Use of a gold-copper alloy nanoparticle in vivo behavior regulation system

Abstract

This invention discloses the application of a gold-copper alloy nanoparticle in vivo behavior regulation system in nanomedicine carriers. The system comprises gold, copper, and ligands; wherein the ligands are selected from compact small-molecule thiol ligands or thiolized polyethylene glycol ligands, with the thiolized polyethylene glycol ligands having a molecular weight of 800–2000 Da. The system is prepared via a one-step reduction method, a mild and simple process. Studies show that, compared to single gold nanoparticles, this alloy system significantly prolongs the blood circulation half-life and improves the enrichment efficiency in tumor tissues while maintaining excellent biocompatibility primarily through renal clearance. This invention solves the problem of limited efficacy of ultrasmall nanomedicines due to rapid metabolism, providing a scientific basis for the rational design of drug carriers for tumor diagnosis and treatment.

Description

Technical Field

[0001] This invention belongs to the field of nanomaterials and nanomedicine, specifically relating to a gold-copper alloy nanoparticle behavior regulation system in vivo and its application in nanomedicine carriers. Background Technology

[0002] Nanomaterials, due to their unique properties, exhibit revolutionary potential in the biomedical field. Their particle size typically ranges from 1 to 100 nm, a size effect that endows them with exceptional biological barrier penetration capabilities. Their high surface area to volume ratio provides ample sites for surface functionalization, enabling efficient drug loading and precise targeted delivery through ligand-receptor-mediated active targeting mechanisms, significantly enhancing bioactivity and specificity. The physicochemical parameters of nanomaterials (including shape regulation, surface charge modification, and hydrophilicity-hydrophobicity balance) can be finely designed to optimize their pharmacokinetic properties.

[0003] Although nanoparticles with a hydrated particle size below the glomerular filtration threshold can be efficiently cleared via the kidneys, thereby reducing the risk of long-term organ accumulation and improving biosafety, this rapid renal clearance mechanism directly leads to a significant shortening of their half-life in the bloodstream. This means that nanomedicines are metabolized before reaching the target site, severely reducing their total accumulation in the lesion area.

[0004] Among numerous nanoparticles, ultrasmall gold nanoparticles (AuNPs, with a core diameter less than 3 nm) have attracted widespread attention in the biomedical field due to their excellent biocompatibility, abundant surface ligand modifications, and diverse alloying or doping control methods. Alloying can not only modulate the electronic structure and catalytic performance of nanoparticles but is also considered to have the potential to optimize their in vivo distribution. However, in practical applications, the effectiveness of alloying strategies is often affected by the surface chemical environment. Moreover, due to the lack of a systematic understanding of the synergistic mechanism between the metal core and surface ligands, current nanomaterial design still relies mainly on empirical optimization, making it difficult to achieve rational control and design of in vivo behavior.

[0005] Therefore, it is necessary to construct a gold-copper alloy nanoparticle system with in vivo behavior regulation function, so as to provide a scientific basis for the rational design of ultrasmall nanomedicines. Summary of the Invention

[0006] The purpose of this invention is to overcome at least one deficiency of the prior art and to provide a gold-copper alloy nanoparticle behavior regulation system in vivo and the application of the system in nanomedicine carriers.

[0007] The technical solution adopted in this invention is: In a first aspect, the present invention provides an application of a gold-copper alloy nanoparticle in vivo behavior regulation system in a nanomedicine carrier, wherein the gold-copper alloy nanoparticle in vivo behavior regulation system comprises gold, copper and ligands. The ligand is selected from compact small molecule thiol ligands or thiolized polyethylene glycol ligands, wherein the molecular weight of the thiolized polyethylene glycol ligand is 800-2000 Da.

[0008] In some embodiments, the proportion of copper in the total molar amount of copper and gold is 1-20%.

[0009] In some embodiments, the compact small molecule thiol ligand is selected from glutathione.

[0010] In some embodiments, the preparation method of the gold-copper alloy nanoparticle in vivo behavior regulation system is as follows: 1) Mix gold precursor and copper salt to form a mixed metal precursor; 2) Add ligands to the mixed metal precursor and mix thoroughly, then add a reducing agent to carry out a reduction reaction, and finally purify to obtain the target gold-copper alloy nanoparticle in vivo behavior regulation system.

[0011] In some embodiments, the copper salt is selected from at least one of copper nitrate, copper sulfate, or copper chloride.

[0012] In some embodiments, the gold precursor is selected from chloroauric acid or gold salt.

[0013] In some embodiments, the reducing agent is selected from lithium borohydride, sodium borohydride, or potassium borohydride.

[0014] In some embodiments, the reaction time of the reduction reaction is 4 to 8 hours.

[0015] In some embodiments, the nanomedicine is a tumor diagnostic and therapeutic drug.

[0016] In some embodiments, the metal core of the gold-copper alloy nanoparticle in vivo behavior regulation system has a gold-copper alloy structure and a core particle size of less than 2 nm.

[0017] The beneficial effects of this invention are: 1) The technical solution proposed in this invention can provide a reference for the rational design and screening of ultra-small nano-drug carriers, reduce trial and error, and improve R&D efficiency; 2) While maintaining the in vivo metabolic characteristics of nanoparticles, which are mainly cleared by the kidneys, we can achieve a moderate extension of circulation time and an improvement in the ability to accumulate in tumor tissue, thus balancing biosafety and application potential.

[0018] 3) The system of the present invention can be constructed by conventional wet chemical methods. The process conditions are mild and the operation is simple, with good reproducibility and scalability. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the synthesis of gold-copper alloy nanoparticles in Example 1.

[0020] Figure 2 The image shows the morphology and size characterization of the nanoparticles obtained in Example 1.

[0021] Figure 3 The image shows the XPS analysis of the nanoparticles obtained in Example 1.

[0022] Figure 4 This is the in vivo pharmacokinetic curve.

[0023] Figure 5 It is the result of 7 days of metabolism and accumulation in major organs.

[0024] Figure 6 This is a diagram showing the distribution of tumor tissue. Detailed Implementation

[0025] The following disclosure provides many different implementations or examples for different ways of implementing the present invention. Example 1: A general preparation method for AuNPs / AuCuNPs (GSH / PEG system)

[0026] like Figure 1 As shown, this embodiment prepared paired control nanoparticle systems, including GS-AuNPs, GS-AuCuNPs, PEG-AuNPs, and PEG-AuCuNPs, using the following methods: 1) Preparation of metal precursor solution At room temperature (25 °C), 286 μL of 20 mM chloroauric acid solution was added to 4.415 mL of deionized water; when preparing AuCuNPs, in addition to adding chloroauric acid, 39 μL of 20 mM copper chloride solution was also added. The resulting solution was stirred under magnetic stirring (600-1500 rpm) for 5 min to form a homogeneous metal precursor solution.

[0027] 2) Ligand introduction Add the ligand solution to the above metal precursor solution and continue magnetic stirring for 10 min to allow the ligand to fully complex with the metal ions. The ligand solution is selected as follows: Compact small molecule thiol ligand system (GSH system): 260 μL 0.1 M glutathione solution; Thiolized polyethylene glycol ligand system (PEG system): thiol polyethylene glycol (PEG-SH, molecular weight approximately 1000 Da) in an equimolar amount with the GSH system described above.

[0028] 3) Reduction reaction Under continuous stirring, 312.5 μL of freshly prepared sodium borohydride solution (8 mg / mL) was rapidly added to initiate the reduction reaction, and the reaction was continued to be stirred at 25 °C for 30 min.

[0029] 4) End of reaction and purification After stirring was stopped, the reaction system was allowed to stand at 25 °C for 5 h to further homogenize the size of the nanoparticles. The reaction solution was then transferred to a 10 kDa molecular weight cutoff ultrafiltration tube, centrifuged at 4000 rpm for 15 min, the filtrate was discarded, and the solution was resuspended in deionized water. The ultrafiltration operation was repeated three times to remove unreacted metal ions and free ligands, resulting in a purified nanoparticle solution.

[0030] 5) Sample correspondence The following samples can be obtained using the methods described above: Nanoparticle system 1: GS-AuNPs, which is without added copper chloride and has GSH as the surface ligand; Nanoparticle system 2: GS-AuCuNPs, which contains copper chloride and has GSH as the surface ligand; Nanoparticle system 3: PEG-AuNPs, which are without added copper chloride and have PEG-SH as the surface ligand; Nanoparticle system 4: PEG-AuCuNPs, which contains copper chloride and has PEG-SH as the surface ligand.

[0031] 6) Morphological and dimensional characterization like Figure 2 As shown, transmission electron microscopy results indicate that the metal core diameter of all four samples is approximately 2.0 nm. The GS-AuNPs were 1.9 ± 0.2 nm. The GS-AuCuNPs were 1.9 ± 0.2 nm. The PEG-AuNPs were 1.8 ± 0.2 nm. The PEG-AuCuNPs were 1.8 ± 0.2 nm.

[0032] Dynamic light scattering results showed that the hydrated particle size of the GSH system was approximately 2.3–2.5 nm, while that of the PEG system was approximately 4.8–5.1 nm.

[0033] Within the same ligand system, the changes in the core size and hydrated particle size of nanoparticles before and after the introduction of copper are relatively small.

[0034] 7) Surface chemical analysis like Figure 3 As shown, a significant Cu 2p signal was detected in the XPS overall spectrum of the AuCuNPs sample, while no copper was detected in the AuNPs sample. Further analysis of the Au 4f, S 2p, and O 1s spectra revealed changes in the peak positions and relative intensities after alloying, suggesting that the introduction of copper affects the surface chemical environment of the nanoparticles. Example 2: In vivo pharmacokinetic evaluation

[0035] 1) Drug administration and sampling like Figure 4 As shown, the GS-AuNPs, GS-AuCuNPs, PEG-AuNPs, and PEG-AuCuNPs obtained in Example 1 were injected into Balb / c mice (n = 3) via the tail vein. The single injection volume was 200 μL, and the drug concentration was 10 mg / mL.

[0036] Blood samples were collected at 2, 3.5, 5, 10, and 30 min and at 1, 3, 5, 8, 12, 24, 48, and 72 h after drug administration. The collected blood samples were digested with aqua regia, and the gold content was determined by inductively coupled plasma mass spectrometry (ICP-MS). Blood drug concentration-time curves were then plotted.

[0037] 2) Model Fitting Method The obtained blood drug concentration-time data were subjected to nonlinear fitting using Origin software, and the double exponential decay model ExpDec2 was selected for fitting. Its mathematical expression is as follows: y = y0 + A1 e -t / t1 + A2 e -t / t2 ; The parameters in this expression have the following meanings: y represents the gold content in the blood at different times; t represents time; y0 is the baseline term; A1 and A2 are the magnitude parameters of the two exponentially decaying terms; t1 and t2 are the time constants for the fast distribution phase and the slow elimination phase, respectively.

[0038] This model is used to describe the biphasic decay behavior of nanoparticles in vivo. All data sets are well-fitted by the model.

[0039] 3) Pharmacokinetic parameters Based on the model fitting results above, the main pharmacokinetic parameters were calculated, including blood exposure (AUC) and elimination half-life (t).1 / 2β The results of the analysis of the clearance rate (CL) are shown in Table 1.

[0040] Table 1. Pharmacokinetic parameters of different nanoparticles (n = 3, Mean ± SD)

[0041] Combination Figure 4 As can be seen from Table 1: In different alloy systems, compared with GS-AuNPs and PEG-AuNPs respectively, GS-AuCuNPs and PEG-AuCuNPs both showed higher AUC values ​​and longer elimination half-lives, while the scavenging rate was reduced, suggesting that gold-copper alloying under this ligand condition helps to prolong the in vivo circulation time of nanoparticles. Example 3: Evaluation of metabolism and organ accumulation in normal mice over 7 days

[0042] 1) Dosing regimen Healthy Balb / c mice were selected, and the GS-AuNPs and GS-AuCuNPs obtained in Example 1 were injected via the tail vein, respectively. The administration volume was 200 μL, and the administration concentration was 10 mg / mL.

[0043] 2) Collection of urine and feces Mice were housed individually in metabolic cages. Urine and fecal samples were collected at 6 h, 12 h, 1 d, 2 d, 3 d, 5 d, and 7 d after drug administration. The collected urine and fecal samples were digested with aqua regia, and the gold content was determined by inductively coupled plasma mass spectrometry (ICP-MS). The cumulative excretion amount at each time point is expressed as %ID.

[0044] 3) Organ distribution and accumulation Mice were sacrificed on day 7 after drug administration, and major organ tissues, including heart, liver, spleen, lung, and kidney, were collected. After weighing each organ sample, they were digested with aqua regia, and the gold content was determined by ICP-MS. The distribution in different organs is expressed as %ID / g.

[0045] 4) Results Analysis The results are as follows Figure 5 As shown: Within 7 days after administration, the main excretion route for both nanoparticle samples was urine. The gold content detected in urine was significantly higher than that detected in feces, suggesting that it was mainly cleared via the kidneys. The relatively low gold content detected in the feces suggests that a limited proportion is excreted via the hepatobiliary-intestinal route. Compared with GS-AuNPs, the residual amount of GS-AuCuNPs in some organs was slightly increased on day 7, but the overall amount was still within the range of detectable and gradually cleared, and no abnormal enrichment trend was observed.

[0046] Based on the combined results of urine and fecal excretion and organ distribution, it can be seen that while prolonging blood circulation time, gold-copper alloy nanoparticles still maintain the in vivo metabolic characteristics of being mainly cleared by the kidneys, and show a gradual clearance trend within 7 days after administration, suggesting that their long-term organ accumulation risk is low. Example 4: Tumor tissue distribution

[0047] 1) Animal model construction and drug administration A subcutaneous tumor-bearing mouse model of MDA-MB-231 was constructed. After the tumor volume grew to the predetermined range, the GS-AuNPs and GS-AuCuNPs obtained in Example 1 were injected into the tumor-bearing mice via the tail vein. The administration volume was 200 μL, and the administration concentration was 10 mg / mL.

[0048] 2) Sampling and testing methods Mice were sacrificed at 1 h and 12 h after drug administration, and tumor tissue and major organ tissues were collected. The collected tissue samples were weighed, digested with aqua regia, and the gold content was determined by ICP-MS. The distribution in different tissues is expressed as %ID / g.

[0049] 3) Results Analysis The results are as follows Figure 6 As shown: At 1 h and 12 h after administration, the cumulative amount of GS-AuCuNPs in tumor tissue was higher than that of GS-AuNPs. At the aforementioned time points, the tumor / liver ratio of GS-AuCuNPs was greater than 1, while the corresponding ratio of GS-AuNPs was significantly lower than that of the GS-AuCuNPs group, suggesting that gold-copper alloying is beneficial to improving the relative enrichment of nanoparticles in tumor tissue under the GSH ligand system.

[0050] The above results indicate that, under the condition of small molecule thiol ligands, AuCuNPs exhibit a higher tissue enrichment trend in tumor models compared to AuNPs, which is more conducive to their application in nanomedicine design research.

[0051] The above is a further detailed description of the present invention and should not be considered as a limitation on the specific implementation of the present invention. For those skilled in the art, simple deductions or substitutions without departing from the concept of the present invention are all within the protection scope of the present invention.

Claims

1. The application of a gold-copper alloy nanoparticle in vivo behavior regulation system in nanomedicine carriers, characterized in that, The in vivo behavior regulation system of the gold-copper alloy nanoparticles includes gold, copper, and ligands. The ligand is selected from compact small molecule thiol ligands or thiolized polyethylene glycol ligands, wherein the molecular weight of the thiolized polyethylene glycol ligand is 800-2000 Da.

2. The application according to claim 1, characterized in that, The proportion of copper in the total molar ratio of copper and gold is 1-20%.

3. The application according to claim 1, characterized in that, The compact small molecule thiol ligand is selected from glutathione.

4. The application according to claim 1, characterized in that, The preparation method of the gold-copper alloy nanoparticle in vivo behavior regulation system is as follows: 1) Mix gold precursor and copper salt to form a mixed metal precursor; 2) Add ligands to the mixed metal precursor and mix thoroughly, then add a reducing agent to carry out a reduction reaction, and finally purify to obtain the target gold-copper alloy nanoparticle in vivo behavior regulation system.

5. The application according to claim 4, characterized in that, The copper salt is selected from at least one of copper nitrate, copper sulfate, or copper chloride.

6. The application according to claim 4, characterized in that, The gold precursor is selected from chloroauric acid or gold salt.

7. The application according to claim 4, characterized in that, The reducing agent is selected from lithium borohydride, sodium borohydride, or potassium borohydride.

8. The application according to claim 4, characterized in that, The reduction reaction takes 4 to 8 hours.

9. The application according to claim 1, characterized in that, The nanomedicine is a tumor diagnostic and treatment drug.

10. The application according to claim 1, characterized in that, The metal core of the in vivo behavior regulation system of gold-copper alloy nanoparticles has a gold-copper alloy structure and a core particle size of less than 2 nm.

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