Introduction

Over the past few decades, materials with sizes ranging from 1 to 100 nm have attracted increasing attention and research in the scientific field due to their unique properties. Research conducted on nanoparticles and their subsequent development have revealed their innovative applications. Nanoparticle applications can lead to rapid advances in food, agriculture, and biomedical sciences, particularly to a boom in the field of biomedical applications1,2,3. Noble-metal nanoparticles have attracted the attention of researchers owing to their unique properties, such as plasmon resonance, large specific surface area, and accessible surface functionalisation4. Following the discovery made by Falk and Taylor in 1971 that gold nanoparticles (AuNPs) could be used for immunolabeling, many excellent properties of AuNPs have been discovered and widely applied in biomedical fields. AuNPs are considered considerably effective and safe for tumour treatment and drug delivery and thus have great potential5. Mahmoudi et al.6 enhanced the cellular uptake and antioxidant properties of curcumin to produce cytotoxic and apoptotic effects on cancer cells. AuNPs serve as excellent drug delivery carriers in cancer treatment have also attracted significant research interest. They have great potential for application in biopharmaceuticals, anticancer therapy, and bioimaging and are the most used nanoparticles in biomedicine7,8. AgNPs incorporated into denture base polymers and tissue conditioners have shown excellent antibacterial activity and ability to prevent stomatitis9. AgNPs have been reported to serve as antiviral nanoparticles that can reduce the coronavirus infection rate by interacting with viral surface proteins10. In recent years, the emerging inorganic hydrotalcite-layered nanoparticles (LDHs) have been extensively explored by researchers owing to their excellent biocompatibility, high specific surface area, and unique layered structure properties and have been gradually and widely used in the biomedical field11. The layered structure of LDHs comprises several specific cations and adjustable interlayer ions. Therefore, the LDHs cause different biological effects and play different roles in the regulation of cellular functions. In addition, LDHs can also be used as excellent nanodrug carriers owing to their unique controlled release properties in biomedical applications, according to different pH environments12. Shun et al.13 constructed a polypyrrole-arsenic LDH film on the surface of a nickel-titanium alloy in situ for use in cancer treatment. LDHs serving as drug carriers effectively kill tumour cells when used in high-temperature combined chemotherapy.

Nanoparticles have contributed to biomedicine significantly. They enable accurate drug delivery targeting, safe and effective cancer treatment, and precise and noninvasive organ imaging. Thus, they have minimised patient suffering and ushered in a new era for the medical field4,14. The optimal synthesis of nanoparticles has also become a research focus in nanotechnology development. Obtaining nanoparticles with excellent properties has sparked a research boom in the synthesis of nanoparticles through different pathways, while different nanoparticle preparation methods are constantly emerging. The development of new methods for nanoparticle synthesis is important for promoting research on the use of nanoparticles and their application in biomedicine and other fields. These new methods of synthesising nanoparticles would enable improved preparation efficiency, performance regulation, new functional characteristics, and industrial-scale production in relation to nanoparticles. Additionally, they can not only improve the therapeutic effect produced by nanoparticles in biomedical applications but also reduce the environmental and health risks of nanoparticles8. Andrey et al.15 reviewed the superiority of CuNPs in biomedical applications and expounded the necessity for developing new synthesis methods. The new methods should enable more precise control of the characteristics of nanoparticles and improve their stability and biocompatibility, which are critical for their safety and effectiveness in clinical applications. Additionally, with the increasing use of nanoparticles, developing industrial methods for the large-scale production of nanoparticles has become important. This can not only meet market demand, but also reduce costs and make the application of particles more flexible. Moreover, in contrast to the existing preparation methods, the new methods would produce green, low-cost, and high-quality nanoparticles, thereby enhancing their applicability. Methods such as physical grinding, chemical reduction, coprecipitation, and electrochemical catalysis have limitations, hindering the development of nanoscience. For example, the physical grinding method requires large equipment, which takes a long time for material preparation, consumes a large amount of energy, and leads to uneven particle distribution. The chemical reduction method limits the application scope of the nanoparticles. The coprecipitation method requires the addition of dispersants and organic solvents during washing and precipitation processes to produce uniformly sized particles. The method is complex and could pose a threat to the environment. Therefore, a new energy-efficient, harmless, rapid method that can be used for the preparation of a large number of sterile nanoparticles with uniform particle size and good stability has become necessary.

Moreover, most nanoparticle applications in the biomedical field are implantable in vivo. The premise of ensuring their availability is sterilizing them to prevent microorganisms (spores, fungi, bacteria, and viruses) from invading the human body, and biosafety is particularly important16. At present, the sterilization methods mainly include autoclaving, sterile filtration, and ionizing radiation. Among them, autoclaving is the gold standard recognised internationally17. However, these sterilisation methods, including autoclaving, tend to negatively affect the morphology and physicochemical properties of nanoparticles, hindering their effective application and safety18. Therefore, how these problems could be solved to make the best use of nanoparticles with excellent performance is worth exploring.

Based on previous research findings, this study considered a one-step method for the preparation of sterile nanoparticles with excellent performance. The combination of the preparation step and final sterilisation procedure is realized compared with the existing nanoparticle preparation method. The sterile nanoparticles prepared using the one-step method exhibited excellent performance. Currently, there are no reports on this aspect. The high-pressure sterilisation method used in this study has certain research value and is innovative.

In this study, we innovatively used a high-pressure sterilisation method to prepare nanoparticles. Our method enables one-pot preparation of metal nanoparticles and LDHs. The morphology and size of the nanoparticles were characterised using transmission electron microscopy (TEM) and Malvern particle size analysis. The unique plasmon resonance absorption peak (SPR) of the nanoparticles were studied using the UV-vis absorption spectra (UV-vis spectra). Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) results revealed the surface functional groups and crystal structure of nanoparticles. The characterisation results obtained for the nanoparticles prepared in this study are similar to the characterisation results obtained for the nanoparticles prepared using traditional methods, which can provide an alternative way for nanotechnology synthesis. Thus, by using the nanoparticle preparation method introduced in this study, a large number of high-performance nanoparticles could be prepared, and the prepared nanoparticles could be subjected to sterilisation treatment, forming an efficient, economical, and scalable integrated solution.

Results

Characterization of noble metal source nanoparticles

The solutions of AuNPs and AgNPs obtained in this study are typical wine red and yellow19,20. Based on the unique SPR phenomena that occur in noble-metal nanoparticles, the AuNPs and AgNPs prepared in this study were characterised using UV-vis spectroscopy. The SPR characteristic absorption peak provides useful information on the size and shape of the synthesised nanoparticles to verify the synthesised nanoparticles preliminarily. In addition, the SPR effect determines the colour of the reaction solution containing noble metal nanoparticles. The AuNPs and AgNPs exhibited strong SPR effects. When the nanoparticle size was within a particular range, the SPR absorption peaks were located within the visible light region, causing certain wavelengths of light to be strongly absorbed by the nanoparticles. The nanoparticles simultaneously reflected or scattered their complementary colour. The reaction solutions of the noble metal nanoparticles exhibited specific colours. The illustration in Fig. 1a shows a standard wine-red AuNP reaction solution prepared under natural light using our method, indicating the formation of AuNPs. To verify AuNPs stability, we left AuNPs for some time. The reaction solution remained transparent and uniform, with a wine-red colour, indicating excellent stability. The UV-vis spectrum further confirmed AuNPs synthesis. The absorption spectra of the synthesised AuNPs had the largest SPR absorption peaks in the range of 300–700 nm, with the maximum absorption peak appearing at 520 nm (Fig. 1a), which is the characteristic absorption peak of AuNPs21. The characteristic absorption peak was narrow, indicating that the synthesised AuNPs had uniform particle size and good stability. The UV-Vis spectra of the AgNPs showed that the nanoparticles had a narrow SPR peak at 436 nm (Fig. 1b), which proved that the synthesised AgNPs had a uniform particle size and good dispersion22. The illustration in Fig. 1b showed an orange-yellow AgNPs reaction solution prepared under natural light. It fully supporting the UV-vis spectra characterisation results.

Fig. 1
figure 1

Characterization results of noble metal source nanoparticles. (a) UV-vis spectrum of AuNPs (SPR: 520 nm); The illustration of (a) is the wine-red AuNPs reaction stock solution and particle size distribution; (b) UV-vis spectrum of AgNPs; The illustration in (b) are an orange-yellow AgNPs reaction solution prepared under natural light and particle size distribution; (c) TEM characterization results of AuNPs; (d) TEM characterization results of AgNPs.

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TEM can be used to visually analyse the size, dispersion, and morphology of the synthesised nanoparticles. As Fig. 1c shows, the synthesised AuNPs exhibited a spherical, partially elliptical, well-dispersed morphology with a uniform particle size. The AuNPs particle diameter was approximately 21 nm (Fig. 1c Illustration). The morphology of the AgNPs was ellipsoidal, particle size was uniform, and the agglomeration is less (Fig. 1d). The AgNPs particle diameter was approximately 25 nm (Fig. 1d Illustration). The synthesised particles were characterised by dynamic light scattering (DLS) to confirm their sizes, and the results are shown in Fig. 2a, b, which display average particle sizes of 25 and 38 nm, respectively. The particle-size distributions of the two nanoparticles were concentrated. The average nanoparticle size of TEM (Nano Measurer 1.2) was often smaller than the corresponding DLS. This difference is generally attributed to the fact that the DLS particle size represents the hydrodynamic diameter of the nanoparticle with a capping agent coating on its surface23. The FTIR spectra of the AuNPs and AgNPs were similar (Fig. 2c, d). The broad peaks of AuNPs at 3435 cm−1 and AgNPs at 3434 cm−1 match the stretching vibration absorption peaks of alcohol hydroxyl groups24. The absorption peak around 2923 cm−1 can be attributed to the stretching vibration of the carboxyl group. The peak at 2854 cm−1 corresponds to the stretching vibration of the –CH bond25, whereas the weak absorption peak observed at about 1725 cm−1 and the peak around 1619 cm−1 are typically caused by C=O vibration, which is associated with the presence of carbonyl groups26. The peaks of the AuNPs and AgNPs observed at 1108 and at 1100 cm−1, respectively, were related to the asymmetric C–O–C stretching vibration27. Table 1 lists results of these analyses. After characterising the morphology and surface functional groups of the nanoparticles, their elemental compositions were studied using Energy Dispersive Spectroscopy (EDS). Figure 3 shows the elemental mapping images of AuNPs and AgNPs, showing the elemental maps of Au and Ag in orange and pink, indicating their uniform distribution on the nanoparticles.

Fig. 2
figure 2

Characterization results of noble metal source nanoparticles. (a) The particle size distribution of AuNPs; (b) The particle size distribution of AgNPs; (c) FTIR characterization results of AuNPs; (d) FTIR characterization results of AgNPs.

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Table 1 Infrared vibrational wavenumber (cm−1) of AuNPs and AgNPs.
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Fig. 3
figure 3

The corresponding SEM-EDS mapping of the noble metal source nanoparticles. (a, b) The Au element mapping of AuNPs; (c, d) The Ag element mapping of AgNPs.

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Characterization analysis of hydrotalcite nanoparticles

LDHs comprise octahedral hydroxyl groups and two metal ions that form positively charged layers and interlayer anions. Different types of active LDHs can be obtained by adjusting the chemical compositions between layers28. The substitutional properties of the interlayer anions enable most anionic drugs to enter the hydrotalcite interlayer through substitution. Thus, LDHs have become important drug carriers in biomedicine and offer great research value. In this study, LDHs with Co and Al cations were prepared using high-pressure sterilisation, while anionic-intercalated Mg2Al-LDH was prepared with nitrate ions as interlayer anions. The morphology, structure, and surface functional groups of the synthesised nanoparticles were analysed.

The TEM results of the Mg2Al-NO3-LDHs and Co2Al-LDHs demonstrated that the synthesised nanoparticles were in the form of hexagonal nanosheets (Fig. 4a, b). The illustrations in the upper-right corners of Fig. 4a, b show the milky white and bright pink clear reaction solutions of Mg2Al-NO3-LDHs and Co2Al-LDHs, respectively, under natural conditions. The reaction solution was maintained at room temperature for a specific period of time with no colour change or precipitation. The two LDHs exhibited good dispersion and no substantial agglomeration. The illustrations in the lower-left corners of Fig. 4a, b show the average particle sizes of the two LDHs measured according to the TEM results. The apparent diameters of Mg2Al-NO3-LDHs and Co2Al-LDHs were 75 and 83 nm, respectively. The DLS characterisation results of the two LDHs are shown in Fig. 4c, d. The average particle size of the Mg2Al-NO3-LDHs was 82 nm, its particle size distribution was concentrated. The average particle size of the Co2Al-LDHs was 90 nm, with a narrow particle size distribution and good dispersion. Similarly, the DLS of the Mg2Al-NO3-LDHs the Co2Al-LDHs were slightly larger than its apparent TEM particle size. Typically, DLS measures the hydrodynamic diameter of nanoparticles in solution. In addition to water molecules adsorbed on the surface of nanoparticles and surface modification, it also has a certain relationship with the measured concentration and shape of nanoparticles29. The nanoparticles prepared in this study belong to polygon, which may cause differences in particle size distribution. Considering the particle size distribution of nanoparticles, DLS can better reflect the overall particle size and stability of nanoparticles.

Fig. 4
figure 4

Characterization results of LDH nanoparticles. (a) TEM characterization results of Mg2Al-NO3-LDHs; The illustrations of (a) are the milky white Mg2Al-NO3-LDHs reaction stock solution and particle size distribution; (b) TEM characterization results of Co2Al-LDHs; The illustrations of (b) are the bright pink Co2Al-LDHs reaction stock solution and particle size distribution; (c) The particle size distribution of Mg2Al-NO3-LDHs; (d) The particle size distribution of Co2Al-LDHs; (e) FTIR characterization results of Mg2Al-NO3-LDHs and Co2Al-LDHs; (f) XRD characterization results of Mg2Al-NO3-LDHs (JCPDS no. #35-0965) and Co2Al-LDH (JCPDS no. #51-0045).

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Figure 4e shows the FTIR spectra of the surface functional groups of the two LDHs. The broad absorption peak of Mg2Al-NO3-LDHs at 3480 cm−1 and that of Co2Al-LDHs at 3433 cm−1 are attributed to the stretching vibration of the hydroxyl group of the LDH sheet and the OH bond in the H2O interlayer (Fig. 4e)30. The weak peaks of the two LDHs at approximately 2920 cm−1 and the bending vibration of the Mg2Al-NO3-LDHs peak at 1632 cm−1 are related to intercalated H2O, which could be related to the lack of dryness in the test sample31. The strong and weak absorption peaks of the Mg2Al-NO3-LDHs at 1384 and 834 cm−1, respectively, are matched to the antisymmetric stretching vibration32 of NO3−. The peaks of Co2Al-LDHs at 1354 cm−1 and 796 cm−1 are attributed to the stretching vibration of CO32− as a compensatory intercalation anion33. Other peaks below 800 cm−1 in the FTIR spectra are generally caused by the stretching vibration and bending oscillation of metal-oxygen (M-O) and metal-oxygen-hydrogen (M-O-H) bonds34. The absorption peaks of Co2Al-LDHs match the stretching vibration characteristics of Co-O and Al-O bonds at 614 cm−1 and 550 cm−1. The absorption peaks of Mg2Al-NO3-LDHs attributed to the stretching vibration characteristics of Mg-O and Al-O bonds are located35 at 661 cm−1 and 626 cm−1. Table 2 lists the results of these analyses. Then, the crystal structure of the prepared samples was studied by an X-ray diffractometer. The XRD results indicate that the Co2Al-LDHs and Mg2Al-NO3-LDHs prepared have the characteristic diffraction peaks (003), (006), (012), and (110) (Fig. 4(f)). Among them, the 2θ values of the corresponding diffraction peaks of Mg2Al-NO3-LDHs (JCPDS no. 51-0045) were 11.3° (003), 22.8° (006), 34.7° (012), 39.1° (015), 46.4° (018), 60.6° (110), and 61.9° (113). The 2θ values of the corresponding diffraction peaks of Co2Al-LDHs (JCPDS no. 35–0965) were 11.5° (003), 23.3° (006), 34.5° (012), and 60.2° (110). The ground-state diffraction peaks of the (003) and (006) crystal planes are consistent with the typical diffraction peaks of the LDHs crystal structure. Thus, using a high-pressure sterilisation method, nanoparticles with a unique layered structure could be synthesised successfully36. The sharp diffraction peaks observed in the XRD patterns also indicated the high crystallinity and good layered characteristics of the two LDHs. However, compared with the Mg2Al-NO3-LDHs, the Co2Al-LDHs exhibited a lower crystal structure strength.

Table 2 Infrared vibrational wavenumber (cm−1) of Mg2Al-NO3-LDHs and Co2Al-LDHs.
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The elemental composition of the two LDHs was analyzed by EDS. According to the element mapping shown in Fig. 5a–e, the anion-intercalated Mg2Al-NO3-LDHs comprised Mg, Al, N, and O. According to the EDS spectra, except O, the signal of Mg and Al in LDHs were strong (Fig. 5f). The cation-intercalated Co2Al-LDHs comprised Co, Al, C, and O, with the strongest signals from Co and Al (Fig. 6a–f). The element mapping images of the Mg2Al-NO3-LDHs and Co2Al-LDHs show the distribution of different elements in the LDHs, proving that anions and cations had been successfully inserted into the LDHs layers37.

Fig. 5
figure 5

The corresponding SEM-EDS mapping of the Mg2Al-NO3-LDHs. (a–e) The element mapping of Mg, Al, N, O in the Mg2Al-NO3-LDHs; (f) The EDS spectrum of Mg2Al-NO3-LDHs.

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Fig. 6
figure 6

The corresponding SEM-EDS mapping of the Co2Al-LDHs. (a–e) The element mapping of Co, Al, C, O in the Co2Al-LDHs; (f) The EDS spectrum of Co2Al-LDHs.

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Microbial limit experiment (MLT)

In the previous characterisation, we proved the feasibility of the high-pressure sterilisation method. The innovation of this method also lies in the ability to obtain sterile nanoparticles. Therefore, we conducted MLT to verify the sterility of the nanoparticles. First, according to the results listed in Table 3, the recovery rates for all samples were between 0.50 and 2.00. Therefore, the counting method of the nanoparticle test solution in MLT is suitable for counting the total number of aerobic bacteria, molds and fungi in the sample, as well as subsequent sterility tests. According to the results (Tables 4 and 5), the culture media coated with four commercially available nanoparticles showed positive results in MLT validation experiments, and all of them had colony counts. Compared to commercially available nanoparticles prepared by different methods, the culture medium coated with the four types of nanoparticles prepared by the new method showed no colony growth, indicating a negative result. And the control group also showed negative results. In addition, the validation results of the bacterial control method also showed that the nano particle LB culture tube in this study did not exhibit colony growth, just like the negative control (Table 6). The positive control group culture medium showed significant bacterial growth. Therefore, the analysis of the experimental results comparing the experimental group and the control group confirms that the nanoparticles prepared by this research method have high-quality sterility.

Table 3 Recovery ratio of total number of aerobic bacteria, mold and fungi.
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Table 4 Aerobic bacteria sterility verification.
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Table 5 Anaerobic bacteria sterility verification.
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Table 6 Control bacterial examination method (E. coli).
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Discussion

As nanomaterial applications increase, nanoparticles are becoming more and more accurate and widely used in the biomedical field. Nanotechnology has also developed various synthesis methods that benefit the application of nanomaterials. The aim is to synthesize nanoparticles with specific morphology, particle size, and physicochemical properties to continuously approach the standard of the synthesis process. Current nanoparticle preparation methods widely used include mechanical, chemical reduction, hydrothermal methods, and several other physical and chemical preparation methods7,38,39,40. Most synthesis methods have defects in the reaction system or require improvements. Different nanoparticle synthesis methods and reaction equipment are constantly emerging, along with continuous innovations and research in the field of nanotechnology, to optimise the synthesis conditions and methods of nanoparticles. From inefficient and costly physical approaches to high-yield and stable chemical methods, nanoparticles are continuously synthesized and applied. However, in the face of the biocompatibility of nanoparticles, the application field of nanoparticles obtained by commonly used methods is limited41. Researchers have therefore proposed more and more environmentally friendly and biosafe methods for synthesising nanoparticles42. This study used a high-pressure steam steriliser as a reaction device. Following the general sterilisation procedure, the reaction system was increased to a fixed temperature and pressure as the reaction equipment heated up. At this point, the nanoparticle reaction system is synthesised from an ionic state into nanoparticles with a specific morphology. This synthesis method can adjust the proportions of various reactants in the reaction system according to the initial chemical state of nanoparticles to obtain the type of nanoparticles required in the application field. The method was simple, easy to use, and ensured the stability and repeatability. The stability and scalability of nanomaterial synthesis methods have become a challenge in the research of nanotechnology development43. Most methods used for preparing noble metal nanoparticles and hydrotalcite are bottom-up synthesis strategies. During preparation, reaction conditions, such as reaction temperature and rotation speed, have to be controlled strictly41,44. Any influencing factor can lead to poor repeatability, requiring increased scientific research efforts to solve the resulting problems.

The high-pressure sterilization synthesis method developed in this study was originally designed to obtain large quantities of nanoparticles in a simple, fast, and green manner at once. It fully utilizes conventional sterilisation instruments in biological laboratories as reaction equipment and uses a sterilised vessel as the reaction container. The reaction principle is that the steam in the high-pressure steam sterilization pot transfers heat, causing the reaction system in the reaction vessel to rapidly heat up due to the rapid transfer of water vapor heat. The evenly distributed water vapour heated the liquid in the reaction vessel and made the reaction complete. Compared with single heating stirring at the bottom of the constant temperature heating plate of the magnetic stirrer, the system inside the reaction vessel reacted more fully, and the resulting products were more controllable. Secondly, the traditional hydrothermal preparation of nanoparticles often results in solvent pollution, makes the cleaning of the reaction vessels cumbersome, and product-quality stability across different batches poor45. High-pressure sterilisation synthesis method avoids these issues, reduces experimental costs, and enhances experimental safety. Moreover, the novelty is that the nanoparticles prepared by this method meet sterility requirements of in vivo cell or tissue applications. Ensuring sterility is a necessary processing procedure for implantable nanoparticles in all biological studies46,47. Previous studies have reported that sterilisation methods could affect the surface chemical properties, shape, and size of nanoparticles46,48. The functionality of nanoparticles depends on their morphologies, particle sizes, and physical and chemical properties49. Any changes in these determinants could affect the efficacy and utilisation rate of in vivo nanoparticles. Most sterilisation methods lead to changes in the surface properties and internal structure of nanoparticles, most intuitively manifested as particle aggregation. This is very unfavourable for the biomedical applications of nanoparticles. Compared with other nanoparticle preparation methods, the method used in this study did not require strictly controlled reaction conditions. The mixed reaction precursors can be directly added into autoclavable vessels and placed in the high-pressure steam sterilisation pot. The nanoparticles with excellent properties and sterility were then synthesised under the standard autoclave procedure (121 °C for 20 min). Moreover, many studies are currently focused on reducing the low nanoparticle utilisation rates caused by sterilisation procedures. The innovative method developed in this study can prevent the adverse effects of secondary sterilisation on the nanoparticles, allowing the obtained nanoparticles to retain their properties as much as possible, maintaining their full functionality. Plant extracts were selected as reducing agents and stabilisers in the reaction system of noble metal nanoparticles. This strategy circumvents the potentially toxic side effects associated with chemical-reducing agents and realizes the green synthesis of economic and environmental protection.

Generally, the success of any nanoparticle preparation can be determined during the reaction process based on colour changes. The high-pressure sterilisation synthesis method in this study obtained wine-red AuNPs, yellow AgNPs, pale pink Co2Al-LDHs, and milky white Mg2Al-NO3-LDHs reaction solutions. Thus, the method is feasible. The UV-vis spectra of the nanoparticles can be used to preliminarily judge their morphologies, sizes, and stabilities50. The TEM results intuitively showed the morphology of the synthesised nanoparticles49. Combined with TEM and UV-vis spectra results, the nanoparticles prepared by the method have good dispersion and uniform particle size, which can increase nanoparticle applicability. The FTIR, XRD, and EDS results showed the surface groups, crystal structure, and elemental composition of the nanoparticles38,51. In this study, AuNPs and AgNPs were prepared using sodium salts obtained from the same plant extract as reducing and stabilizing agents. According to TEM and FTIR results, the morphologies and surface functional groups of the AuNPs and AgNPs were similar, which could be attributed to the sodium malate capping on their surfaces. The average particle size of 25 nm and 38 nm and the corresponding SPR peak at 520 nm and 436 nm, respectively, align with the AuNPs in the 500–600 nm prominent absorption peak and AgNPs in the 400–500 nm absorption peak38. The results are similar to the characterization of green synthesized AuNPs and AgNPs using peppermint essential oil, Callistemon viminalis extract, and Peganum harmala extract in previous studies23,52,53. The SPR of a noble-metal nanoparticle strongly depends on its particle size, morphology, and stabiliser coating thickness. Although different green synthesises of noble-metal nanoparticles, there may be differences in these characteristics, their performance both in their noble metal nanoparticles SPR characteristics of typical range50. This study also successfully prepared anion-intercalated Mg2Al-NO3-LDHs and cation-intercalated Co2Al-LDHs. Through characterisation, the absorption peak positions, elemental compositions, and crystal structures of the prepared nanoparticles were observed. FTIR and EDS results revealed the position of the absorption peaks and the composition of the elements, showing that the absorption peaks of the surface functional groups and metal element mappings were observed. The prominent diffraction peaks of Co2Al-LDHs (JCPDS No. 35-0965) and Mg2Al-NO3-LDHs (JCPDS No. 51-0045) were obtained based on the XRD crystal structure data, which conformed to the classical crystal plane structure of hydrotalcite. These findings are consistent with previous characterisation results obtained for the LDHs synthesised using the coprecipitation method54,55,56. The characterisation results of the nanoparticles synthesised in this study were consistent with those of the nanoparticles prepared using the conventional method. The unique physical and chemical properties of the nanoparticles had not been destroyed, thereby ensuring the application value of the materials used. The MLT study verified that the prepared nanoparticles satisfied the sterility requirements, which aligned with the high sterilisation performance of high-pressure steam sterilisation equipment. The results confirmed the effectiveness of the research methodology and demonstrated the potential use and reliability of high-pressure sterilisation synthesis in nanoparticle preparation. Thus, this novel nanoparticle preparation method warrants further study.

Conclusion

In summary, we developed a novel high-pressure sterilisation synthesis method to prepare nanoparticles. The noble-metal and hydrotalcite nanoparticles that were first synthesised exhibited good dispersibility, uniformity, and sterility. The preparation method was simple and had a low time cost. The method would have direct applications in fields such as in vivo imaging, photothermal therapy, and drug delivery. Our study preliminarily verified the feasibility of the high-pressure sterilization synthesis method for the preparation of nanoparticles. We will, in future, explore the safety and application differences of the nanoparticles in organisms.

However, the methods currently used in nanoparticle synthesis have certain limitations. Under conventional conditions, the reaction temperature and stirring speed are key factors affecting nanoparticle morphology and particle size. Our method only has a limited ability to regulate the reaction temperature and stirring speed. Moreover, nanoparticles with excellent properties can be developed only after a comprehensive exploration. Therefore, we plan to extend our research by optimising the reaction system to overcome its limitations and expand the application potential of the nanoparticles. To achieve this goal, we will focus on the concentration ratio and selection of various reactive monomers and reducing agents in the reaction system to prepare nanoparticles with different physicochemical properties. Our high-pressure sterilization synthesis method offers a new and scalable approach for nanotechnology development. It also offers innovative solutions in the biomedical field, which could promote the development of related fields.

Experimental

Experimental materials

Tetrahydrate chloroauric acid (HAuCl4·4H2O) was purchased from China National Pharmaceutical Group Chemical Reagent Co., Ltd. (Shanghai, China). Silver nitrate (AgNO3) was purchased from Tianjin Yongsheng Fine Chemical Co., Ltd. (Tianjin, China). Disodium malate is provided by Bailingwei Chemical Reagent Co., Ltd. (Huai’an, China). Aluminum nitrate (Al(NO3)3·9H2O) and methanol were both purchased from Tianjin Guangfu Fine Chemical Research Institute (Tianjin, China). Magnesium nitrate hexahydrate (Mg(NO3)2·6H2O) and cobalt chloride hexahydrate (CoCl2·6H2O) were purchased from Shanghai Yuanye Biological Co., Ltd. (Shanghai, China). Sodium hydroxide is provided by McLean Biotech (Shanghai, China). Tryptone and Yeast extract were purchased from AoBoXing Biotech Co., Ltd. (BeiJing). Nacl was purchased from ShengAo Co., Ltd. (Tianjin). Ager was purchased from Coolaber Co., Ltd. (BeiJing). The laboratory prepares ultrapure water for future use. Staphylococcus flavus [CMCC(B)26003], Bacillus subtilis [CMCC(B)63501], Escherichia coli [CMCC(B)] 44102], Candida albicans [CMCC(F)98001], Aspergillus Niger [CMCC(F)98003] and Pseudomonas aeruginosa [CMCC(B)10104 were all derived from China National Institute of Food and Drug Control (Beijing). Qingdao Haibo Biotechnology Co., Ltd. (Shandong) provides the material and medium for Microbial limit test (MLT), including pancreatic cheese Soy Peptone agar medium (TSA), pancreatic cheese Soy Peptone liquid medium (TSB), Sabouraud glucose agar medium (SDA), Sabouraud glucose liquid medium (SDB), McConkey liquid medium, McConkey agar medium (MAC), sterile sodium chloride-peptone buffer (pH = 7.0), polysorbate 80.

Instruments and equipment

A conventional high-pressure steam sterilisation pot in a biological laboratory was used as reaction and sterilisation equipment for the culture medium. An ultrasonic cleaner was used to disperse and mix the reaction mixture. An ultrapure water meter (milliqA10) was used to prepare the water required for the reaction. A TEM instrument (Hitachi HT7700) was used to observe the morphology of the noble metal nanoparticles and layered hydrotalcite nanomaterials. The samples were prepared on a copper mesh (carbon film) using the drop-casting method. A Malvern laser particle-size analyser (EetastEcyNancEs) was used to determine the average particle size of the nanoparticles. The SPR peaks of the AuNPs and AgNPs were characterised using an enzyme-linked immunosorbent assay reader (SpectrumMax M2). Sample suspensions of the AuNPs and AgNPs (3 mL) were placed in quartz cuvettes to determine their absorbance values. Field-emission scanning electron microscopy (FE-SEM) and EDS were used to study the elemental compositions of the prepared nanoparticles. The structures of the metal and non-metal nanomaterials prepared were characterised using an X-ray diffractometer (D8 ADVANCE). An FTIR spectrometer (Bruker VERTEX 70v) was used to characterise the surfaces of the two types of nanoparticles prepared. A clean bench was used to inoculate the plate with nanomaterials. A constant temperature incubator was used as the cultivation plate.

Preparation of noble metal source nanoparticles by high-pressure sterilization synthesis method

The high-pressure sterilization synthesis method to prepare nanoparticles, a high-pressure steam sterilisation pot was used as the reaction device for synthesising sterile-grade nanoparticles that could be directly applied to a human body in one step. This study used 1% chloroauric acid and silver nitrate as noble metal salts to prepare AuNPs and AgNPs. The following specific experimental steps were followed: A specific quantity (0.27 mL) of 1% silver nitrate (or 1% chloroauric acid) and 0.5 mL of 1% malic acid disodium aqueous solution were added to a conical flask containing 24.23 mL of ultrapure water and ultrasonically mixed for 30 s. The resulting mixed solution was placed in a pressure steam sterilisation pot, and the reaction conditions were set at the conventional sterilisation temperature (121 °C) and time (20 min). During this process, the temperature of the reaction equipment gradually increased from room temperature to 121 °C, and the pressure gradually increased to 103.4 kPa. After 20 min, the reaction was stopped, and the solution was cooled to room temperature to obtain the AuNPs and AgNPs.

Preparation of hydrotalcite nanoparticles by high-pressure sterilization synthesis method

Anionic (cationic)-intercalated LDHs prepared using high-pressure sterilisation, along with noble metal nanoparticles. The preparation of LDHs was based on the improved classical hydrothermal–coprecipitation method. The specific experimental steps followed were as follows: First, 1.538 g of Mg(NO3)2·6H2O and 0.75 g of Al(NO3)3·9H2O were dissolved in 10 mL of methanol for later use. Then, 0.64 g of NaOH was weighed and dissolved in 15 mL of methanol. Methanolic salt solution (10 mL) containing Mg(NO3)2·6H2O (2.0 mM) and Al(NO3)3·9H2O (1.0 mM) was slowly dropped into the 15-mL methanolic solution containing NaOH (16 mM), with vigorous stirring for 15 min to mix. The mixed reaction system was centrifuged at 6000 rpm for 10 min to collect the precipitate, washed three times, and resuspended in a conical flask containing 40 mL of methanol solution. The resuspended methanol solution was placed in a pressure steam sterilisation pot for the reaction to occur. The temperature of the solution was gradually increased from room temperature to 121 °C, while the pressure was gradually increased to 103.4 kPa. After 20 min, the reaction was stopped, and the reaction system was cooled to room temperature to obtain Mg2Al-NO3-LDHs with a mass concentration of 8.0 mg mL−1. The preparation procedure of cationic intercalated LDH is similar to that of anionic intercalated LDH: 20 mL of a salt solution containing CoCl2 (2.0 mM) and AlCl3 (1.0 mM) and 20 mL of a mixed aqueous solution containing NaOH (0.15 M) and anhydrous Na2CO3 (0.013 M) were vigorously stirred in an N2 atmosphere to obtain the reaction solution. The generated precipitate was washed twice for 10 min at 6000 rpm, dispersed in a conical flask containing 40 mL of water, and allowed to react in a high-pressure steam sterilisation pot. The treatment conditions were the same as those applied in the previous experiments, and Co2Al-LDHs with a mass concentration of 5.0 mg mL−1 were finally obtained.

Microbial limit experiment (MLT)

To study whether the nanoparticles prepared using the high-pressure sterilisation synthesis method were sterile, we conducted an MLT, as stated in Chinese Pharmacopoeia, with some modifications. We used four types of commercially available nanoparticles as control groups to validate the advantages of our approach. Among them, commercially available AuNPs and AgNPs are obtained by chemical synthesis, while Mg2Al-NO3-LDHs and Co2Al-LDHs are prepared by conventional hydrothermal co-precipitation method. First, the suitability and reliability of MLT needs to be verified by colony recovery results for aerobic bacteria, molds and fungi. If the recovery rate is 0.50–2.00%, the preparation method and counting method of the nanoparticle test solution used can be used to count the total number of aerobic bacteria and fungi in the sample. These colony recovery results were further used for subsequent sterility tests to verify the sterility performance of the nanoparticles. Colony recovery rate (%) = (Colony count of the sample group − Colony count of the blank control group)/Colony count of the bacterial solution group × 100%. Through the analysis of the growth of all culture media, the superiority of the high-pressure sterilisation method was determined.

Because the AuNPs and AgNPs prepared in this study exhibited antibacterial properties, different methods were chosen for the MLT. The total number of fungi was determined using the conventional Petri dish method, while the total number of aerobic bacteria was determined using membrane filtration, along with a neutralising agent (1% polysorbate 80). The specific steps involved in counting the total number of aerobic bacteria were as follows: a 10.0 mL sample was prepared and diluted with a sterile sodium chloride-peptone buffer (pH 7.0) to obtain a 1:50 test solution, and a neutralising agent was added and mixed to eliminate antibacterial activity in the solution. Subsequently, 9.9 mL of the test solution was added to the appropriate concentration of the bacterial suspension and mixed well to bring down the bacterial content of the test solution to a value below 100 CFU mL−1. The solution was thereafter filtered using a thin film (1 mL), rinsed using sterile sodium chloride-peptone buffer (0.01% polysorbate 80), and applied to evenly coat into TSA medium. Two types of LDHs with no antibacterial properties were cultivated using the conventional Petri dish method. A sample solution of 10.0 mL was diluted with sterile sodium chloride-peptone buffer (pH 7.0) to obtain a 1:50 test solution. Then, 9.9 mL of the test solution was added to the appropriate concentration of the bacterial suspension and mixed well to make the bacterial content of the test solution to fall below 100 CFU mL−1. Thereafter, 1 mL of the test solution was evenly applied to the TSA medium. All treatment groups were incubated at a constant temperature (35 °C) for 3 d, and the colony results were observed. Sample processing of the total fungal count method was same as that of the conventional Petri dish method. Finally, 1 mL of the test solution was evenly coated on the SDA medium and cultured at constant temperature (25 °C) for 5 d, and the colony results were observed. To ensure the accuracy of the experiment, the bacterial solution, neutraliser, and blank control groups were set up in parallel.

After determining the applicability of the MLT, 1 mL of each of the four nanoparticle test solutions prepared using the new method was taken and evenly coated on a LB medium (37 °C, 48 h) to observe the growth of bacterial colonies. Commercially available AuNPs, AgNPs, Mg2Al-NO3-LDHs, and Co2Al-LDHs were used as controls and treated identically. The blank control group consists of empty plates containing only the culture medium, and the culture medium coated with a mixture of neutralizing agent and buffer solution was used as the negative control group.

In order to verify the reliability of the results of the sterility of the nanoparticles in this study, a bacterial control method validation experiment was conducted using Escherichia coli (E. coli) as a positive control. Take 1mL of each of the four types of nanoparticle test solutions obtained by high-pressure sterilization synthesis method, and add them to sterile LB liquid culture medium as experimental groups. LB liquid culture medium was used as a negative control. Among them, the concentration of E. coli bacterial solution in the positive control should not exceed 100 CFU mL−1.