Ultrasmall Silica Nanoparticles: Synthesis, Functionalization and Biomedical Application

Ultrasmall Silica Nanoparticles: Synthesis, Functionalization and Biomedical Application | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Ultrasmall Silica Nanoparticles: Synthesis, Functionalization and Biomedical Application Feihu Cui, Lishuo Qu, Yao Gong, Yijun Xie, Qing Chang, Christian Celia, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3825399/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Ultrasmall silica nanoparticles (USNPs) with a size ＜20 nm exhibit unique advantages such as low toxicity, surface modification, and efficient renal clearance, making them highly promising in the fields of bioimaging, disease detection, gene delivery, and drug delivery. In this short review, synthesis, functionalization, and biomedical applications of USNPs are discussed. First, the different synthetic methods for fabricating hollow USNPs and solid USNPs. Then, surface modification methods are described in detail. Finally, the biomedical application progress and toxicity of USNPs encapsulated with fluorescent and drug molecule are discussed. Silica Nanoparticles functionalization diagnosis and therapy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1 Introduction Silica nanoparticles (SNPs) have broad prospects in the biomedical field, such as in medical diagnosis, photodynamic therapy, and drug delivery, due to their advantages of controllable particle size, large surface area, easy surface modification, and good biocompatibility [ 1 – 3 ]. However, as an inorganic material, SNPs are very slowly metabolized in the body, and prolonged residence in the body may potentially lead to long-term toxicity. The size of nanoparticles can critically affect their pharmacokinetics in the biological system. Smaller particles are more easily and rapidly cleared through the kidneys, resulting in shorter residence in the body [ 4 ]. Chen et al. investigated the distribution and metabolism of fluorescent SNPs with sizes of 118 nm, 72 nm, 47 nm, and 27 nm in mice. The results showed that smaller SNPs had more pronounced systemic distribution and were more easily metabolized through the kidneys and excreted in urine. Choi et al. [ 5 ] indicated that nanoparticles with a size of ca 10 nm could escape the renal glomerulus. Therefore, to synthesize SNPs that do not persist in the body for long period, more and more researchers are focusing on the synthesis of ultrasmall silica nanoparticles (USNPs) with sizes below or about 20 nm and exploring their biomedical applications. In this short review, we will summarize and outline the progress of USNPs in the preparation, modification, and biomedical application, and provide an outlook on the future of USNPs. 2 Synthesis of hollow USNPs The most used method for synthesizing hollow USNPs is the soft-template approach, where surfactants and other organic additives first form a template, and then the precursor molecules self-assemble and undergo chemical reactions on the template surface. After the reaction is complete, the template is removed, resulting in the formation of hollow USNPs. Figure 1 illustrates the synthesis process of hollow USNPs using the soft-template method [ 6 ]. Common surfactants can be categorized into two types: non-ionic surfactants and ionic surfactants, which are detailed below. 2.1 Non-ionic surfactants as templates The frequently used non-ionic surfactant is Pluronic F127, which is a poly (ethylene oxide)-poly (propylene oxide)-poly (ethylene oxide) triblock copolymer consisting of hydrophilic polyethylene oxide (PEO) and hydrophobic polypropylene oxide (PPO) segments [ 7 ]. When its concentration exceeds the critical micelle concentration, F127 forms micelles in aqueous solution with a PPO core and a PEO shell. Under room temperature conditions, hydrolysis and condensation of the silica source occur on the surface of the micelles by adding hydrochloric acid solution. By introducing a silane coupling agent as a terminator, the number of silanol groups on the surface of particles can be reduced, resulting in uniformly dispersed USNPs. Huo et al. [ 8 ] first utilized F127 as a template agent, with tetraethyl orthosilicate (TEOS) as the main silicon source, hydrochloric acid as the hydrolysis catalyst, while dimethyldiethoxylsilane (DEDMS) were added as a terminator. The resulting USNPs had a size of approximately 10 nm. This method was subsequently widely adopted by other groups [ 9 – 13 ]. Chi et al. [ 14 ] employed F127 as a template and investigated the influence of different silane terminator agents on the particle size and morphology of USNPs. The results indicated that the higher the number of methyl groups in the silane terminator, the greater the steric hindrance, leading to smaller particle size. Compared to DEDMS, USNPs prepared using trimethylethoxysilane (TMES) as a terminator had a smaller particle size. This is attributed to the reaction between the organic silane and the silanol groups on the surface of nanoparticles, effectively preventing nanoparticle growing and aggregation. Additionally, examining the length of the carbon chain in the organic silane showed that longer carbon chains were more favorable for forming highly dispersed nanoparticles. Furthermore, if the terminator contained amino groups, it would enhance the stability and dispersion of the nanoparticles [ 15 ]. This is due to the repulsion of the positive charge in the amino groups. Tan et al. [ 16 ] employed a new synthesis method by dissolving F127 in tetrahydrofuran (THF), slowly adding tetramethoxysilane (TMOS) at room temperature, and subsequently introducing deionized water. TMOS formed the shell of USNPs at the interface of F127 micelles. TEM results showed that the USNPs had a particle size of approximately ca 14 nm (Fig. 2 ). Zanarini et al. [ 17 ] encapsulated fluorescent dyes within the core of USNPs, resulting in the 10-nm fluorescent USNPs. Wang et al. [ 18 ] used a similar approach where, under acidic conditions, they employed F127 as a template, TEOS as the silicon source, encapsulating a hydrophobic oxygen-sensitive probe within the hydrophobic core, while a hydrophilic pH probe was attached to the polyethylene glycol (PEG) on the outer shell. They used DMDMS as a terminator and one-step synthesis to produce stable and relatively small (12 nm) USNPs, which exhibited dual functionality in detecting both oxygen levels and pH values. Chi et al. [ 19 ] further manipulated the ratio and amount of 1,3,5-trimethylbenzene (TMB) in conjunction with F127 to synthesize hollow SNPs ranging from 10 to 90 nm in size (Fig. 3 ). TEM results indicated that lower TMB concentrations led to smaller SNP sizes, and higher reaction temperatures resulted in smaller sizes and pore diameters of the SNPs. 2.2 Ionic surfactants as templates Yamada et al. [ 4 ] employed hexadecyltrimethylammonium bromide (CTAB) as a template agent and triethanolamine (TEA) as a catalyst. They conducted a stirring reaction at 80 o C for 6 h to investigate the influence of different alkoxy silanes (TMOS, TEOS, TPOS, or TBOS) on the particle size of SNPs. The results revealed that TMOS had the fastest hydrolysis rate, resulting in the smallest particle size, approximately 20 nm. To synthesize USNPs with a size smaller than 10 nm, Ma et al. [ 20 ] made improvements to Yamada's method. They used ammonia solution (NH 3 ·H 2 O) as a catalyst, maintained the pH of the reaction system at 8, and introduced PEG-silane to terminate the reaction [ 21 ]. This modification resulted in well-dispersed hollow USNPs with a size of 10 nm (Fig. 4 ). The choice of NH 3 ·H 2 O as a catalyst in the preparation process is primarily because NH 3 ·H 2 O is a small molecule that does not adsorb on the surface of hollow USNPs, thereby avoiding an impact on the particle morphology [ 22 , 23 ]. Moller et al. [ 24 ] utilized hexadecyltrimethylammonium chloride (CTAC) as a template and investigated the impact of temperature and the ratio of TEOS/TEA on the particle size and morphology of USNPs. The results showed that at a TEOS/TEA volume ratio of 1:3, the synthesized hollow USNPs had a particle size range of 20–70 nm (Fig. 5 A). Simultaneously, it was observed that with a constant temperature, an increase in the TEOS/TEA ratio led to an increase in the USNPs' particle size. Pan et al. [ 25 ] improved upon Moller's method by using water as the solvent, CTAC as the surfactant, and TEOS as the silicon source. They controlled the system pH by adjusting the mass of TEA, resulting in hollow USNPs with a size of approximately 25 nm (Fig. 5 B). 3 Preparation of Solid USNPs The commonly used method for synthesizing solid USNPs is the sol-gel method. Typically, TEOS is used as the raw material, and ethanol or methanol serves as the medium, with alkaline substances acting as catalysts. There are two commonly used alkaline catalysts: NH 3 ·H 2 O and amino acids. 2.1 Ammonium Hydroxide as the Catalyst Giesche et al. [ 26 ], using NH 3 ·H 2 O as a catalyst and controlling the drop rate of TEOS, successfully prepared solid USNPs with a size of 17.5 ± 3 nm (Fig. 6 a). Kim et al. [ 27 ], by changing the solvent (methanol and ethanol) and using NH 3 ·H 2 O as a catalyst, synthesized monodisperse solid USNPs with sizes ranging from 5 to 450 nm. They found that compared to ethanol, the use of methanol as a solvent resulted in smaller silica sphere sizes (Fig. 6 b). Ow et al. [ 28 ] covalently attached organic fluorescent dyes to USNPs, synthesizing USNPs with a size of 20–30 nm and high fluorescence intensity. Larson et al. [ 29 ] and Herz et al. [ 30 – 32 ], following Ow's synthesis method, prepared fluorescent USNPs with a size of 10–15 nm by adding different fluorescent molecules, using ethanol as the medium and NH 3 ·H 2 O as a catalyst. Compared to free fluorescent molecules, the absorption and emission peaks of fluorescent USNPs did not change, but the fluorescence intensity increased. This enhancement is primarily attributed to the rigid environment provided by the USNPs shell, which increases the quantum yield of the fluorescent molecules. To further investigate the reasons for the fluorescence enhancement of dye molecules encapsulated in USNPs, Cohen et al. [ 33 ] synthesized fluorescent USNPs with sizes of 20 and 30 nm, containing 4 and 7 fluorescent molecules of DY630 in the core, respectively. Compared to free DY630, the fluorescence quantum yield of encapsulated DY630 increased by 13 and 15 times, mainly because USNPs provided a local environment for DY630, reducing its interactions with solvent molecules and fluorescent molecules, thereby improving the quantum yield. 2.2 Amino Acids as the Catalysts Davis et al. [ 34 ] first used L-lysine as a catalyst to synthesize USNPs with a size of 5 nm. The method involved dissolving lysine in deionized water, slowly adding an appropriate amount of TEOS, and stirring the reaction for 24 h. TEOS gradually hydrolyzed, forming USNPs. Simultaneously, Yokoi et al. [ 35 ] also used L-lysine as a catalyst to synthesize ordered arrangements of USNPs with a size of 12 nm. The interaction between protonated amino groups in L-lysine and anionic silicate groups allows lysine to cover the surface of USNPs. Simultaneously, due to the presence of hydrogen bonds between lysine molecules, a closely packed structure is easily formed, thereby controlling the particle size. Yokoi et al. [ 36 ] and Wang et al. [ 37 ], by altering synthesis conditions such as reaction time, stirring rate, composition of reactants, surface potential, pH value, and TEOS concentration, prepared USNPs with sizes ranging from 8 to 35 nm. They found that lysine not only acted as a weak alkaline catalyst, slowing down the hydrolysis of TEOS, but also served as a buffer, maintaining a constant pH in the system. Additionally, lysine prevented USNPs from aggregating, and by changing the stirring rate/reactant composition, the size of USNPs could be precisely controlled. Hartlen et al. [ 38 ] used arginine as a catalyst to synthesize highly uniform-sized USNPs in cyclohexane or vegetable oil solvents. Watanabe et al. [ 39 ] modified the reaction conditions, using arginine as a catalyst, TEOS as the silicon source, maintaining a pH of 9–10, and conducting the reaction at 70°C for 24 h in an oil/water biphasic environment. They successfully synthesized USNPs with a size of 12 nm. 4 Removal of the Template Agents An essential step in preparing hollow USNPs is the removal of organic template agents. During this removal process, the hollow structure of USNPs is prone to collapse, and aggregation between USNPs may occur, forming large aggregates. Generally, there are three methods for removing template agents from hollow USNPs: high-temperature calcination, dialysis, and extraction. Shimogaki et al. [ 40 ] employed a calcination method to remove the surfactant from the pores of SNPs. The advantage of high-temperature calcination is easy operation. However, the drawback is that the hollow structure of USNPs is prone to collapse, and excessively high temperatures can lead to sintering of USNPs. Dialysis for removing template agents is a relatively mild method that effectively avoids the collapse of the hollow structure and high-temperature sintering of USNPs. Yamada et al. [ 4 ] used dialysis bags with a molecular weight cutoff of 8000–14000 to remove the CTAB template from USNPs using acetic acid and ethanol solutions. Results showed that CTAB was completely removed after three rounds of dialysis [ 20 , 41 ]. Extraction can also be employed to remove template agents from USNPs. Pan et al. [ 25 ] performed extraction by stirring the reaction product in a sodium chloride-methanol solution at room temperature for 3 h to remove the surfactant CTAC. Infrared spectroscopy results showed that CTAC could be completely removed. The advantage of this method is that the template agent can be recycled after extraction, but the drawback is that multiple extractions may be required to completely remove the template agent. Moller et al. [ 24 ] chose different solvents (ammonium nitrate or hydrochloric acid) and stirred at 60°C for 2–20 h, extracting the CTAC template from the solution. After two to three extractions, the template agent could be completely removed. 5 Functionalization of USNPs USNPs have a silicon-oxygen bond-supported framework, with a large number of silicon hydroxyl groups on the inner and outer surfaces. These groups can be easily further modified with active functional groups, thereby altering the properties of USNPs and expanding their applications. Non-surface modification refers to the incorporation of two or more silicon sources during the synthesis of USNPs, introducing organic functional groups into the silica framework. Li et al. [ 42 ] introduced APTES during the formation of silica spheres, preparing amino-modified USNPs. Subsequently, by electrostatic interaction, these USNPs were covered on the surface of Layered Double Hydroxide (LDH) to form a composite material (NH 2 -SiO 2 @LDH). This composite material could stably exist in culture media or PBS buffer solutions. Cell experiments indicated that, compared to LDH, NH 2 -SiO 2 @LDH composites were evenly distributed within cells and could be used as carriers to deliver siRNA546 into cells. Surface modification involves modifying the surface of USNPs after their synthesis. For example, Ma et al. [ 43 ] modified fluorescent USNPs with PEG and aluminosilicate. PEG increased the colloidal stability of USNPs, while aluminosilicate enhanced the encapsulation rate of fluorescent molecules and increased the intensity of the fluorescent signal. Additionally, various active groups (hydroxyl/amine/thiol/carboxyl groups) can be introduced to USNPs through microemulsion methods. These modified USNPs can then be combined with various active molecules, such as transferrin, monoclonal antibodies, etc., to enhance their targeting capabilities. 6 Biomedical Applications of USNPs Due to their excellent biocompatibility, degradability, and non-toxicity, USNPs have been extensively investigated and applied in controlled release carriers for genes and drugs, as well as in cell imaging [ 44 ], disease diagnosis [ 45 ] and treatment [ 46 – 48 ]. 6.1 Gene Carrier RNA interference technology, an efficient and specific method for blocking the expression of endogenous homologous genes, is a promising biotechnological approach. However, the siRNA molecules that generate RNA interference are challenging to enter cells and are easily cleared by the body. Thus, a suitable carrier is needed, and USNPs are one such carrier. Yu et al. [ 49 ] successfully used 10-nm USNPs as carriers to deliver siRNA into tumor cells (Fig. 7 ). They first modified the surface of USNPs with positively charged polyethyleneimine (PEI), which not only increased the loading capacity of siRNA but also protected siRNA from degradation by nucleases. Li et al. [ 42 ] also constructed a novel NH 2 -SiO 2 @LDH nanocomposite that could serve as a carrier to deliver siRNA into cells. 6.2 Drug Carrier Due to the drug resistance of certain cancer cells, some anticancer drugs, such as doxorubicin (DOX), cannot maintain activity for an extended period [ 50 ]. Therefore, it is necessary to find suitable carriers. USNPs, as carriers, have several advantages, such as: larger surface area, enabling the loading of more anticancer drugs; efficient renal clearance, preventing long-term retention and toxicity; easily modifiable surface for targeting ligands, enhancing drug targeting; and more [ 51 ]. Huo et al. [ 8 ] compared the loading and release behavior of the drug molecule paclitaxel by 10 nm USNPs and polymeric F127 micelles. The results indicated that USNPs exhibited a significantly higher drug loading capacity than F127 micelles, and USNPs showed a slow release rate, while USNPs also extended the circulation time of the drug in the body. Pan et al. [ 25 ] modified USNPs with TAT peptide to facilitate efficient cellular uptake and delivery of DOX into the cell nucleus, thereby improving the bioavailability of DOX. 6.3 Imaging Kumar et al. [ 52 ] synthesized 20-nm USNPs modified with near-infrared (NIR) fluorescent molecules. Through NIR imaging and radioisotope labeling experiments, it was observed that USNPs primarily distributed in the liver and spleen of mice (Fig. 8 ). They were excreted through the hepatobiliary pathway, and histopathological analysis indicated that USNPs did not exhibit any potential toxicity. Therefore, USNPs can be used as biocompatible fluorescence probes for in vivo imaging [ 53 – 55 ]. Turker et al. [ 56 ] synthesized NIR fluorescent USNPs with sizes of 3.3 nm and 6.0 nm. In vivo imaging results revealed that smaller-sized USNPs were more easily excreted from the kidneys. Simultaneously, surface modification with PEG-silane effectively prevented the adsorption of serum proteins, enabling efficient excretion through urine. 6.4 Diagnosis Benezra et al. [ 23 ] synthesized fluorescent USNPs with a size of 7 nm that could target cancer cells. The USNPs were modified with RGD peptide and radioactive iodine, enhancing their selectivity for tumor cells, improving the tumor/blood ratio, and facilitating renal clearance. This USNPs have been approved for human clinical trials [ 57 ]. Melanoma is considered the most common cancer in the world, and early diagnosis and treatment can reduce mortality. The spread of melanoma to lymph nodes is a crucial indicator of cancer development. Bradbury et al. [ 58 – 60 ] synthesized 10-nm fluorescent USNPs (124I-cRGDY-PEG-C dots) for detecting the metastasis of melanoma in lymph nodes using positron emission tomography (PET) and optical imaging techniques. Compared to conventional clinical use of 18 F-FDG, these fluorescent USNPs efficiently located and monitored cancer cells, serving as fluorescent probes for monitoring cancer cell metastasis. 7 Toxicity of USNPs The translational potential of USNPs from the laboratory to clinical use is highly dependent on toxicological assessments [ 61 – 64 ]. Generally, amorphous silica is recognized as safe by numerous regulatory authorities such as Therapeutic Goods Administration Australia (TGA), European Medicines Agency and United States Food and Drug Administration, and many studies show that SNPs have minimal toxicity in vivo [ 65 ]. Kim et al. investigated the oral toxicity of colloidal SNP of 20 and 100 nm in rats over a period of 90 days, and found that doses of up to 2000 mg kg-1 showed no signs of toxicity [ 66 ]. Akhtar et al. [ 67 ] investigated the toxicity of 10 nm and 80 nm USNPs in A549 cells, finding that cell viability decreased with increasing USNPs concentration. However, there was no significant size-dependent effect on cell toxicity under the same concentration conditions. Similarly, Lin et al. [ 68 ] examined the toxicity of 15 nm and 46 nm SNPs, observing no significant differences in cell toxicity despite the difference in SNPs size. The main reason is that smaller-sized USNPs tend to aggregate, resulting in size-independent toxicity to cells. Bradbury group and their collaborator studied the toxicity profiles of USNPs with size of 6–7 nm in animal models. Upon these limited toxicological evaluation, the main organs such as liver, lung, kidney and heart, demonstrated no abnormalities or tissue damages at the dose of applied in cancer therapy [ 69 – 71 ]. Hameed et al. [ 72 ] synthesized porphyrin-based USNPs with size of 6.2 nm and used for fluorescence imaging-guided cancer photodynamic therapy, where organ toxicology and blood analysis also show no abnormalities after 24 h post-injection. 8 Conclusion and Prospects USNPs have advantages such as high specific surface area, good biocompatibility, low toxicity, and rapid excretion through urine. These properties make them widely applicable as carriers for drug or gene delivery and for the diagnosis and treatment of tumors. However, as a novel nanomaterial, there are still challenges in the preparation and application of USNPs. Firstly, large-scale synthesis of USNPs remains challenging, and scaling up experiments may affect their morphology and size. Secondly, there are many unresolved clinical issues, such as long-term toxicity of USNPs. Therefore, extensive research is needed before clinical use, including studies not only on acute toxicity but also on long-term toxicity, such as genetic toxicity. Yamashita et al. [ 73 ] reported that intravenous injection of pregnant mice with USNPs might lead to pregnancy complications, highlighting the need for detailed research on the in vivo toxicity of USNPs. Declarations Author contributions Feihu Cui: writing original draft and editing; Lishuo Qu and Yao Gong: figure and table generation and conceptualization of the manuscript; Xiaoyong Deng, Christian Celia, Yijun Xie and Qing Chang revised the paper. Funding This study is supported by National Natural Science Foundation of China (No. 21371118). The research activity of Christian Celia was supported by the Overseas Visiting Fellow Program 2022, Shanghai University, China. Christian Celia acknowledges financial support funded by the European Union – Next Generation EU, under the National Recovery and Resilience Plan (NRRP), Mission 4 Component 2 - M4C2, Investiment 1.5 – Call for tender No. 3277 of 30.12.2021, Italian Ministry of University, Award Number: ECS00000041, Project Title: ‘‘Innovation, digitalisation and sustainability for the diffused economy in Central Italy’’, Concession Degree No. 1057 of 23.06.2022 adopted by the Italian Ministry of University. CUP: D73C22000840006. Availability of data and materials: The collected datasets during the current study can be obtained from the corresponding author on reasonable request. Ethics approval and consent to participate No ethics approval was involved for this work. 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ACS Nano. 2022;16:2002-20033. https://doi.org/10.1021/acsnano.2c05342 Aragon-Sanabria V, Aditya A, Zhang L, Chen F, Yoo B, Cao T, Madajewski B, Lee R, Turker MZ, Ma K, Monette S, Chen P, Wu J, Ruan S, Overholtzer M, Zanzonico P, Rudin CM, Brennan C, Wiesner U, Bradbury MS. Ultrasmall nanoparticle delivery of doxorubicin improves therapeutic index for high-grade glioma. Clin Cancer Res. 2022;28:2938-52. https://doi.org/10.1158/1078-0432.CCR-21-4053 Hameed S, Bhattarai P, Gong ZR, Liang XL, Yue XL, Dai ZF. Ultrasmall porphyrin-silica core-shell dots for enhanced fluorescence imaging-guided cancer photodynamic therapy. Nanoscale Adv. 2023;5:277. https://doi.org/10.1039/d2na00704e Yamashita K, Yoshioka Y, Higashisaka K, Mimura K, Morishita Y, Nozaki M, Yoshida T, Ogura T, Nabeshi H, Nagano K. Silica and titanium dioxide nanoparticles cause pregnancy complications in mice. Nat Nanotechnol. 2011;6(5):321-28. https://doi.org/10.1038/nnano.2011.41 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3825399","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":265687223,"identity":"fcc7fa68-b465-4335-9872-734fbced00f4","order_by":0,"name":"Feihu Cui","email":"","orcid":"","institution":"Shanghai University","correspondingAuthor":false,"prefix":"","firstName":"Feihu","middleName":"","lastName":"Cui","suffix":""},{"id":265687224,"identity":"5b87a262-7103-476f-b60f-d2e17b2b011e","order_by":1,"name":"Lishuo Qu","email":"","orcid":"","institution":"Shanghai University","correspondingAuthor":false,"prefix":"","firstName":"Lishuo","middleName":"","lastName":"Qu","suffix":""},{"id":265687225,"identity":"ccd0a5f1-b56c-49d1-b95e-e36505145b5b","order_by":2,"name":"Yao Gong","email":"","orcid":"","institution":"Shanghai University","correspondingAuthor":false,"prefix":"","firstName":"Yao","middleName":"","lastName":"Gong","suffix":""},{"id":265687226,"identity":"a6f7049c-a6e2-4de5-9d32-212cb5129f7c","order_by":3,"name":"Yijun Xie","email":"","orcid":"","institution":"Shanghai University","correspondingAuthor":false,"prefix":"","firstName":"Yijun","middleName":"","lastName":"Xie","suffix":""},{"id":265687227,"identity":"85997a75-1cdf-42a0-9751-bbb28c6b76c4","order_by":4,"name":"Qing Chang","email":"","orcid":"","institution":"Shanghai University","correspondingAuthor":false,"prefix":"","firstName":"Qing","middleName":"","lastName":"Chang","suffix":""},{"id":265687228,"identity":"08899d4c-2573-4bca-938e-ac029bd9fe2c","order_by":5,"name":"Christian Celia","email":"","orcid":"","institution":"Gabriele d'Annunzio University of Chieti and Pescara: Universita degli Studi Gabriele d'Annunzio Chieti Pescara","correspondingAuthor":false,"prefix":"","firstName":"Christian","middleName":"","lastName":"Celia","suffix":""},{"id":265687229,"identity":"559b6820-8bb9-4754-bc28-e9319c817f46","order_by":6,"name":"Xiaoyong Deng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzElEQVRIiWNgGAWjYHACAyC2AZMMPCRoSSNdy2EStBjcSN74uODXeXtziQTGB2/bGOTNCWmRnJFWbDyz73bizhkJzIZz2xgMdzYQ0MIvkWMmzdtzO8HgRgKbNG8bQ4LBAQJa2CRyzH/z9pyzB2ph/02UFpAtzDw/DjBuANrCTJQWyZ5nxdK8DcmJG848bJacc07CcAMhLQbHkzd+5vljZw9kHPzwpsxGnqAtDAIJDAyMbSAWYwOQkCCkHgj4QYb+IULhKBgFo2AUjFwAAPYpP2BH+cwZAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-6967-7050","institution":"Shanghai University","correspondingAuthor":true,"prefix":"","firstName":"Xiaoyong","middleName":"","lastName":"Deng","suffix":""}],"badges":[],"createdAt":"2023-12-31 07:55:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3825399/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3825399/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":49374849,"identity":"b0f5bdba-6079-45d8-942d-9877d48e191b","added_by":"auto","created_at":"2024-01-09 15:42:46","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":435939,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic illustration of the soft-templating route for preparing hollow-structured USNPs [6]\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3825399/v1/34708acbf893bf8a2688adcd.jpg"},{"id":49374847,"identity":"40cc2145-c7e3-4b6d-a0da-754f23571d75","added_by":"auto","created_at":"2024-01-09 15:42:46","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":90666,"visible":true,"origin":"","legend":"\u003cp\u003eSynthesis scheme for the preparation of the hollow-structured USNPs (A) and transmission electron microscopy (TEM) image of USNPs (B) [16]\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3825399/v1/16fbc3299295772850eb945b.jpg"},{"id":49374142,"identity":"6a0a2620-9500-47ca-9bdf-f03aacc4ae95","added_by":"auto","created_at":"2024-01-09 15:26:46","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":176097,"visible":true,"origin":"","legend":"\u003cp\u003eTEM picture of hollow-structured SNPs with different size: (a) 10 nm TMB/F127=0, 22 ℃；(b) 12.3 nm TMB/F127=0.04, 13 ℃；(c) 14.7 nm TMB/F127=0.12, 22 ℃；(d) 33 nm TMB/F127=0.57, 3 ℃. [19]\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3825399/v1/6c608a9399b7e6291662838f.jpg"},{"id":49374542,"identity":"f7efa083-a305-4812-8bbb-c7b02868daaf","added_by":"auto","created_at":"2024-01-09 15:34:46","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":114230,"visible":true,"origin":"","legend":"\u003cp\u003eSynthesis scheme and transmission electron microscopy (TEM) for the preparation of hollow-structured USNPs. [20]\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3825399/v1/4440c382423b4d6fdf2db63a.jpg"},{"id":49375081,"identity":"aaa504e4-585a-410b-b0cb-33437070fe45","added_by":"auto","created_at":"2024-01-09 15:50:46","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":54330,"visible":true,"origin":"","legend":"\u003cp\u003eTEM image of hollow-structured USNPs synthesized by cationic surfactant (CTAC) templating approach: (A) ethanol as solvent; [24] (B)water as solvent. [25]\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3825399/v1/a0450d054322e1746f24eb20.jpg"},{"id":49374146,"identity":"6adf44ed-acc7-4d7c-8289-0cb30c6d6976","added_by":"auto","created_at":"2024-01-09 15:26:46","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":89557,"visible":true,"origin":"","legend":"\u003cp\u003eTEM microphotographs of the monodispersed USNPs synthesized by using (a) ethanol as a solvent [26] (b) methanol as a solvent [27]\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3825399/v1/9238039a582c569644ca1cf4.jpg"},{"id":49374140,"identity":"f085e04e-3268-4158-b7a9-25bad88e222f","added_by":"auto","created_at":"2024-01-09 15:26:46","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":82197,"visible":true,"origin":"","legend":"\u003cp\u003eIllustration of the Polyethyleneimine Conjugation Process on the Surface of ultrasmall USNPs, Followed by the siRNA Delivery into Cells. [49]\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3825399/v1/0cf4e9a25305145b397ebdda.jpg"},{"id":49374541,"identity":"b5fd1551-6ecc-42a6-8228-9ca89ca34f1d","added_by":"auto","created_at":"2024-01-09 15:34:46","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":81793,"visible":true,"origin":"","legend":"\u003cp\u003ePET image of the mice injected with 124I-USNPs (a) 2 h postinjection (b) 24 h postinjection (c) quantitative radioactivity measurement from the individual organs of the mice. [52]\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3825399/v1/6f812f6d22e3b2c131b5b635.jpg"},{"id":50576973,"identity":"684f61c0-682d-4314-ba31-f269aadff2cf","added_by":"auto","created_at":"2024-02-02 17:55:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":934976,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3825399/v1/567239b3-82e1-4510-93a7-02dd528e270f.pdf"}],"financialInterests":"","formattedTitle":"Ultrasmall Silica Nanoparticles: Synthesis, Functionalization and Biomedical Application","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eSilica nanoparticles (SNPs) have broad prospects in the biomedical field, such as in medical diagnosis, photodynamic therapy, and drug delivery, due to their advantages of controllable particle size, large surface area, easy surface modification, and good biocompatibility [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. However, as an inorganic material, SNPs are very slowly metabolized in the body, and prolonged residence in the body may potentially lead to long-term toxicity.\u003c/p\u003e \u003cp\u003eThe size of nanoparticles can critically affect their pharmacokinetics in the biological system. Smaller particles are more easily and rapidly cleared through the kidneys, resulting in shorter residence in the body [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Chen et al. investigated the distribution and metabolism of fluorescent SNPs with sizes of 118 nm, 72 nm, 47 nm, and 27 nm in mice. The results showed that smaller SNPs had more pronounced systemic distribution and were more easily metabolized through the kidneys and excreted in urine. Choi et al. [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] indicated that nanoparticles with a size of \u003cem\u003eca\u003c/em\u003e 10 nm could escape the renal glomerulus. Therefore, to synthesize SNPs that do not persist in the body for long period, more and more researchers are focusing on the synthesis of ultrasmall silica nanoparticles (USNPs) with sizes below or about 20 nm and exploring their biomedical applications.\u003c/p\u003e \u003cp\u003eIn this short review, we will summarize and outline the progress of USNPs in the preparation, modification, and biomedical application, and provide an outlook on the future of USNPs.\u003c/p\u003e"},{"header":"2 Synthesis of hollow USNPs","content":"\u003cp\u003eThe most used method for synthesizing hollow USNPs is the soft-template approach, where surfactants and other organic additives first form a template, and then the precursor molecules self-assemble and undergo chemical reactions on the template surface. After the reaction is complete, the template is removed, resulting in the formation of hollow USNPs. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e illustrates the synthesis process of hollow USNPs using the soft-template method [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Common surfactants can be categorized into two types: non-ionic surfactants and ionic surfactants, which are detailed below.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Non-ionic surfactants as templates\u003c/h2\u003e \u003cp\u003eThe frequently used non-ionic surfactant is Pluronic F127, which is a poly (ethylene oxide)-poly (propylene oxide)-poly (ethylene oxide) triblock copolymer consisting of hydrophilic polyethylene oxide (PEO) and hydrophobic polypropylene oxide (PPO) segments [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. When its concentration exceeds the critical micelle concentration, F127 forms micelles in aqueous solution with a PPO core and a PEO shell. Under room temperature conditions, hydrolysis and condensation of the silica source occur on the surface of the micelles by adding hydrochloric acid solution. By introducing a silane coupling agent as a terminator, the number of silanol groups on the surface of particles can be reduced, resulting in uniformly dispersed USNPs. Huo et al. [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] first utilized F127 as a template agent, with tetraethyl orthosilicate (TEOS) as the main silicon source, hydrochloric acid as the hydrolysis catalyst, while dimethyldiethoxylsilane (DEDMS) were added as a terminator. The resulting USNPs had a size of approximately 10 nm. This method was subsequently widely adopted by other groups [\u003cspan additionalcitationids=\"CR10 CR11 CR12\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Chi et al. [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] employed F127 as a template and investigated the influence of different silane terminator agents on the particle size and morphology of USNPs. The results indicated that the higher the number of methyl groups in the silane terminator, the greater the steric hindrance, leading to smaller particle size. Compared to DEDMS, USNPs prepared using trimethylethoxysilane (TMES) as a terminator had a smaller particle size. This is attributed to the reaction between the organic silane and the silanol groups on the surface of nanoparticles, effectively preventing nanoparticle growing and aggregation. Additionally, examining the length of the carbon chain in the organic silane showed that longer carbon chains were more favorable for forming highly dispersed nanoparticles. Furthermore, if the terminator contained amino groups, it would enhance the stability and dispersion of the nanoparticles [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. This is due to the repulsion of the positive charge in the amino groups. Tan et al. [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] employed a new synthesis method by dissolving F127 in tetrahydrofuran (THF), slowly adding tetramethoxysilane (TMOS) at room temperature, and subsequently introducing deionized water. TMOS formed the shell of USNPs at the interface of F127 micelles. TEM results showed that the USNPs had a particle size of approximately \u003cem\u003eca\u003c/em\u003e 14 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eZanarini et al. [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] encapsulated fluorescent dyes within the core of USNPs, resulting in the 10-nm fluorescent USNPs. Wang et al. [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] used a similar approach where, under acidic conditions, they employed F127 as a template, TEOS as the silicon source, encapsulating a hydrophobic oxygen-sensitive probe within the hydrophobic core, while a hydrophilic pH probe was attached to the polyethylene glycol (PEG) on the outer shell. They used DMDMS as a terminator and one-step synthesis to produce stable and relatively small (12 nm) USNPs, which exhibited dual functionality in detecting both oxygen levels and pH values. Chi et al. [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] further manipulated the ratio and amount of 1,3,5-trimethylbenzene (TMB) in conjunction with F127 to synthesize hollow SNPs ranging from 10 to 90 nm in size (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). TEM results indicated that lower TMB concentrations led to smaller SNP sizes, and higher reaction temperatures resulted in smaller sizes and pore diameters of the SNPs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Ionic surfactants as templates\u003c/h2\u003e \u003cp\u003eYamada et al. [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] employed hexadecyltrimethylammonium bromide (CTAB) as a template agent and triethanolamine (TEA) as a catalyst. They conducted a stirring reaction at 80 \u003csup\u003eo\u003c/sup\u003eC for 6 h to investigate the influence of different alkoxy silanes (TMOS, TEOS, TPOS, or TBOS) on the particle size of SNPs. The results revealed that TMOS had the fastest hydrolysis rate, resulting in the smallest particle size, approximately 20 nm. To synthesize USNPs with a size smaller than 10 nm, Ma et al. [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] made improvements to Yamada's method. They used ammonia solution (NH\u003csub\u003e3\u003c/sub\u003e\u0026middot;H\u003csub\u003e2\u003c/sub\u003eO) as a catalyst, maintained the pH of the reaction system at 8, and introduced PEG-silane to terminate the reaction [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. This modification resulted in well-dispersed hollow USNPs with a size of 10 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The choice of NH\u003csub\u003e3\u003c/sub\u003e\u0026middot;H\u003csub\u003e2\u003c/sub\u003eO as a catalyst in the preparation process is primarily because NH\u003csub\u003e3\u003c/sub\u003e\u0026middot;H\u003csub\u003e2\u003c/sub\u003eO is a small molecule that does not adsorb on the surface of hollow USNPs, thereby avoiding an impact on the particle morphology [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMoller et al. [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] utilized hexadecyltrimethylammonium chloride (CTAC) as a template and investigated the impact of temperature and the ratio of TEOS/TEA on the particle size and morphology of USNPs. The results showed that at a TEOS/TEA volume ratio of 1:3, the synthesized hollow USNPs had a particle size range of 20\u0026ndash;70 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Simultaneously, it was observed that with a constant temperature, an increase in the TEOS/TEA ratio led to an increase in the USNPs' particle size. Pan et al. [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] improved upon Moller's method by using water as the solvent, CTAC as the surfactant, and TEOS as the silicon source. They controlled the system pH by adjusting the mass of TEA, resulting in hollow USNPs with a size of approximately 25 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"3 Preparation of Solid USNPs","content":"\u003cp\u003eThe commonly used method for synthesizing solid USNPs is the sol-gel method. Typically, TEOS is used as the raw material, and ethanol or methanol serves as the medium, with alkaline substances acting as catalysts. There are two commonly used alkaline catalysts: NH\u003csub\u003e3\u003c/sub\u003e\u0026middot;H\u003csub\u003e2\u003c/sub\u003eO and amino acids.\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Ammonium Hydroxide as the Catalyst\u003c/h2\u003e \u003cp\u003eGiesche et al. [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], using NH\u003csub\u003e3\u003c/sub\u003e\u0026middot;H\u003csub\u003e2\u003c/sub\u003eO as a catalyst and controlling the drop rate of TEOS, successfully prepared solid USNPs with a size of 17.5\u0026thinsp;\u0026plusmn;\u0026thinsp;3 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). Kim et al. [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], by changing the solvent (methanol and ethanol) and using NH\u003csub\u003e3\u003c/sub\u003e\u0026middot;H\u003csub\u003e2\u003c/sub\u003eO as a catalyst, synthesized monodisperse solid USNPs with sizes ranging from 5 to 450 nm. They found that compared to ethanol, the use of methanol as a solvent resulted in smaller silica sphere sizes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOw et al. [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] covalently attached organic fluorescent dyes to USNPs, synthesizing USNPs with a size of 20\u0026ndash;30 nm and high fluorescence intensity. Larson et al. [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] and Herz et al. [\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], following Ow's synthesis method, prepared fluorescent USNPs with a size of 10\u0026ndash;15 nm by adding different fluorescent molecules, using ethanol as the medium and NH\u003csub\u003e3\u003c/sub\u003e\u0026middot;H\u003csub\u003e2\u003c/sub\u003eO as a catalyst. Compared to free fluorescent molecules, the absorption and emission peaks of fluorescent USNPs did not change, but the fluorescence intensity increased. This enhancement is primarily attributed to the rigid environment provided by the USNPs shell, which increases the quantum yield of the fluorescent molecules. To further investigate the reasons for the fluorescence enhancement of dye molecules encapsulated in USNPs, Cohen et al. [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] synthesized fluorescent USNPs with sizes of 20 and 30 nm, containing 4 and 7 fluorescent molecules of DY630 in the core, respectively. Compared to free DY630, the fluorescence quantum yield of encapsulated DY630 increased by 13 and 15 times, mainly because USNPs provided a local environment for DY630, reducing its interactions with solvent molecules and fluorescent molecules, thereby improving the quantum yield.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Amino Acids as the Catalysts\u003c/h2\u003e \u003cp\u003eDavis et al. [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] first used L-lysine as a catalyst to synthesize USNPs with a size of 5 nm. The method involved dissolving lysine in deionized water, slowly adding an appropriate amount of TEOS, and stirring the reaction for 24 h. TEOS gradually hydrolyzed, forming USNPs. Simultaneously, Yokoi et al. [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] also used L-lysine as a catalyst to synthesize ordered arrangements of USNPs with a size of 12 nm. The interaction between protonated amino groups in L-lysine and anionic silicate groups allows lysine to cover the surface of USNPs. Simultaneously, due to the presence of hydrogen bonds between lysine molecules, a closely packed structure is easily formed, thereby controlling the particle size. Yokoi et al. [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] and Wang et al. [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], by altering synthesis conditions such as reaction time, stirring rate, composition of reactants, surface potential, pH value, and TEOS concentration, prepared USNPs with sizes ranging from 8 to 35 nm. They found that lysine not only acted as a weak alkaline catalyst, slowing down the hydrolysis of TEOS, but also served as a buffer, maintaining a constant pH in the system. Additionally, lysine prevented USNPs from aggregating, and by changing the stirring rate/reactant composition, the size of USNPs could be precisely controlled. Hartlen et al. [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] used arginine as a catalyst to synthesize highly uniform-sized USNPs in cyclohexane or vegetable oil solvents. Watanabe et al. [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] modified the reaction conditions, using arginine as a catalyst, TEOS as the silicon source, maintaining a pH of 9\u0026ndash;10, and conducting the reaction at 70\u0026deg;C for 24 h in an oil/water biphasic environment. They successfully synthesized USNPs with a size of 12 nm.\u003c/p\u003e \u003c/div\u003e"},{"header":"4 Removal of the Template Agents","content":"\u003cp\u003eAn essential step in preparing hollow USNPs is the removal of organic template agents. During this removal process, the hollow structure of USNPs is prone to collapse, and aggregation between USNPs may occur, forming large aggregates. Generally, there are three methods for removing template agents from hollow USNPs: high-temperature calcination, dialysis, and extraction. Shimogaki et al. [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] employed a calcination method to remove the surfactant from the pores of SNPs. The advantage of high-temperature calcination is easy operation. However, the drawback is that the hollow structure of USNPs is prone to collapse, and excessively high temperatures can lead to sintering of USNPs. Dialysis for removing template agents is a relatively mild method that effectively avoids the collapse of the hollow structure and high-temperature sintering of USNPs. Yamada et al. [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] used dialysis bags with a molecular weight cutoff of 8000\u0026ndash;14000 to remove the CTAB template from USNPs using acetic acid and ethanol solutions. Results showed that CTAB was completely removed after three rounds of dialysis [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Extraction can also be employed to remove template agents from USNPs. Pan et al. [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] performed extraction by stirring the reaction product in a sodium chloride-methanol solution at room temperature for 3 h to remove the surfactant CTAC. Infrared spectroscopy results showed that CTAC could be completely removed. The advantage of this method is that the template agent can be recycled after extraction, but the drawback is that multiple extractions may be required to completely remove the template agent. Moller et al. [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] chose different solvents (ammonium nitrate or hydrochloric acid) and stirred at 60\u0026deg;C for 2\u0026ndash;20 h, extracting the CTAC template from the solution. After two to three extractions, the template agent could be completely removed.\u003c/p\u003e"},{"header":"5 Functionalization of USNPs","content":"\u003cp\u003eUSNPs have a silicon-oxygen bond-supported framework, with a large number of silicon hydroxyl groups on the inner and outer surfaces. These groups can be easily further modified with active functional groups, thereby altering the properties of USNPs and expanding their applications.\u003c/p\u003e \u003cp\u003eNon-surface modification refers to the incorporation of two or more silicon sources during the synthesis of USNPs, introducing organic functional groups into the silica framework. Li et al. [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] introduced APTES during the formation of silica spheres, preparing amino-modified USNPs. Subsequently, by electrostatic interaction, these USNPs were covered on the surface of Layered Double Hydroxide (LDH) to form a composite material (NH\u003csub\u003e2\u003c/sub\u003e-SiO\u003csub\u003e2\u003c/sub\u003e@LDH). This composite material could stably exist in culture media or PBS buffer solutions. Cell experiments indicated that, compared to LDH, NH\u003csub\u003e2\u003c/sub\u003e-SiO\u003csub\u003e2\u003c/sub\u003e@LDH composites were evenly distributed within cells and could be used as carriers to deliver siRNA546 into cells.\u003c/p\u003e \u003cp\u003eSurface modification involves modifying the surface of USNPs after their synthesis. For example, Ma et al. [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] modified fluorescent USNPs with PEG and aluminosilicate. PEG increased the colloidal stability of USNPs, while aluminosilicate enhanced the encapsulation rate of fluorescent molecules and increased the intensity of the fluorescent signal. Additionally, various active groups (hydroxyl/amine/thiol/carboxyl groups) can be introduced to USNPs through microemulsion methods. These modified USNPs can then be combined with various active molecules, such as transferrin, monoclonal antibodies, etc., to enhance their targeting capabilities.\u003c/p\u003e"},{"header":"6 Biomedical Applications of USNPs","content":"\u003cp\u003eDue to their excellent biocompatibility, degradability, and non-toxicity, USNPs have been extensively investigated and applied in controlled release carriers for genes and drugs, as well as in cell imaging [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], disease diagnosis [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e] and treatment [\u003cspan additionalcitationids=\"CR47\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e6.1 Gene Carrier\u003c/h2\u003e \u003cp\u003eRNA interference technology, an efficient and specific method for blocking the expression of endogenous homologous genes, is a promising biotechnological approach. However, the siRNA molecules that generate RNA interference are challenging to enter cells and are easily cleared by the body. Thus, a suitable carrier is needed, and USNPs are one such carrier. Yu et al. [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e] successfully used 10-nm USNPs as carriers to deliver siRNA into tumor cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). They first modified the surface of USNPs with positively charged polyethyleneimine (PEI), which not only increased the loading capacity of siRNA but also protected siRNA from degradation by nucleases. Li et al. [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] also constructed a novel NH\u003csub\u003e2\u003c/sub\u003e-SiO\u003csub\u003e2\u003c/sub\u003e@LDH nanocomposite that could serve as a carrier to deliver siRNA into cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e6.2 Drug Carrier\u003c/h2\u003e \u003cp\u003eDue to the drug resistance of certain cancer cells, some anticancer drugs, such as doxorubicin (DOX), cannot maintain activity for an extended period [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Therefore, it is necessary to find suitable carriers. USNPs, as carriers, have several advantages, such as: larger surface area, enabling the loading of more anticancer drugs; efficient renal clearance, preventing long-term retention and toxicity; easily modifiable surface for targeting ligands, enhancing drug targeting; and more [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Huo et al. [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] compared the loading and release behavior of the drug molecule paclitaxel by 10 nm USNPs and polymeric F127 micelles. The results indicated that USNPs exhibited a significantly higher drug loading capacity than F127 micelles, and USNPs showed a slow release rate, while USNPs also extended the circulation time of the drug in the body. Pan et al. [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] modified USNPs with TAT peptide to facilitate efficient cellular uptake and delivery of DOX into the cell nucleus, thereby improving the bioavailability of DOX.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e6.3 Imaging\u003c/h2\u003e \u003cp\u003eKumar et al. [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e] synthesized 20-nm USNPs modified with near-infrared (NIR) fluorescent molecules. Through NIR imaging and radioisotope labeling experiments, it was observed that USNPs primarily distributed in the liver and spleen of mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). They were excreted through the hepatobiliary pathway, and histopathological analysis indicated that USNPs did not exhibit any potential toxicity. Therefore, USNPs can be used as biocompatible fluorescence probes for \u003cem\u003ein vivo\u003c/em\u003e imaging [\u003cspan additionalcitationids=\"CR54\" citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Turker et al. [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e] synthesized NIR fluorescent USNPs with sizes of 3.3 nm and 6.0 nm. In vivo imaging results revealed that smaller-sized USNPs were more easily excreted from the kidneys. Simultaneously, surface modification with PEG-silane effectively prevented the adsorption of serum proteins, enabling efficient excretion through urine.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e6.4 Diagnosis\u003c/h2\u003e \u003cp\u003eBenezra et al. [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] synthesized fluorescent USNPs with a size of 7 nm that could target cancer cells. The USNPs were modified with RGD peptide and radioactive iodine, enhancing their selectivity for tumor cells, improving the tumor/blood ratio, and facilitating renal clearance. This USNPs have been approved for human clinical trials [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Melanoma is considered the most common cancer in the world, and early diagnosis and treatment can reduce mortality. The spread of melanoma to lymph nodes is a crucial indicator of cancer development. Bradbury et al. [\u003cspan additionalcitationids=\"CR59\" citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e] synthesized 10-nm fluorescent USNPs (124I-cRGDY-PEG-C dots) for detecting the metastasis of melanoma in lymph nodes using positron emission tomography (PET) and optical imaging techniques. Compared to conventional clinical use of \u003csup\u003e18\u003c/sup\u003eF-FDG, these fluorescent USNPs efficiently located and monitored cancer cells, serving as fluorescent probes for monitoring cancer cell metastasis.\u003c/p\u003e \u003c/div\u003e"},{"header":"7 Toxicity of USNPs","content":"\u003cp\u003eThe translational potential of USNPs from the laboratory to clinical use is highly dependent on toxicological assessments [\u003cspan additionalcitationids=\"CR62 CR63\" citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. Generally, amorphous silica is recognized as safe by numerous regulatory authorities such as Therapeutic Goods Administration Australia (TGA), European Medicines Agency and United States Food and Drug Administration, and many studies show that SNPs have minimal toxicity in vivo [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. Kim et al. investigated the oral toxicity of colloidal SNP of 20 and 100 nm in rats over a period of 90 days, and found that doses of up to 2000 mg kg-1 showed no signs of toxicity [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. Akhtar et al. [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e] investigated the toxicity of 10 nm and 80 nm USNPs in A549 cells, finding that cell viability decreased with increasing USNPs concentration. However, there was no significant size-dependent effect on cell toxicity under the same concentration conditions. Similarly, Lin et al. [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e] examined the toxicity of 15 nm and 46 nm SNPs, observing no significant differences in cell toxicity despite the difference in SNPs size. The main reason is that smaller-sized USNPs tend to aggregate, resulting in size-independent toxicity to cells. Bradbury group and their collaborator studied the toxicity profiles of USNPs with size of 6\u0026ndash;7 nm in animal models. Upon these limited toxicological evaluation, the main organs such as liver, lung, kidney and heart, demonstrated no abnormalities or tissue damages at the dose of applied in cancer therapy [\u003cspan additionalcitationids=\"CR70\" citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. Hameed et al. [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e] synthesized porphyrin-based USNPs with size of 6.2 nm and used for fluorescence imaging-guided cancer photodynamic therapy, where organ toxicology and blood analysis also show no abnormalities after 24 h post-injection.\u003c/p\u003e"},{"header":"8 Conclusion and Prospects","content":"\u003cp\u003eUSNPs have advantages such as high specific surface area, good biocompatibility, low toxicity, and rapid excretion through urine. These properties make them widely applicable as carriers for drug or gene delivery and for the diagnosis and treatment of tumors. However, as a novel nanomaterial, there are still challenges in the preparation and application of USNPs. Firstly, large-scale synthesis of USNPs remains challenging, and scaling up experiments may affect their morphology and size. Secondly, there are many unresolved clinical issues, such as long-term toxicity of USNPs. Therefore, extensive research is needed before clinical use, including studies not only on acute toxicity but also on long-term toxicity, such as genetic toxicity. Yamashita et al. [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e] reported that intravenous injection of pregnant mice with USNPs might lead to pregnancy complications, highlighting the need for detailed research on the in vivo toxicity of USNPs.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFeihu Cui: writing original draft and editing; Lishuo Qu and Yao Gong: figure and table generation and conceptualization of the manuscript; Xiaoyong Deng, Christian Celia, Yijun Xie and Qing Chang revised the paper.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study is supported by National Natural Science Foundation of China (No. 21371118). The research activity of Christian Celia was supported by the Overseas Visiting Fellow Program 2022, Shanghai University, China. Christian Celia acknowledges financial support funded by the European Union \u0026ndash; Next Generation EU, under the National Recovery and Resilience Plan (NRRP), Mission 4 Component 2 - M4C2, Investiment 1.5 \u0026ndash; Call for tender No. 3277 of 30.12.2021, Italian Ministry of University, Award Number: ECS00000041, Project Title: \u0026lsquo;\u0026lsquo;Innovation, digitalisation and sustainability for the diffused economy in Central Italy\u0026rsquo;\u0026rsquo;, Concession Degree No. 1057 of 23.06.2022 adopted by the Italian Ministry of University. CUP: D73C22000840006.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials:\u0026nbsp;\u003c/strong\u003e \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe collected datasets during the current study can be obtained from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo ethics approval was involved for this work. In addition, the authors did not conduct any animal or human studies for this article\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors confirm that they have carefully reviewed the final version of the manuscript and agree to submit it for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eZhang L, Aragon-Sanabria V, Aditya A, Marelli M, Cao T, Chen F, Yoo B, Ma K, Zhuang L, Cailleau T. Engineered ultrasmall nanoparticle drug-Immune conjugates with \u0026quot;Hit and Run\u0026quot; tumor delivery to eradicate gastric cancer. 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Nat Nanotechnol.\u003cem\u003e \u003c/em\u003e2011;6(5):321-28. https://doi.org/10.1038/nnano.2011.41\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Silica Nanoparticles, functionalization, diagnosis and therapy","lastPublishedDoi":"10.21203/rs.3.rs-3825399/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3825399/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eUltrasmall silica nanoparticles (USNPs) with a size ＜20 nm exhibit unique advantages such as low toxicity, surface modification, and efficient renal clearance, making them highly promising in the fields of bioimaging, disease detection, gene delivery, and drug delivery. In this short review, synthesis, functionalization, and biomedical applications of USNPs are discussed. First, the different synthetic methods for fabricating hollow USNPs and solid USNPs. Then, surface modification methods are described in detail. Finally, the biomedical application progress and toxicity of USNPs encapsulated with fluorescent and drug molecule are discussed.\u003c/p\u003e","manuscriptTitle":"Ultrasmall Silica Nanoparticles: Synthesis, Functionalization and Biomedical Application","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-09 15:26:41","doi":"10.21203/rs.3.rs-3825399/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"dbe4dd02-1b32-401f-9430-0955cd9d27bc","owner":[],"postedDate":"January 9th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-02-02T17:47:07+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-09 15:26:41","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3825399","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3825399","identity":"rs-3825399","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}