a supramolecular assembled surfactant that functions as a targeting moiety [25]. MSN-based biological applications have shown to be highly promising (Figure 3). The advantages of MSNs include: (i) pore channels with a vast surface area and pore volume have a lot of potential for drug adsorption and loading; (ii) compared to other nanostructures, the mesoporous silica has a large drug-loading capacity and release kinetics because of its porous nature and tunable pore size; (iii) the therapeutic effectiveness of drugs is improved, and toxicity is reduced when delivered via a surface that can be easily adjusted for the regulation and targeted drug administration [58,59]. In vivo biosafety studies for cytotoxicity, biodegradation, biodistribution, and excretion have given positive results. (iv) Drug delivery and bioimaging can be performed simultaneously when magnetic and/or luminous substances are used; (v) bioactive materials with good surface characteristics and porosity have proven to be good options for bone regeneration [60]. The number of studies on mesoporous silica materials has risen considerably as a result of these distinct properties. Additional research on MSNs, which are covalently attached to dipalmitoyl molecules with the support of phosphorylated lipids, were effectively produced into a controlled release mechanism. Through a chemically induced disulfide reduction, the system may release fluorescein moieties from the porous structure of MSNs coated with the lipid bilayer (LB-MSNs) [61]. According to the system, LB-MSNs can be employed to create a controlled release of drug delivery system.

5.2. Photodynamic Therapy

PDT is an emerging noninvasive therapeutic strategy based on the activation of a photosensitizer (PS) with the help of light in a specific wavelength. In the process of illumination, the excited PS transfers its energy to the molecular oxygen, leading to the generation of reactive oxygen species (ROS). The dioxygen, otherwise called singlet oxygen produced, can efficiently oxidize the main cellular macromolecules that result in vascular closure and tumor cell death [52]. PDT does not pose harmful risks to the biological system, as in the case of ionizing radiation therapy that causes damage to the surrounding normal cells.

It is clear that, compared with traditional treatment modalities, PDT has its advantages due to its limited invasiveness and negligible cumulative toxicity. Therefore, PDT aims to boost the quality of life of cancer patients. PDT is highly successful in treating lung, skin, head, and neck cancers [62]. PDT has not yet been accepted as a first-line cancer treatment process due to the lack of perfect PS and difficulties experienced during the PS formulation. Nanostructured micelles, liposomes, polymers, proteins, or metals are currently available for pharmaceutical purposes in various forms. PS-loaded silica nanoparticles are shown to be potential singlet oxygen-producing platforms for increasing therapeutic-possessing photoactive activity and improves selectivity and solubility [63,64,65]. The photosensitizers can be either tagged or encapsulated onto silica nanoparticles in their inner or outer surfaces. As a result, loading the photosensitizer into the nanomaterial provides outstanding photostability while restraining oxygen species diffusion.

The surface functionalized silica nanoparticles exhibit a greater therapeutic profile in photodynamic therapy against cancer. The porphyrin dye used in PDT suffers from low solubility and selectivity. SNPs are widely used to overcome the issues faced by the PS. The anticancer effect of core-shell nanoparticles where the core was made up of silica and coated with xylan bearing 5-(4-hydroxyphenyl)-10,15,20-triphenylporphyrin (TPPOH) dye was investigated. The prepared hybrid particle was then characterized to assess the pharmacokinetic profile. The in vitro analysis showed better anticancer activity in colorectal cancer compared to the dye alone. The photodynamic activity of mesoporous silica nanoparticle coated with two different photosensitizers, namely, indocyanine Green and Chlorin e6, against prostate cancer was also reported. The formulations have enhanced cancer killing capacity compared to the nude dye. The dual combination of dyes exhibits better therapeutic efficiency when nanoformulated using SNPs.

5.3. Photothermal Therapy

Photothermal therapy (PTT) utilizes a photothermal agent (PA) to convert heat from light energy. It is a noninvasive therapeutic modality with a minimum side effect and is widely used for the treatment of drug-resistant tumors. Mesoporous silica nanoparticles are the most efficient drug delivery agent used for the codelivery of PA and chemotherapeutics in combination therapy [66]. Wang et al. developed NIR-triggered photothermal nanoparticles incorporating indocyanine green into amino-group-modified silica nanoparticles [67]. They have successfully demonstrated that the synthesized photothermal nanoparticles have the potential for enhanced cancer cell killings. Wang et al. formulated multifunctional Janus nanoparticles combining gold triangle and mesoporous silica nanoparticles as drug delivery vehicles to deliver hypoxia-active prodrugs for topical PTT [68]. The folate functionalization on the pegylated Janus structure helps in targeting cancer cells. Therefore, MSN is useful in cancer therapy and a widely used system for theranostics.

5.4. Imaging and Monitoring

Recent studies have explored the usage of SNPs in imaging modalities and found that contrast agents encapsulated in SNPs produce high-resolution images compared to conventional imaging techniques [69]. These investigations revealed that SNPs could deliver contrast agents with high specificity and sensitivity for targeting and better imaging contrast. In addition, SNPs have a controlled release in the body because they have a substantial delay. In biomedical imaging, tumor imaging is an essential factor. Other findings indicate that intravenous administrations of core-shell nanoparticles (silica core and metallic shells) result in an improvement in computed tomography (CT) and magnetic resonance imaging (MRI). By using MSNs as a multimodal strategy, researchers successfully detected tumor blood capillaries in live rat model organisms according to near-infrared (NIR) imaging and positron emission tomography (PET) and other techniques [70]. The results showed that this combination of devices improves optical and PET imaging resolutions. Furthermore, fluorescently tagged mesoporous silica nanoparticles were utilized as an endoscopic contrast medium to diagnose abnormal cells of the gut. The researchers developed a new type of Magnus nano-bullet (Mn-DTPA-F-MSNs), which consists of a head with magnetic property (Fe₃O₄-NPs) and mesoporous silica (SiO₂) body [71]. Researchers redesigned the mesoporous SiO₂ before loading it with Mn²⁺. Both in vitro and in vivo studies demonstrated that the Magnus nano-bullet accelerated the T1-weighted MR images. The magnus nano-bullets showed enhancement in the detection of GSH, a biomarker responsible for redox responsive T1 MRI. A macrophage-driven PET imaging tracker was engineered using aza-dibenzocyclooctyne-tethered PEGylated MSNs (DBCO-MSNs) combining short-lived ¹⁸F labeled radiotracer. The in vivo cancer cell tracking and imaging were performed by the PET imaging protocol with the movement of macrophage cells into the tumor site by the bio-orthogonal SPAAC reactions to form ¹⁸F-labeled aza-dibenzocycloocta-triazolic MSNs inside RAW 264.7 cells [12]. In response, the macrophage was more capable of moving towards the tumor effectively. Finally, the tissue radioactive dispersion measurements were consistent with the PET imaging data. A novel cup-shaped SNP, which was effective for ultrasonic imaging, was also discovered.

Using an emulsification slicing process, researchers developed exosomes-like silica nanoparticles (ELS), or cup-shaped drops were constructed [72]. Researchers discovered that ELS was more effective at enhancing particle echogenicity and cell tagging when compared to SNPs shaped like cup-shaped drops. The study demonstrated that stem cells can be used in ultrasound imaging in the future and that SNPs can improve the echogenicity of stem cells in vitro and in vivo [73]. Furthermore, they described a biodegradable ultrasound-capable material. These SNPs did not witness cytotoxic effects at the 250 µg/mL concentration necessary for tagging and then were removed from cells within three weeks [73]. SNP-based imaging techniques have both advantages and disadvantages. Simple optical imaging with a modest imaging resolution is limited, whereas high-quality CT imaging may contaminate the body with radiation. Researchers show that multimodal imaging is capable of enhancing individual imaging methods. Long-term imaging of sentinel lymph nodes draining tumor utilizing mesoporous nanosilica-based triple-modal probes was demonstrated in a study [74]. Due to the excellent stability and resilience of nanoprobes, imaging results from various modalities are coherent and comparable. Furthermore, biocompatibility is a crucial aspect of the use of SNPs in nanomedicine and imaging techniques. Nanomaterials tagged with multimodal imaging nanoprobes were successfully identified using optical imaging, as well as MRI, and also provide more outstanding biocompatibility [74].

5.5. Protein Recognition and Isolation

The versatile properties of silica set it apart from other materials. The ease of synthesis, high surface area, low cost, and surface functionalization are the major reasons for their biomedical applications. Other than drug delivery and diagnostic applications, they can be used to recognize and separate proteins from the sample. Researchers are keen on the application of silica, since the toxicity inside the biological system is negligible. In order to afford simple and quick separation of proteins, iron oxide nanoparticles (IONP) can be coated with silica [75]. The magnetic property of IONP is used to achieve the isolation process. The silica nanoparticle allows surface imprinting, which is an efficient strategy to obtain superior adsorption affinity as the initial step for separation. Typically, the nanostructures are fabricated employing iron oxide nanoparticles as a core, which is then laminated with silica to accomplish quick biomolecule separation. The core responds to external magnetic fields, whereas the shell serves as a spot for protein entrapment, providing biocompatibility and stability [76]. Core-shell nanoparticles of this sort are implemented in bioseparation, biosensors, immunoassay analysis, nucleic acid detection, and other applications. Mesoporous silica has a higher porous architecture, which offers greater adsorption of the adsorbates. The surface charge of silica nanoparticles is a significant issue. Pure mesoporous silica is said to have a neutral charge. This neutral structure of silica may be susceptible to leaching risks. As a result, chemical modification with tagging of the organic group is used to achieve stability [77].

5.6. Nucleic Acid Detection and Purification

The nucleic acid detection technique is performed in wide areas, including clinical diagnosis, food technology, and environmental safety monitoring [78]. Since nucleic acid is a fundamental component for storing and transmitting genetic information, scientists are focusing their efforts on constructing a facile method for detecting and purifying it in preparation for future research. Three main factors that facilitate DNA adsorption are hydrogen bond formation, electrostatic repulsion, and dehydration. Currently, existing approaches have many implications [79]. The main concern with the conventional approach is that they require pipetting procedures, which apparently reduce sensitivity. To achieve the desired sensitivity, additional treatment, such as amplification, is required. However, the amplification stage frequently produces aerosol, which is detrimental to the environment. The technique is quite complicated and necessitates a high level of expertise. Researchers must exercise extra precautions when working with extremely contagious viruses. In order to upgrade the method, silica-coated magnetic nanoparticles-driven separation was established [80,81,82]. Several investigations suggest that integrating magnetic nanoparticles (MNPs) as a core with a silica shell could be very effective. Such modified core-shell-structured silica-coated MNPs, were used to extract nucleic acid and amplify them. Therefore, silica shells can be employed to perform simultaneous, automated, and precise nucleic acid sequence detection.

5.7. Gene Therapy

Nanotechnology-based gene therapy is now emerging as a possible method of delivering genes to treat a wide variety of genetic complications, including cancer. Nanoparticles are expected to be vital vehicles that could carry genetic molecules into cells. Highly porous nanostructures exhibit high loading volume for incorporation of target moiety. Among all the other nanoparticles, the most preferred carrier vehicle is mesoporous silica nanoparticles (MSNs), since they possess a large surface area and high pore volume [83]. Silica is considered a nonviral vector that could effectively deliver genetic molecules to the target site. To achieve targeted release, receptor-mediated endocytosis predominates to deliver the cargo or payloads to the diseased site. Different bioconjugation and chemical modification can further upregulate the function of silica inside the biological system. Polymers are generally utilized for surface modification to improve performance while avoiding cytotoxicity [84]. In vivo evaluation of chemical modification of silica with sodium chloride or sodium iodide has recently proven to be a promising method for gene transfer without causing any pathological alterations in the cells [85]. Since the DNA is negatively charged, the carrier molecule has to be prepared with a positive surface charge. However, the bonding strength should be weak between the carrier molecules and the targeting moiety as the gene involved in breakage and release is weak [86]. Strong adhesion might lead to release in undesired sites, or may undergo digestion with no release. Hence, the particle with payload has to be tailored to achieve satisfactory results. The control over the morphology of nanomaterial during fabrication serves the purpose [87].

5.8. Vaccine Delivery

Vaccines were at the frontline to maintain human wellbeing and have been a cost-effective solution to prevent serious infections. Concerns rising regarding conventional viral systems are their immunogenicity and toxicity, which are the primary reasons for gaining research interest for designing novel vaccination methods. The present focus of research seems to be on the development and implementation of effective carriers for the delivery of the genetic payload to the target tissue [88]. Three major things to be considered during the formulation of vaccines are effective adjuvant, appropriate antigen, and route of administration. The antigen is used to activate the immunological response, whereas the adjuvant is used to support the antigen to elicit the immune response. Vaccination can be performed either by introducing a protein antigen or by using RNA/DNA that encodes an antigenic protein. Developing a vehicle that can carry the genetic material to the site free of toxicity was a major hurdle for researchers. MSNs are being used in vaccinations that serve as both a carrier and an adjuvant (

Table 2. Summary of relevant theranostic applications of silica nanoparticles.

Sl.No	Type of Silica	Modification	Disease/Disorder	Ref.
1.	Biodegradable mesoporous silica nanoparticles	Encapsulated vaccines	SARS-CoV-2	[90]
2.	Hollow mesoporous silica nanoparticles with extra-large mesopores	Core-shell with Poly(ethylenimine) coating (PEI)	Malignancy	[91]
3.	Mesoporous Silica Nanoparticles	Carboxylic acids	Tuberculosis	[92]
4.	Silica nanoparticles	Mincle agonists	tuberculosis	[93]
5.	Dendrimer-like mesoporous silica nanoparticles	Foot-and-mouth disease VLPs (virus-like particles)	Foot-and-mouth disease	[94]
6.	Rambutan-Like Mesoporous Silica Nanoparticles	DNA vaccine with PEI coating	Chronic infections and cancers.	[95]
7.	Extra-large pore MSNs (XL-MSNs)	Cancer antigen with Amine modification	Malignancy	[96]
8.	Mesoporous SBA-16 and silanized SBA-16 (APTES-SBA-16) nanoparticles	(3-Aminopropyl) triethoxysilane (APTES)	Paracoccidioidomycosis	[97]
9.	MCM-41 type silica nanoparticles	Polymer and amine	Oral protein-based vaccine	[98]
10.	Spherical MSNs	Recombinant EspA loaded	Enterohemorrhagic Escherichia coli	[99]

). MSNs as a carrier can transport RNA/DNA to the target tissue, promoting protein antigen synthesis, and acts as an adjuvant to induce both cell-mediated and humoral immune responses [89]. Several in vitro studies have shown that the vaccines can be delivered based on the acid–base triggered system. Since lymph nodes are the major site for vaccine targets, they need to undergo lymphatic drainage to be taken up by dendritic cells for further action. The surface modification with negative groups makes them a complete lymph-nodes-targeted vehicle. The current pandemic situation due to COVID-19 has evidenced the importance of vaccines. Oligonucleotides-based vaccines are the primary and only therapeutic option available for now. Researchers working on COVID-19 vaccinations are now concentrating on the development of vaccines with nanocarrier for RNA/DNA delivery that encodes specific spike protein [90]. The stability, biocompatibility, and release kinetic profile of MSNs makes them a potent tool for antigen carrier and adjuvant.

5.9. Other Applications

Besides the applications listed above, SNPs are also essential to prevent bacterial infections, speed up wound healing, and promote bone regeneration [100,101]. To enhance the benefits of antibacterial infections, nanocarrier-encapsulated antibiotics have been used to enhance the efficiency of antibacterial treatment with the ability to penetrate bacterial walls [12]. SNP characteristics, such as huge surface-to-volume ratio, pore size, and controlled loading, and drug release of antimicrobial agents are remarkable in this regard. When antibacterial peptides such as LL-37, non-steroidal anti-inflammatory medications, and antibiotics using a combination of levofloxacin-loaded MSNs and polycationic dendrimers are added to the mix, the antibiotics are effective at combating Gram-negative bacterial biofilm. The results of the study show Gram-negative E. coli and Gram-positive Staphylococcus aureus do not grow in the presence of SNPs loaded with captured bacteria [12]. Another study examined Gram-negative bacteria that were driven by targeted QS into virulence. The unit of study was to test the bacterial communication or quorum quenching (QQ) capacity of metals and metal oxide nanoparticles. Miller created bifunctional SNPs with the quenching molecule cyclodextrin [12,102]. These results suggest that using functionalized cyclodextrin SNPs, remain in the bacterial microenvironment and inhibit extracellular bacterial communication proteins from seeping out, resulting in antibiotic activity. The study investigated that SNPs were shown to influence migration and invasion of human skin fibroblasts in a local wound healing model (CCD-25SK). The silicic acid is generated by the fibroblasts when the silica nanoparticles become dissolved after absorption in the cells and stimulates wound healing [103,104]. According to the study, researchers found the biocomposite treatment shortened wound epithelialization time in Wistar rats compared with the control group. Furthermore, the composite has antibacterial properties, good drug availability, and a high absorption rate, which could make SNP biocomposite a potential wound healing material in the future [105]. Moreover, silica at the nanoscale has a crucial role in bone regeneration.

The findings also reveal that SNPs are an essential component of bone grafts in this study. SNPs were found to inhibit osteoclasts and activate osteoblasts in vitro. In mice, SNPs improved bone mineral density significantly [60]. In mice in vivo models, it was also found that the 50 nm SNPs also seemed to promote osteoblast proliferation while inhibiting osteoclast differentiation, thereby enhancing bone density as well as biochemical indicators of bone formation. SNPs play a key role in bone regeneration. Researchers discovered that magnetic nanoparticle (Fe₃O₄)-coated mesoporous silica nanoparticles (M-MSNs) could stimulate bone regeneration in rodents during distraction osteogenesis (DO) in mice [106]. A system was constructed using MSCs encapsulated with polymer to stimulate osteogenic differentiation and bone regeneration. SOST siRNA suppressed the osteoporosis-subjecting gene SOST, which lowers osteoblastic development, resulting in increased bone formation. A team of researchers synthesized bioactive glass nanoparticles with monodispersed strontium using improved Stöber’s method. SNPs with diameter 90 ± 10 nm were formed prior to incorporation of calcium and strontium [107]. MC3T3-E1 cells from mice were labeled with antibodies against colla1, osteocalcin (OSC), and osteopontin (OSP) for immunohistochemistry to determine the protein expression in response to nanoparticles after three weeks of growth. Furthermore, 14 percent Sr-BGNPs also notably demonstrate a high expression of markers, OSC and OSP, which is representative for osteogenic differentiation compared to zero percent Sr-BGNPs. These results indicate that the Sr-BGNPs serve as an effective agent for bone regeneration [107]. All the biomedical applications are summarized in

Table 3. Summary of important silica nanoparticles (SNPs) in theranostics.

Types of SNPs/Synthetic Routes	Surface Chemistry/Modifications	Biomedical Applications	Ref.
Fractal SNP/surfactant-free Stöber method	Fractal silica nanoparticles	Sharp edge and rough surface with enhanced adhesion towards enzyme	[18]
Solid SNP/Stöber method	Amine group/ polyethylenimine	Mimicking virus surface topology and surface roughness enhances the binding of biomolecules	[53]
MSN/co-condensation	-	Ruthenium polypyridyl delivery for cancer treatment	[54]
MSN	Azobenzene-modified	Lubrication enhancement and drug release for osteoarthritis	[55]
MSN	Carboxyl-functionalized	Controlled delivery of NSAIDs	[56]
Virus like hollow MSN/self-consuming perovskite	Electrostatic adsorption of doxorubicin	Combination of chemotherapy and immunotherapy	[57]
MSN/co-condensation	Covalently bound dipalmitoyl	Controlled release of fluorescein	[61]
Core-shell hybrid SNPs/sol-gel process following modified Stöber method	Silica core with xylan linked 5-(4-hydroxyphenyl)-10,15,20-triphenylporphyrin (TPPOH)	PDT against colorectal cancer cells	[63]
Hollow-MSN/Stöber method	Hyaluronic acid	Cancer chemo-PDT	[64]
MSN/ modified Stöber method	haloBODIPYs, PEG and FA	Targeted cancer PDT	[65]
Silica nanospheres/sol-gel method	Indocyanine green to amino modified surface	PTT against drug resistant tumors	[67]
Janus-structured gold triangle-MSN/ sol-gel method	Amino functionalization and attachment of tirapazamine (TPZ)	Extrinsic radiosensitization, local PTT and hypoxia-specific chemotherapy	[68]
FA-Gd-Tb@SiO2/covalent conjugation	Covalent conjugation of luminescent Tb3+ to Si-O-Si framework followed by attachment of Gd and FA (folic acid)	Targeted cellular time-gated luminescence (TGL) and cancer cell MR imaging	[70]
Janus magnus nano-bullets (Mn-DTPA-F-MSNs)/sol-gel process	Mn-DTPA-functionalized Fe3O4-MSNs	GSH (glutathione) responsive T1/T2 MRI	[71]
Exosomes-like cup-shaped SNP/emulsion template method	-	Ultra sound contrast for stem cell imaging and drug delivery	[72]
Theranostic MSN	Amino-silane conjugation of fluorescein followed by Gd-DOTA	Ultra sound and MR imaging of stem cells and slow-release reservoir of insulin-like growth factor (IGF)	[73]
MSN/sol-gel method	Salicylic acid and ketoconazole	Antifungal and wound healing	[103]
SNP	Carbon quantum dots/silica nanoparticles/silk fibroin nanocomposites	Wound repair	[104]
Rod-like silica nanoparticles/one pot method in presence of PVP	Type I collagen-SiO2@Fe3O4	Cell guidance and drug delivery	[106]

.

6. Biocompatibility

The application and study of SNPs for further research are simplified due to their characteristics, which include huge surface area, facile surface functionalization, and strong biocompatibility. However, there are some safety issues concerning SNPs, the conflicting evidence reported on both in vitro and in vivo research. A wide number of toxicological studies demonstrate that SNPs’ toxicity depends on their surface characteristics, size, concentration, and other parameters. The processes underlying toxicity of SNP must be better understood before their implementation in nanomedicine. The countermeasures regarding the toxicity must be taken to minimize health consequences.

Toxicological Studies

SNPs induce cellular damage in a variety of different ways. Autophagy, oxidative stress, apoptosis, and inflammatory reactions are some of the mechanisms reported to be involved in cytotoxicity. Among those, ROS-mediated effects contribute highly to cellular damage. SNPs were shown to have deleterious effects on cells, even when the level of reactive oxygen species is not elevated [108,109,110]. Various investigations were conducted, which proves that the SNPs can induce cytotoxic effects in other ways by hindering cellular respiration, proliferation, and apoptosis. SNPs, for example, can trigger autophagy by altering the configuration of lysosomes, promoting membrane permeability, cathepsin B, and limiting the secretion of lysosomal proteases, impairing the lysosome function. SNP-induced autophagy is well documented, despite the unclear understanding of the precise mechanisms involved. Other adverse consequences of SNPs include immunotoxicity, genotoxicity, and neurotoxicity. Among these, genotoxicity and immunotoxicity are the two most noticeable toxic effects. SNPs may lead to the development of micronuclei at high cytotoxic concentrations [111]. These toxicity levels are consistent across cell lines and SNPs. SNPs have been found to cause genotoxicity in cancer cell lines, which is thought to be linked to the implementation of cellular damage in recent research. The size of the NPs and the extent of this effect are inversely proportional to one another [112]. Negatively charged SNPs have been shown to have the most immunotoxic activities in lymphocytes when examined in vivo [111]. They suppress the generation of proinflammatory cytokines and nitric oxide, while inhibiting the lymphocyte proliferation and NK cell killing capability.

In vivo investigations typically comprise animal toxicity trials where SNPs are supplied mainly through inhalation, topically, orally, or intravenously [12,113]. SNPs can be found across many regions of the body, including the liver, lungs, spleen, kidney, skin, and brain tissue, which solely depends on the exposure site [114,115,116,117]. NPs can be excreted from the body either through the kidneys, where it is filtered out by urine, or by the liver, through bile secretion in the feces. Researchers used ultrastructural localization and fluorescence methods and discovered that SNPs have distributed across the spleen and liver of mice after one shot as an intravenous injection produced substantial lymphocytic infiltration, degeneration, as well as necrosis [118]. The ease of excretion is decided by the smaller size of the nanoparticle injected. As per the outcomes of numerous scientific experiments on mice, there seems to be no buildup of SNPs on the brain tissue and no adverse consequences have been detected. A number of in vivo and in vitro studies demonstrate that SNPs have high toxicity on neurons. The central nervous system under specific circumstances was used as a reference for potential safety application and development [119]. The summary of toxicological effects of silica nanoparticles and their probable reasons are tabulated in

Table 4. Observed toxicological effects of SNPs.

Types of Particles	Mode of Study	Probable Reasons	Ref.
MSN (20–200 nm)	In vitro	Size and dose dependency, ROS generation, and changes in membrane integrity induced by cellular uptake.	[109]
SNP (62 nm)	In vitro	Induced ROS formation and DNA damage response and caused toxicity to endothelial cells through Chk1-dependant G2/M DNA damage checkpoint.	[110]
Fluorescent MSN	In vivo	More than 80% intravenously administered MSNs deposited in liver, spleen, and lung and shape of the particles play a major role.	[113]
SNPs (46 ± 4.9 & 432.0 ± 18.7 nm) and MSNs (466.0 ± 86.0 nm)	In vivo & ex vivo	Microscopic lesions in liver, kidney, spleen, and lungs. Physicochemical properties, dose, and frequency of administration is responsible.	[114]
SNP	In vivo	Dose-dependent hepatotoxicity and nephrotoxicity in male rats due to oxidative stress and apoptosis.	[115]
MSN (75 nm)	In vivo	Induced intestinal oxidative stress and colonic epithelial cell apoptosis in mice	[116]
SNP (57.66 ± 7.30 nm)	In vitro	Impairment in mitochondrial dynamics and biogenesis leading mitochondrial dysfunction, oxidative stress, and, finally, cardiovascular disease.	[112]
SNP (64.43 ± 10.50)	In vivo	Induce hepatic dysfunction and granuloma formation in liver.	[117]

. Comprehensive and systematic studies are required to clarify the deleterious effects of SNPs. However, further investigations on the role of SNP in the biochemical characteristics in vivo and in vitro are highly needed. Chronic toxicity and health impacts of persistent exposure, including co-exposure issues with nano-silica and its structural composition, must be assessed regularly. Nanotoxicity-related norms for detection techniques of toxicity and their potential implementation also have to be considered. Another technique to minimize possible toxicity is to analyze and synthesize biodegradable nanoparticle materials.

Synthesized SNPs contain unreacted chemicals, such as surfactants, organic solvents, etc., that cause cyto-, immuno-, and genotoxicity. In order to resolve the issue, the end product can be further purified before their use in biological systems. In addition to it, their surface can be functionalized with various functional groups, such as phosphate and amino groups. This functionalization can increase the selectivity and can interact with the diseased site, thereby reducing the dendritic effects caused. The alteration of the surface properties of SNP can also reduce the production of free radicals. Despite the surface chemistry, the shapes also determine the level of toxicity. Modification of the shapes can also be performed to reduce the toxic effects of SNPs.

Despite the significant interest in biomedical applications, the fate of the silica nanoparticles is one of the important aspects to be considered. The chemical transformation, degradation, excretion, and their ecotoxicity needs to be well understood before their potential use. The size, shape, and their surface charge have a great impact on their fate. Several studies indicate that the smaller particles will be subsequently eliminated through urine or feces, whereas the larger particles can be degraded by the liver. However, the ultra-fine particles in the range of approximately 6 nm become immediately eliminated by renal clearance. These types of particles cannot be used as drug carriers, but they have a preferential role in imaging and diagnostics. Research proved that the degradation and elimination rate can be modified according to the application by altering the size distribution, morphology, and surface chemistry.

7. Clinical Trials of Silica Nanoparticles

The US Food and Drug Administration (FDA) has recognized silica as generally recognized as safe (GRAS) under section 201(s) of the FD&C act after completing the clinical trial (NCT03667430) [120]. The European Food Safety Authority has recognized amorphous forms of silica and silicates up to 1500 mg per day as safe ingredients for oral delivery [120]. Several human trials were conducted for silica nanoparticles, as per ClinicalTrails.gov (accessed on 31 March 2022), though there is a lack of significant human trials on mesoporous silica nanoparticles for cancer therapy and diagnosis. Janjua et al. recently reported about 11 clinical trials and 2 clinical studies on humans to evaluate the safety profile of silica nanoparticles [121]. Silica nanoparticles improve the solubility of hydrophobic drugs and alter the pharmacokinetics profile. They show good tolerability with minimum side effects when used as an ingredient for oral delivery agents. Tan et al. reported an almost two-fold increase in the bioavailability of ibuprofen when it was formulated using silica–lipid hybrid nanoparticles [122]. A human trial was conducted on 12 adults to demonstrate the tolerability and pharmacokinetics of novel simvastatin encapsulated in silica–lipid hybrid nanoformulations (ACTRN12618001929291) [123]. The formulations show 3.5-fold increased bioavailability of simvastatin compared to the free drugs. Kharlamov et al. reported silica modified SPION and gold nanoparticles for localized plasmonic photothermal therapy to treat atherosclerosis [124]. A phase I clinical trial successfully established that the silica–metal core-shell nanostructure with a diameter of 90–150 nm could reduce coronary atherosclerosis (NCT01270139) [125]. Similarly, focal therapy, diagnosis, and imaging (MRI/Ultrasound) using silica–gold nanoshell showed better efficacy in malignant prostate cancer (NCT04240639, NCT02680535) [126]. The reference number NCT04656678 shows the preliminary effectiveness of prostate cancer focal US-guided theranostics. The main advantage includes the accumulation of nanoparticles or nanoshells at tumor sites that helps in accurate and predictable ablation therapy for cancer with minimal side effects compared to the conventional therapy. Ultrasmall silica nanoparticles, Cornel dots with a 6–10 nm diameter, can be used as tracers for melanoma or malignant brain tumors (NCT03465618) [127]. In a clinical trial (NCT01266096), PET imaging of a patient with melanoma and a malignant brain tumor was demonstrated using a 124I-labeled cRGDY silica nanomolecular particle tracer to establish the efficacy of the nanosilica [121]. Targeted silica nanoparticles can be used effectively for real-time image-guided mapping of nodal metastases (NCT02106598) [128]. The clinical trial results demonstrated that ultrasmall silica dots are stable, readily taken up by the tumor cells, and well-tolerated, with minimum side effects. Wiesner group has been working on silica-based nanoparticles since 2000 for cancer theranostics. They developed ultrasmall (<10 nm) fluorescent ‘C dots’ (Cornell dots) comprised a silica shell encapsulating Cy5 fluorescent dye coated with polyethylene glycol [129,130]. The particles were decorated with radiolabeled RGDY (¹²⁴I-cRGDY) for integrin targeting. The first-generation C dots have been tested in first-in-human FDA-approved clinical trials. This was the first inorganic–polymer hybrid diagnostic probe used in clinical trials [131]. One can easily conclude that the silica nanoparticles are very promising in theranostics considering these many clinical trials.

8. Conclusions and Future Perspectives

Nanotechnology in medicine has advanced significantly due to the advances in science and technology in a variety of research methodologies. Silica nanoparticles have become one of the inevitable research hotspots in nanomedical applications, owing to a huge surface–volume ratio, simplicity in surface modification, easy fabrication, and amplification with excellent biocompatible properties. MSNs use their intrinsic mesoporous surface and porosity characteristics by altering the size of the pore and adjusting the characteristics of the surface to accomplish the drug delivery. Furthermore, in contrast with traditional SNPs, the refined SNPs being created now offer more significant potential for simultaneous molecular imaging and drug administration. Bioimaging can be used to track drug distribution in real time and enhance therapeutic strategies. SNPs are also useful in a variety of scientific domains, including wound healing, photodynamic therapy, photothermal therapy, antimicrobial infection, and bone regeneration. SNPs are widely employed; however, there are concerns about their biosafety. SNPs have been shown to induce cytotoxic effects, hepatosplenic toxicity, carcinogenicity, and hypersensitivity in both in vitro and in vivo investigations. However, the outcomes among many toxicology studies are uncertain, and the research path is unclear. Therefore, it is predicted that the use of SNPs in therapeutic applications would provide additional health advantages to humans in the future. Besides all these advantages, SNP-based nanomedicines face several challenges in biomedical applications. The fabrication of SNP-based nanomedicines is a multistep procedure that starts with synthesis and ends with surface modifications to serve specific purposes in drug delivery and imaging. These complicated syntheses and fabrications are multistep procedures that can be an issue in reproducibility and scaling-up in a cost-effective manner. The biosafety of SNPs is another challenge that biomedical scientists must address to bring SNP-based theranostics from bench to bedside, where chemical composition and physicochemical properties are critical parameters to maintain. Our intention is not to discourage researchers from stating these limitations and challenges. This field is up and coming, and researchers can address these limitations and challenges to develop new nanoformulations that we need to fight against several life-threatening diseases.