Amino acid and gold particle-based self-assembled nanomaterial and preparation method and application thereof

Abstract

The application relates to the technical field of nanobiomaterial preparation, and discloses an amphiphilic L-arginine (L-Arg) and nano-gold self-assembly material (Fmoc-L-Arg-Au) and application thereof in cancer treatment. The system can co-carry gold nanoparticles and L-Arg; under a tumor microenvironment, the gold nanoparticles catalyze glucose oxidation to generate hydrogen peroxide (H2O2) and gluconic acid; under low pH and high H2O2 concentration, the gluconic acid further reacts with a guanidino group of L-Arg to generate therapeutic gas nitric oxide (NO), and the system has excellent anticancer activity. The nano-drug preparation process is simple, the conditions are mild, the nano-drug is easy to mass-produce, and the nano-drug has good industrialization and practical prospects; the synthesized Fmoc-L-Arg-Au utilizes the characteristics of a tumor microenvironment to generate a cascade reaction, combines starvation therapy and gas treatment, efficiently kills cancer cells, and the amino acid structure of the main body has excellent biocompatibility, which provides favorable conditions for clinical transformation and application.

Description

Technical Field

[0001] This application relates to the fields of nanomaterials and pharmaceutical technology, specifically to a self-assembled nanomaterial simultaneously loaded with gold particles and arginine, its preparation method, and its application. Background Technology

[0002] In clinical treatment, surgery, chemotherapy, and radiotherapy are routine methods for treating cancer. Newer cancer treatment modalities include phototherapy, ultrasound therapy, gas therapy, immunotherapy, and starvation therapy. However, these treatment methods still have limitations and shortcomings. For example, surgery is difficult to remove tiny residual tumor cells, which can easily lead to tumor recurrence and further metastasis. Chemotherapy failure is mainly due to the development of drug resistance in cancer cells. The hypoxic tumor environment also limits the therapeutic effects of radiotherapy, sonodynamic therapy, and photodynamic therapy. Nitric oxide (NO) is a pleiotropic gaseous molecule that exhibits a concentration-dependent dual role in cancer biology. When its concentration is in the range of 1 μM to mM, it can exert anti-cancer effects through oxidative and nitrosogenic stress with relatively few side effects, and has become a very promising area in cancer treatment. However, the potential of commonly used NO donors in clinical applications is limited by two major challenges: first, their chemical structure is unstable, leading to difficulties in in vivo delivery and uncontrollable efficacy; second, toxic byproducts may be generated during metabolism, bringing additional safety risks. Therefore, developing novel nanoparticle delivery systems to achieve safe, continuous, and stable release of nitric oxide at tumor sites has become a highly promising research direction.

[0003] L-arginine (L-Arg) has attracted much attention as a promising NO donor due to its excellent biocompatibility and biosafety. Compared with commonly used donors (such as sodium nitroprusside and S-nitrosothiol), which tend to induce premature NO release and produce toxic byproducts under physiological conditions, L-Arg can generate NO under the catalysis of reactive oxygen species (ROS). However, achieving L-Arg delivery often requires suitable nanocarriers, and the high solubility of L-Arg makes loading it into nanoformulations challenging. Therefore, the rational design of novel carrier-free nanomedicines can help solve the problems of efficient L-Arg delivery and sustained and safe NO release. Summary of the Invention

[0004] To address the aforementioned technical challenges and maximize the synergistic therapeutic efficiency of self-assembled materials based on amphiphilic amino acids and gold nanoparticles, this application provides a self-assembled nanomaterial (Fmoc-L-Arg-Au) simultaneously loaded with L-Arg and gold nanoparticles. Fmoc-L-arginine (Fmoc-L-Arg) is used as the nitric oxide (NO) source and main framework. A chloroauric acid (HAuCl4) solution is added to generate a colloidal solution, which is then reduced using sodium borohydride (NaBH4) to obtain gold particles, ultimately yielding Fmoc-L-Arg-Au nanoparticles. This invention introduces amphiphilic L-arginine (Fmoc-L-Arg) and metal ions that can self-assemble to form nanostructures. Nanomedicines simultaneously loaded with arginine and gold particles are obtained under in-situ reduction with sodium borohydride. The generation of gold nanoparticles can simulate glucose oxidase (GOx) catalyzing the conversion of glucose in the tumor microenvironment into gluconic acid and H2O2, thereby lowering the intratumoral pH and increasing the H2O2 concentration. Under low pH, the guanidinyl group of the amphiphilic amino acid and the ROS reaction continuously produce NO, achieving an anti-cancer effect.

[0005] This application also provides a method for preparing self-assembled nanomaterials simultaneously loaded with L-Arg and gold nanoparticles (Fmoc-L-Arg-Au), including the following steps:

[0006] Step 1: Preparation of Fmoc-HAuCl4 self-assembled sol:

[0007] Fmoc-L-Arg hydrochloric acid solution and HAuCl4 aqueous solution were added together to deionized water and stirred magnetically to obtain Fmoc-HAuCl4 self-assembled sol;

[0008] Step 2: Preparation of Fmoc-L-Arg-Au nanomaterials:

[0009] Under magnetic stirring in an ice bath, sodium borohydride (NaBH4) was added to Fmoc-HAuCl4 sol to generate gold particles in situ. The sample was collected by centrifugation and washed with deionized water to obtain Fmoc-L-Arg-Au nanomaterials.

[0010] As a preferred embodiment, in the preparation method of Fmoc-L-Arg-Au nanomaterials described above, in step one, the final concentration of Fmoc-L-Arg is 2-4 mg / mL, the final concentration of HCl is 10-20 mM, and the final concentration of HAuCl4 is 0.5-1 mM; the stirring temperature is 25-30 ℃, and the stirring time is 1-2 h.

[0011] As a preferred embodiment, in the preparation method of Fmoc-L-Arg-Au nanomaterials described above, in step two, the concentration of the NaBH4 aqueous solution is 1 mg / mL; the reaction temperature is an ice bath; the magnetic stirring time is 1~2 h; and the centrifugation conditions are 8000~10000 rpm for 5~10 min.

[0012] This application also provides the use of Fmoc-L-Arg-Au nanomaterials in the preparation of antitumor drugs. Particularly preferred applications are in drugs for treating breast cancer, pancreatic cancer, gastric cancer, liver cancer, cervical cancer, or colorectal cancer.

[0013] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0014] L-Arg, as a natural NO donor, exhibits excellent biocompatibility and can continuously release NO in the presence of inducible nitric oxide synthase. Furthermore, L-Arg can also be oxidized by H2O2 to produce NO. Therefore, introducing L-Arg into the H2O2-rich tumor microenvironment holds promise for generating large amounts of NO at the tumor site, thereby achieving the goal of gas therapy. This invention utilizes Fmoc-L-Arg and HAuCl4 as self-assembly units, leveraging coordination synergistic hydrophobic interactions to drive their self-assembly into a nanostructure, thus achieving carrier-free, highly efficient loading of L-Arg. However, in the actual tumor microenvironment, despite H2O2 overexpression, its background concentration remains low, insufficient to meet the demand for continuous and efficient NO generation. During tumor growth, glucose serves as an energy source, promoting cancer cell proliferation. Therefore, this invention incorporates NaBH4 into the aforementioned self-assembled sol to generate gold nanoparticles through in-situ reduction. This endows the nanomaterials with excellent enzyme-like activity, exhibiting good catalytic oxidation of glucose, significantly increasing H2O2 concentration while lowering the pH of the tumor microenvironment, further promoting continuous NO generation.

[0015] The advantages of the NO-generating nanoparticles proposed in this invention are mainly reflected in the following aspects: First, in terms of preparation technology, most current NO-generating nanoparticles suffer from limitations such as complex synthesis steps and harsh conditions. However, the self-assembly in-situ reduction preparation method used in this invention does not require additional L-Arg nanocarriers. The selected raw materials are readily available, the synthesis conditions are mild, and the steps are simple, significantly reducing the preparation difficulty and cost, and possessing the potential for large-scale production. Second, in terms of material properties, this nanoparticle system exhibits good biocompatibility, providing an important foundation for its subsequent clinical translation and application. In summary, this invention not only simplifies the process and is easy to implement, but also possesses good biocompatibility and prospects for large-scale production, showing significant potential in industrial and practical applications. Attached Figure Description

[0016] Figure 1This invention illustrates the scanning electron microscope characterization of the Fmoc-L-Arg-Au morphology structure in one embodiment of this application.

[0017] Figure 2 This paper illustrates the transmission electron microscopy characterization of the Fmoc-L-Arg-Au morphology structure in one embodiment of this application. Figure 3 This image shows a transmission electron microscope (TEM) image of Fmoc-L-Arg-Au in one embodiment of this application;

[0018] Figure 4 The image shows the high-angle annular dark-field scanning transmission electron microscopy characterization of Fmoc-L-Arg-Au in one embodiment of this application, and the elemental analysis results of carbon (C), nitrogen (N), oxygen (O), and gold (Au).

[0019] Figure 5 This document shows the EDS energy dispersive spectroscopy analysis results of Fmoc-L-Arg-Au characterized by high-angle annular dark-field scanning transmission electron microscopy in one embodiment of this application;

[0020] Figure 6 This paper shows the X-ray photoelectron spectroscopy detection results of Fmoc-L-Arg-Au in one embodiment of this application;

[0021] Figure 7 The following are the zeta potential analysis results of Fmoc-HAuCl4 and Fmoc-L-Arg-Au in one embodiment of this application;

[0022] Figure 8 This paper shows the DLS particle size analysis results of Fmoc-L-Arg-Au in one embodiment of this application;

[0023] Figure 9 This invention illustrates the NO production results of Fmoc-L-Arg-Au incubated with 10 mM glucose in one embodiment of this application.

[0024] Figure 10 This image shows a confocal fluorescence pattern of NO production in MCF-7 cells by Fmoc-L-Arg-Au nanoparticles prepared in one embodiment of this application.

[0025] Figure 11 This invention illustrates the cytotoxicity of Fmoc-L-Arg-Au nanoparticles prepared in one embodiment of this application against MCF-7 cells.

[0026] Figure 12 The image shows a confocal fluorescence image of cancer cells stained with AM-PI after treatment with Fmoc-L-Arg-Au nanoparticles prepared in one embodiment of this application. Detailed Implementation

[0027] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0028] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0029] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0030] Example 1

[0031] The preparation of a self-assembled nanomaterial based on amino acids and gold particles includes the following steps:

[0032] 1. Preparation of Fmoc-HAuCl4 sol

[0033] Fmoc-L-Arg hydrochloric acid solution and HAuCl4 aqueous solution were added together to deionized water and magnetically stirred. The final concentration of Fmoc-L-Arg was 4 mg / mL, the final concentration of HCl was 10 mM, and the final concentration of HAuCl4 was 1 mM. The mixture was continuously stirred magnetically at 25 °C for 2 h to obtain Fmoc-HAuCl4 sol.

[0034] 2. Preparation of Fmoc-L-Arg-Au nanomaterials

[0035] Under ice bath magnetic stirring, NaBH4 (0.5, 1, and 2 mg / mL) was added to the above Fmoc-HAuCl4 sol to generate different gold particles in situ. The reaction was carried out for 2 hours, and then the samples were collected by centrifugation (8000 r, 6 min) at room temperature. The samples were washed twice with deionized water to obtain three final products (Fmoc-L-Arg-Au nanoparticles), which were stored at 4 °C for subsequent characterization and antitumor performance testing.

[0036] Example 2 Characterization of Fmoc-L-Arg-Au nanoparticles

[0037] 1. Characterization of Fmoc-L-Arg-Au synthesis

[0038] like Figure 1 As shown, scanning electron microscopy (SEM) images reveal that when the concentration of the reducing agent NaBH4 is 0.5 mg / mL ( Figure 1 A), gold reduction is less, when the concentration increases to 1 mg / mL ( Figure 1B) A large number of smaller gold particles can be clearly seen forming in the self-assembled structure, but when the concentration is further increased to 2 mg / mL ( Figure 1 (C) It can be seen that larger gold particles are generated individually in the solution, rather than being reduced in the self-assembled nanoparticles. Therefore, the optimal concentration of NaBH4 was found to be 1 mg / mL. Figure 2 The SEM images show that Fmoc-L-Arg-Au has a good geometry and uniform distribution, and is a sphere with an average diameter of about 200 nm. Figure 3 The transmission electron microscope (TEM) image of Fmoc-L-Arg-Au shows that the reduced nanoparticles contain numerous gold particles. To further confirm the presence of carbon (C), nitrogen (N), oxygen (O), and gold (Au) in the Fmoc-L-Arg-Au structure, elemental mapping analysis was performed. Figure 4 The results showed that C, N, O and Au elements were uniformly distributed within the Fmoc-L-Arg-Au nanoparticles, indicating that amino acids and gold particles could be simultaneously loaded after self-assembly and in-situ reduction.

[0039] like Figure 5 As shown, the EDS-acquired energy spectrum also confirmed the presence of C, N, O, and Au, thus confirming the successful synthesis of Fmoc-L-Arg-Au nanomaterials. Secondly, X-ray photoelectron spectroscopy (XPS) was used to characterize the sample surface to determine its elemental composition, chemical state, and bonding structure. The XPS peak results for the corresponding C1s, O1s, N1s, and Au4f in Fmoc-L-Arg-Au are shown below. Figure 6 This also demonstrates the successful preparation of Fmoc-L-Arg-Au nanomaterials.

[0040] 2. Characterization and NO release performance testing of Fmoc-L-Arg-Au

[0041] like Figure 7 As shown, zeta potential measurements were performed to evaluate the surface electrostatic properties of the nanoparticles. The zeta potential of Fmoc-L-Arg-Au was +48 mV, while after in-situ reduction with NaBH4, the zeta potential of CLBRP was -12 mV, indicating successful reduction of the gold nanoparticles. Figure 8As shown, the hydrodynamic size distribution of Fmoc-L-Arg-Au in the dynamic light scattering (DLS) measurements is relatively narrow, approximately 210 nm, consistent with SEM and TEM characterization. To verify the cascade catalytic effect of the nanoparticles, we tested their ability to generate nitric oxide in the presence of glucose. The experiment used the Griess method: different concentrations of nanoparticles were incubated with 10 mM glucose overnight at 37°C, the supernatant was collected after centrifugation, and a colorimetric reaction was performed. The OD value was measured at 560 nm. Figure 9 As shown, the results clearly confirm that the nanoparticles can successfully catalyze the conversion of glucose into nitric oxide, verifying their designed function and demonstrating their potential in anti-tumor applications.

[0042] Example 3: Application of Fmoc-L-Arg-Au nanoparticles

[0043] 1. The ability of Fmoc-L-Arg-Au prepared according to the method of Example 1 to produce NO in breast cancer MCF-7 cells was detected by using the DFM-DA probe. The procedure was as follows: the cells were prepared at a density of 10T 4 MCF-7 cells were seeded at a density of 10 cells / mL in confocal culture dishes and cultured overnight. Subsequently, the cells were treated with PBS and Fmoc-L-Arg-Au (nanoparticle concentration: 10 μg / mL) for 6 hours, respectively. The culture medium was removed, and the cells were washed three times with PBS. Then, DFM-DA probe (5 μM) was added to each well, and incubation continued for 30 min. The liquid was removed again, and the cells were washed three times with PBS, followed by the addition of serum-free culture medium. Finally, the cell nuclei were stained with Hoechst (1.0 μg / mL), and the release of intracellular NO was observed using an inverted fluorescence microscope. Figure 10 It is evident that the control group produced almost no green fluorescence, while the cells in the group with added nanomaterials exhibited significant green fluorescence, indicating that Fmoc-L-Arg-Au nanoparticles can produce NO within cancer cells.

[0044] 2. To investigate the antitumor activity of the prepared nanoparticles, this example used breast cancer MCF-7, pancreatic cancer PANC-1, and colon cancer CT26 cells as models. The MTT assay was used for detection. The experimental steps are as follows: First, MCF-7, PANC-1, and CT26 cells in logarithmic growth phase were collected, and after adjusting the density, they were seeded in 96-well plates (1×10⁻⁶). 4Cells were cultured in a CO2 incubator for 24 hours to allow complete cell adhesion. After removing the old culture medium, different concentrations of Fmoc-L-Arg-Au nanomaterials (in a medium containing 10 mM glucose) were added to each well, and incubation continued for another 24 hours. After incubation, MTT reagent was added to each well, and culture was terminated after 4 hours. The liquid in the wells was carefully removed, and DMSO was added to dissolve the blue-purple crystals. After shaking and mixing, the optical density (OD value) of each well was measured at a wavelength of 490 nm, and the cell viability was calculated accordingly. The cell viability was calculated using the OD value, and the results are shown below. Figure 11 As shown, the cell death rate of these three types of cells gradually increased with the increase of nanoparticle concentration, reaching more than 70% when the nanoparticle concentration was 60 μg / mL, indicating that the nanoparticles have good in vitro therapeutic effects.

[0045] 3. To further visualize and evaluate the killing effect of Fmoc-L-Arg-Au on MCF-7 cells, Calcein-AM / PI double staining combined with laser scanning confocal microscopy was used for observation. Logarithmic growth phase MCF-7 cells were cultured at 2×10⁻⁶ cells / cells. 4 Cells were seeded at a density of [number] cells / mL in confocal culture dishes and cultured for 24 hours to allow complete cell adhesion. After discarding the culture medium, the experimental groups were treated as follows: the negative control group received serum-free medium; the experimental groups received serum-free medium containing 10 μg / mL and 50 μg / mL of Fmoc-L-Arg-Au nanoparticles, respectively. After 4 hours of culture, the culture medium was aspirated, the cells were washed twice with PBS, and then incubated for another 24 hours with fresh serum-free medium. Subsequently, the culture medium was discarded, and the cells were incubated with Calcein-AM / PI fluorescent dye in the dark for 40 minutes. After gently washing twice with PBS, the cells were added to fresh serum-free medium and immediately imaged under a laser scanning confocal microscope. The results are as follows: Figure 12 As shown, all cells in the control group exhibited green fluorescence, with no dead cells observed; while at lower concentrations of Fmoc-L-Arg-Au material, red and green fluorescence coexisted, indicating partial cell death; and when the concentration of Fmoc-L-Arg-Au nanomaterial reached 50 μg / mL, red fluorescence predominated with only a very small amount of green fluorescence, indicating that the nanoparticles can efficiently kill MCF-7 cells and have a good in vitro anti-tumor effect.

[0046] The above are merely preferred embodiments of the present invention. It should be noted that, for those skilled in the art, numerous improvements and modifications can be made without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing gold nanoparticles and arginine nanomaterials simultaneously, characterized in that, Includes the following steps: Fmoc-L-arginine (Fmoc-L-Arg) was used as the nitric oxide source and main framework. Chloroauric acid (HAuCl4) solution was added to generate a colloidal solution through self-assembly. Gold particles were then obtained by sodium borohydride (NaBH4) reduction, and finally Fmoc-L-Arg-Au nanoparticles were prepared.

2. The method for preparing Fmoc-L-Arg-Au particles according to claim 1, characterized in that, Includes the following steps: Step 1: Preparation of Fmoc-HAuCl4 self-assembled sol: Fmoc-L-Arg hydrochloric acid solution and HAuCl4 aqueous solution were added together to deionized water and stirred magnetically to obtain Fmoc-HAuCl4 self-assembled sol; Step 2: Preparation of Fmoc-L-Arg-Au nanomaterials: Under magnetic stirring in an ice bath, sodium borohydride (NaBH4) was added to the Fmoc-HAuCl4 sol for in-situ reduction to generate gold particles. The sample was collected by centrifugation and washed with deionized water to obtain Fmoc-L-Arg-Au nanomaterials.

3. The method for preparing Fmoc-L-Arg-Au nanomaterials according to claim 2, characterized in that, In step one, the final concentration of Fmoc-L-Arg is 2-4 mg / mL, the final concentration of HCl is 10-20 mM, and the final concentration of HAuCl4 is 0.5-1 mM; the stirring temperature is 25-30 ℃, and the stirring time is 1-2 h.

4. The method for preparing Fmoc-L-Arg-Au nanomaterials according to claim 2, characterized in that, In step two, the concentration of the NaBH4 aqueous solution is 1~2 mg / mL; the reaction temperature is an ice bath; the magnetic stirring time is 1~2 h; and the centrifugation conditions are 8000~10000 rpm for 5~10 min.

5. The Fmoc-L-Arg-Au nanomaterial according to any one of claims 1 to 4 continuously catalyzes a cascade reaction that consumes glucose and produces NO.

6. The use of the Fmoc-L-Arg-Au nanomaterials according to any one of claims 1 to 4 in the preparation of anticancer drugs.

7. The use of the Fmoc-L-Arg-Au nanomaterial according to any one of claims 1 to 4 in the preparation of drugs for treating breast cancer, pancreatic cancer, gastric cancer, liver cancer, cervical cancer, or colorectal cancer.

8. The application according to claim 6 or 7, characterized in that, The concentration range of the Fmoc-L-Arg-Au nanomaterial is 0.1 μg / mL to 50 μg / mL.

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