CN115645548A - Drug-loaded albumin-magnetite nanoparticle drug delivery system and preparation method thereof - Google Patents

Abstract

The invention discloses a drug-loaded albumin-magnetite nanoparticle drug delivery system and a preparation method thereof, belonging to the technical field of drug delivery. The drug-loaded albumin-magnetite nanoparticle drug delivery system provided by the present invention comprises a nanocage with a cavity formed by albumin self-assembly and magnetite nanoparticles located in the cavity of the nanocage, and The surface-loaded drugs of magnetite nanoparticles can effectively integrate the advantages of inorganic nanoparticle-based drug delivery systems and protein carrier-based drug delivery systems, thereby achieving better drug delivery and therapeutic effects, and can be used to prepare anticancer drugs .

Description

A drug-loaded albumin-magnetite nanoparticle drug delivery system and its preparation method

technical field

The invention belongs to the technical field of drug delivery, and in particular relates to a drug-loaded albumin-magnetite nanoparticle drug delivery system (named "nano torpedo") and a preparation method thereof.

Background technique

A safe and efficient drug delivery system (DDS) is expected to prolong the half-life of chemotherapy drugs and reduce toxic side effects. In recent years, with the rapid development of nanotechnology, liposome chemotherapy drugs such as Doxil (doxorubicin liposome) and Onivyd (irinotecan liposome) have been widely used in clinic. However, it is unsatisfactory that the effect of this liposomal chemotherapeutic drug in tumor treatment is only moderate, and its stability is poor, resulting in the leakage of the drug in the blood circulation, which in turn leads to its accumulation in the lesion area The dose is low and the potential risk of cardiotoxicity is high.

In order to overcome the above shortcomings, two important drug delivery systems have been developed: one is an inorganic nanoparticle carrier (such as magnetite nanoparticles or silica nanoparticles) drug delivery system, which can effectively To stabilize chemotherapy drugs and prolong their half-life in blood circulation. In addition, the high specific surface area of nanoparticles can also improve drug loading efficiency, and when functionalized nanoparticles are used as carriers, it can endow drugs with the ability of comprehensive diagnosis and treatment. The other is a protein (such as albumin and ferritin) drug delivery system, which has good biocompatibility and biodegradability, is easy to modify, can improve drug stability, reduce or eliminate immunogenicity and side effect. However, these two drug delivery systems still have their own limitations, mainly manifested in the high biotoxicity of inorganic nanoparticles and the low drug loading efficiency of protein carriers.

Contents of the invention

Aiming at one or more problems in the prior art, one aspect of the present invention provides a drug-loaded albumin-magnetite nanoparticle drug delivery system, which includes:

(1) nanocages with cavities formed by self-assembly of albumin; and

(2) magnetite nanoparticles positioned in the cavity of the nanocage;

Wherein the surface of the magnetite nano particle is coated with medicine.

In some embodiments, in the drug-loaded albumin-magnetite nanoparticle drug delivery system, the diameter of the nanocage is 16.5-20.5 nm.

In some embodiments, in the drug-loaded albumin-magnetite nanoparticle drug delivery system, the diameter of the magnetite nanoparticles is 2.5-4.5 nm.

In some embodiments, in the drug-loaded albumin-magnetite nanoparticle drug delivery system, the cavity has a diameter of 4-12 nm.

In some embodiments, in the cavity of the nanocage, the surface of the magnetite nanoparticle is bound to the residue of the albumin; optionally, the residue of the albumin is selected from the following One or more of: Arg81, Glu82, Asp86, Asp89 and Glu92.

In some embodiments, in the cavity of the nanocage, the drug is chelated with the iron ion on the surface of the magnetite nanoparticle through a chemical bond.

In some embodiments, in the cavity of the nanocage, the group of the drug interacts with the residue of the albumin through a cation-π bond and/or a hydrogen bond; optionally, the drug The groups of are selected from aryl groups, and the residues of albumin are selected from one or more of the following: Arg208, Lys350 and Glu478.

In some embodiments, in the cavity of the nanocage, the drug is surrounded by residues of the albumin to form a hydrophobic bond; optionally, the residues of the albumin are selected from one of the following One or more of: Phe205, Ala209, Ala212, Leu480 and Val481.

In some embodiments, the drug includes anticancer chemotherapy drugs; optionally, the anticancer chemotherapy drugs include hydrophobic anticancer chemotherapy drugs and hydrophilic anticancer chemotherapy drugs; further optionally, the anticancer chemotherapy drugs Chemotherapy drugs are selected from one or more of the following: Adriamycin, Adriamycin Hydrochloride, Fluorouracil, Mitomycin, Actinomycin, Pingyangmycin, Cisplatin, Carboplatin, Epirubicin, A aminopterin and cytarabine.

In some embodiments, the albumin is selected from one or more of the following: bovine serum albumin, and human serum albumin.

In some embodiments, the drug is doxorubicin or doxorubicin hydrochloride, the albumin is selected from one or more of the following: bovine serum albumin, and human serum albumin, and the nanocage Formed by self-assembly of 6 albumin molecules, the surface of the magnetite nanoparticles is coated with 72 drug molecules.

Another aspect of the present invention provides a method for preparing a drug-loaded albumin-magnetite nanoparticle drug delivery system, which includes the following steps:

(S1) premixing the drug solution with the iron salt solution to obtain a premixed solution;

(S2) Add the albumin solution to the premix solution obtained in step (S1), and add a slightly excess NaOH solution relative to the iron ions in the premix solution, stir, filter, and dialyze to obtain the drug-loaded albumin solution. Protein-magnetite nanoparticle drug delivery system;

Wherein step (S1) and step (S2) are both carried out under the protection of inert gas.

In some embodiments, in step (S2), the amount of albumin solution added meets: the molar ratio of albumin to iron in the premixed solution is 1:(110-220); optionally 1 :(110-165).

In some embodiments, in step (S2), the amount of albumin solution added is such that the molar ratio of the drug to the albumin is 1.5 or more, optionally 3 or more.

In some embodiments, in the step (S1), in the premixed solution, the molar ratio of the drug to iron is ≥1.5:(110-220); optionally ≥1.5:(110-165).

In some embodiments, in step (S1), Fe ³⁺ /Fe ²⁺ =(1.5-2.5):1 in the iron salt solution.

Another aspect of the present invention provides the application of the above drug-loaded albumin-magnetite nanoparticle drug delivery system in the preparation of anticancer drugs.

The drug-loaded albumin-magnetite nanoparticle drug delivery system provided based on the above technical scheme includes a nanocage with a cavity formed by self-assembly of albumin and magnetite nanoparticles located in the cavity of the nanocage, And the drug (such as anticancer chemotherapeutics, including hydrophobic anticancer chemotherapeutics and hydrophilic anticancer chemotherapeutics) is loaded on the surface of magnetite nanoparticles, the examples prove that the drug delivery system of this structure provided by the present invention It can effectively integrate the advantages of the existing inorganic nanoparticle-based drug delivery system and protein carrier-based drug delivery system, and further improve the drug-loading efficiency of the drug delivery system, as well as further improve its biocompatibility and biosafety. On this basis, the drug loading stability of the drug delivery system can be further improved, effectively reducing the drug leakage during drug delivery, thereby prolonging the half-life of the drug in the blood circulation, and facilitating the delivery of the drug delivery system in lysosomes. Drug release in the medium, as well as penetration and accumulation of drugs in the tumor area, can achieve better disease (such as cancer) treatment effect. In addition, compared with the drug delivery system in the prior art, the preparation method of the drug-loaded albumin-magnetite nanoparticle drug delivery system provided by the present invention has the advantage of simple operation, which can improve the synthesis efficiency of the drug delivery system, Contribute to its application in drug development and delivery in the field of nanomedicine.

Description of drawings

Figure 1 is the characterization of Dox@BMNT synthesized under different FR conditions in Example 1, (a-e) SEC analysis results; (f-j) and (k-o) are the negative staining TEM of Dox@BMNT synthesized under different FR conditions and TEM images (the inset is a 50X magnified HRTEM image); (p) the size distribution of protein cages and magnetite obtained from negative-stained TEM and HRTEM images; (q) HD and zeta potential of Dox@BMNT; (r) Dox loading efficiency under different FR conditions.

Figure 2 shows the model structure analysis of Dox@BMNT, (a) Negative stain TEM image of Dox@BMNT; (b) 2D structural features of the four main classes of Dox@BMNT; (c) Dox@BMNT under different viewing angles Structural model and surface charge distribution; (d) internal slice of Dox@BMNT model; (e) interface information between Dox and BSA in the protein cage cavity, and interfacial interaction between BSA and magnetite.

Figure 3 shows the Dox release curves of Dox@BMNT in serum over time obtained under different molar ratios of Fe and BSA.

Figure 4 is the characterization of Dox·Hcl@BMNT, (A) and (B) negative-stained TEM images of Dox·Hcl@BMNT; (C) is the negative-stained electron microscope, Dox·Hcl@BMNT with a cage structure; (D) SEC analysis column absorption peak; (E) and (F) TEM characterization of Dox·Hcl@BMNT and HRTEM characterization of the presence of magnetite; (G) Inorganic core particle size statistics of Dox·Hcl@BMNT; (H) and (I) are the Zeta-potential and hydrated particle size DLS of Dox·Hcl@BMNT, respectively.

Figure 5 shows the characterization of Dox@HMNT, (A) and (B) Dox@HMNT negative-stained TEM images; (C) negative-stained electron microscope, the particle size statistics of cage-like structure Dox@HMNT; (D) SEC analysis column absorption Peaks; (E) TEM characterization of Dox@HMNT; and (F) inorganic core particle size statistics of Dox@HMNT.

Figure 6 shows the characterization of Dox·Hcl@HMNT, (A) and (B) Dox·Hcl@HMNT negative staining TEM images; (C) negative staining electron microscopy, Dox·Hcl@HMNT with a cage structure; (D) SEC Analysis column absorption peak; (E) TEM characterization of Dox·Hcl@HMNT; and (F) particle size statistics of Dox·Hcl@HMNT.

Figure 7 shows the in vitro controlled release of Dox@BMNT and the evaluation of its killing effect on tumor cells, (ad) the cumulative release of Dox from Dox@BMNT at different pH conditions; (e) the total Dox of Dox@BMNT at different pH conditions within 24 h Statistical analysis of release amount; (f) Dox release of Dox@BMNT in serum over time; (g) and (h) the cytotoxicity and IC ₅₀ values of Dox-BSA, Doxil and Dox@BMNT on 4T1 cell line, respectively .

Figure 8 shows the effect of Dox@BMNT on the viability of 293T and HL7702 cells.

Figure 9 shows the cell uptake and retention analysis of Dox-BSA, Doxil and Dox@BMNT, (a) the distribution of Dox in the cells after 2 h of culture; (b) the distribution of Dox in the cells after 4 h of drug removal; (c-d) The average fluorescence intensity of Dox in lysosome and nucleus; (e) the total fluorescence intensity of Dox in lysosome and nucleus; (f) the decrease of total fluorescence intensity after drug removal for 4 hours.

Figure 10 shows the in vivo MRI and anti-tumor efficacy of Dox@BMNT, (a) schematic diagram of anti-tumor treatment schedule; (b) dynamic t1-weighted MRI tracking drug distribution in the tumor area after intravenous injection of Dox@BMNT; (c) CNR corresponding to MRI Ratio; (d) body weight changes of mice during treatment and (e) tumor growth curve; (f) survival rate of mice; (g) pictures of tumors after treatment; (h) pictures of mice before and after treatment; (i) Histological analysis of tumor tissues after treatment.

Figure 11 is the in vivo biocompatibility evaluation of Dox@BMNT, (a) iron content in urine samples collected at different times after injection of Dox@BMNT and PBS; (b-d) TEM images of urine 6h and 24h after injection; (e-l ) blood biochemical examination results; (m) H&E analysis of major organs of mice after treatment.

Detailed ways

In view of the high biological toxicity of the drug delivery system based on nanoparticles and the low drug loading efficiency of the drug delivery system based on protein carriers in the prior art, the inventors were inspired by natural biomineralization and used biological Biomimetic synthesis of macromolecular templates (such as proteins) to construct an albumin-magnetite nanoparticle drug delivery system with low biological toxicity and high drug loading efficiency. The advantages of the drug system and the drug delivery system based on the protein carrier, on the one hand, can improve the drug loading efficiency based on the high specific surface area of the inorganic nanoparticles, and stably assemble the drug on the surface of the nanoparticles (for example, through chemical bonds and metal ions on the surface of the nanoparticles). Chelation); on the other hand, the outer layer of the drug delivery system is a protein with a cage structure, which has good biocompatibility and biodegradability, and can reduce or eliminate the immunogenicity and side effects of the drug delivery system. Then the nanoparticles coated with drugs can be further coated to reduce the exposure of drugs and improve the stability of the entire drug delivery system; more importantly, the special structure of the drug delivery system provided by the present invention (that is, to load the drug in the The surface of magnetite nanoparticles in the cavity of protein nanocages) can effectively solve the leakage problem of small drug molecules during the delivery process, and further prolong their half-life in blood circulation, and it is more conducive to the drug delivery system in the dissolution process. The drug is released from the enzyme body and penetrates and accumulates in the tumor area. The invention also provides a preparation method of the drug delivery system.

Below in conjunction with specific embodiment, the present invention is further elaborated. It should be understood that the specific examples are only used to further illustrate the present invention, rather than limit the content of the present invention.

The methods used in the following examples are conventional methods unless otherwise specified. For specific steps, please refer to: "Molecular Cloning Experiment Guide" ("Molecular Cloning: A Laboratory Manual" Sambrook, J., Russell, David W., Molecular Cloning: A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring Harbor).

The acquisition methods of various biological materials described in the examples are only to provide an experimental acquisition method to achieve the purpose of specific disclosure, and should not be a limitation on the source of the biological materials in the present invention. In fact, the sources of the biological materials used are extensive, and any biological materials that can be obtained without violating laws and ethics can be replaced according to the tips in the examples. Unless otherwise specified, the experimental materials used in the following examples are conventional biochemical reagents, which can be purchased through commercial channels.

The materials used in the following examples were obtained from bovine serum albumin (BSA), human serum albumin (HSA), FeSO ₄ .7H ₂ O, FeCl ₃ .6H ₂ O, doxorubicin (Dox), Phosphate buffered saline (PBS), dimethylsulfoxide (DMSO) and NaOH were purchased from Sigma-Aldrich (USA); cell counting kit-8 (CCK-8) was purchased from Beijing Sun Biotechnology Co., Ltd.; DAPI, Lysosome-tracker Green fluorescent staining solution, hematoxylin and eosin (H&E) and paraformaldehyde (4%) were purchased from Sangon Biotechnology (Shanghai, China); Doxil hydrochloride liposomes (Doxil) were purchased from Sequuspharmaceuticals ticaks (U.S).

The synthesis method of Dox-BSA used in the following examples is as follows: Dox (4 mg) was dissolved in DMSO, then BSA (10 mg) was added, stirred at 37° C. in the dark for 12 hours (under N ₂ atmosphere protection). The formed Dox-loaded BSA (Dox-BSA) was collected, filtered through a 0.22 μm filter, and finally dialyzed against PBS.

Example 1: Preparation and Characterization of Drug-Loaded Albumin-Magnetite Nanoparticle Drug Delivery System

In this example, a bovine serum albumin (BSA)-magnetite (Fe ₃ O ₄ ) nanoparticle drug delivery system (named Dox@BMNT) loaded with Dox (a hydrophobic chemotherapeutic drug) was prepared, and its The characterization specifically includes the following steps.

1.1. Under dark conditions, dissolve Dox (0mg, 0.25mg, 0.5mg, 1mg, or 2mg) in DMSO, and mix with iron salt solution (iron prepared from FeSO ₄ 7H ₂ O and FeCl ₃ 6H ₂ O Salt solution, wherein Fe ³⁺ /Fe ²⁺ can be (1.5-2.5): 1, as long as the main part of the nanoparticles obtained by it finally (after adding NaOH solution dropwise in step 1.2) is Fe ₃ O ₄ Get final product, In this embodiment, it is specifically Fe ³⁺ /Fe ²⁺ =2/1, and the nanoparticles obtained after adding the NaOH solution dropwise in the following step 1.2 are magnetite (Fe ₃ O ₄ ) nanoparticles) premixed (wherein The molar ratios of Dox and iron are respectively: 0/165, 0.75/165, 1.5/165, 3/165, and 6/165) to obtain premixed solutions containing different amounts of Dox. This process is carried out under the protection of _N2 atmosphere and stirring in a water bath at about 37°C (eg, 37±2°C).

1.2, the bovine serum albumin (BSA) solution is added to the different premixed solutions obtained in step 1.1 respectively with the molar ratio of BSA and iron as 1/165, so that the feed ratio (molar ratio, FR) of Dox and BSA is respectively 0/1, 0.75/1, 1.5/1, 3/1, and 6/1 (corresponding to premix solutions containing different amounts of Dox). Then slowly dropwise slightly excess NaOH solution (100mM) relative to iron ions (wherein the NaOH equilibrium addition amount is calculated by the chemical equation 2Fe ³⁺ +Fe ²⁺ + ^8OH- =Fe ₃ O ₄ +4H ₂ O), stirring for about After 3 h, the products were collected separately. This process is carried out under the protection of _N2 atmosphere and stirring in a water bath at about 37°C (eg, 37±2°C).

1.3. Filter the product obtained in step 1.2 through a 0.22 μm filter membrane, and finally dialyze with PBS to obtain BSA-magnetite nanoparticle drug delivery systems synthesized under different FR ratio conditions, which are named Dox@BMNT (FR: 0/1 ), Dox@BMNT (FR: 0.75/1), Dox@BMNT (FR: 1.5/1), Dox@BMNT (FR: 3/1) and Dox@BMNT (FR: 6/1).

1.4. Characterization of Dox@BMNT:

(1) Based on the size exclusion chromatography (SEC) method, the Dox@BMNT loaded with different amounts of Dox obtained in the above step 1.3 were first dispersed in PBS through a concentrator tube (100K, Merck Millipore, USA), and then loaded into Superose 6Increase 10 UV absorbance monitoring was performed at 280 nm and 480 nm on a G/300GL column (GE Healthcare). The results are shown in Figure 1 a-e, which are the SEC analysis results of Dox@BMNT synthesized under different FR ratio conditions, in which 280nm is the specific absorption peak (higher peak) of BSA nanocage, and 480nm is the specific absorption peak of Dox It can be seen that Dox@BMNT synthesized under different FR conditions were all eluted to obtain a 280nm absorption peak with a volume of about 12.8ml, indicating the formation of BSA nanocages. With the increase of Dox in the synthesis system, Absorption at 480 nm increased, indicating successful Dox loading.

与磁铁矿(311)平面的标准晶格常数相对应，表明在纳米笼空腔中均形成了磁铁矿纳米颗粒。(2) Synthesis under different FR (0/1, 0.75/1, 1.5/1, 3/1, and 6/1) conditions using transmission electron microscopy (TEM, JEM2100) and high-resolution transmission electron microscopy (HRTEM, JEM2100) The Dox@BMNT was further characterized, and the results are shown in fo in Figure 1, where fj in Figure 1 is the negative staining TEM image, and ko is the TEM image (the inset in the TEM image is a HRTEM image magnified 50 times), it can be seen that the acetic acid After uranium negative staining, the BSA protein in Dox@BMNT synthesized under different FR conditions was mineralized to form a clear cage-like nanostructure (i.e., nanocages with cavities), and the inorganic core particles in the cavities of the nanocages all showed The small size and uniformity (ko in Fig. 1) can be attributed to the spatial confinement and homogeneity of the BSA nanocages. In addition, the HRTEM images of ko in Figure 1 show that the lattice spacing of the inorganic core particles in the nanocage cavity of Dox@BMNT synthesized under different FR conditions is about

Corresponding to the standard lattice constant of magnetite (311) planes, it indicates that magnetite nanoparticles are formed in both nanocage cavities.

(3) Based on the TEM images of the transmission electron microscope, the sizes of the nanocages and magnetite nanoparticles formed by the BSA protein in the Dox@BMNT synthesized under different FR conditions were calculated. The results are shown in p in Figure 1. It can be seen that although the reaction process Different Dox to BSA feed ratios FR were used, but the final synthesized Dox@BMNTs showed relatively uniform and stable magnetite cores and protein cages with sizes of about 2.5-4.5nm and 16.5-20.5nm, respectively.

(4) For Dox@BMNT synthesized under different FR conditions, its hydrodynamic diameter (HD) and zeta potential were detected by a dynamic light scattering instrument (MalvernZetasizer Nano ZS, UK). The results are shown in q in Figure 1, and different Both HD and zeta of Dox@BMNT synthesized under FR conditions have good consistency, especially HD (ranging from 19-22 nm) does not change more than 10%, showing high homogeneity and applicability. In contrast, Dox-BSA exhibits a larger size (average about 35.3 nm) and poorer uniformity (varies in the interval of about 15–70 nm), which is mainly due to the lack of spatial confinement of nanocages. This shows that in the Dox@BMNT synthesized in the present invention, Dox is not involved in the formation of BSA nanocages, but is combined with Fe ions and loaded into the cavity of the nanocages through magnetite mineralization, indicating that the present invention is based on BSA nanocages Biomimetic synthetic approaches to regulate biomineralization have general applicability. In addition, it can also be known from the results shown in q in Figure 1 that the zeta potential of Dox@BMNT synthesized under different FR conditions is about -27mV (about -24 to -31mV) (while the zeta potential of Dox-BSA is about -12.8 mV), it can be considered to have sufficient charge repulsion in aqueous solution to maintain stability, which is helpful to improve the biological safety and stability of Dox@BMNT.

(5) For Dox@BMNT synthesized under different FR conditions, the Dox loading efficiency was also detected (for Dox@BMNT synthesized under different FR conditions, based on the SEC analysis results shown in Figure 1 a-e (280nm and 480nm Absorption peak), the concentration of Dox was detected with a fluorescence spectrophotometer, and the concentration of BSA was measured with a BCA protein assay kit (Thermo, USA), and the Dox loading efficiency was calculated from this), the results are shown as r in Figure 1, and it can be seen that As the FR of Dox to BSA increases, the loading efficiency of Dox increases obviously and finally tends to be saturated when the FR is 3/1. The fitting curve showed that when the loaded Dox reached saturation, the molar ratio of Dox to BSA in the synthesized Dox@BMNT was about 12, that is, each BSA protein participated in the loading of 12 Dox molecules on average. Therefore when preparing Dox@BMNT, it is preferable to control the feed ratio of Dox and BSA to be more than 1.5/1, preferably more than 3/1, which can improve the loading efficiency of Dox; , fluorouracil, mitomycin, actinomycin, pingyangmycin, cisplatin, carboplatin, epirubicin, methotrexate, and cytarabine) and proteins (such as human serum albumin (HSA)) , those skilled in the art can determine through the method of this embodiment.

1.5. Structural analysis of Dox@BMNT:

To determine the structure of Dox@BMNT, Dox@BMNT (FR: 3/1) purified by SEC was negatively stained with uranyl acetate and studied under TEM, as shown in Figure 2a. More than 10,000 particles were then selected from a set of photomicrographs to perform an iterative reference-free two-dimensional alignment and classification procedure using Imagic (van Heel, et al. A new generation of the IMAGIC image processing system. J.Struct.Biol.116, 17–24 (1996)), where the alignment and classification process was iterated 15 times to converge to a final class mean. For each representative class mean, particles were randomly assigned to two sets of equal size, the class mean was calculated, and the Fourier ring correlation between the two class means was determined. Finally 4 main classes are visualized and divided by overall size and shape, as shown in b in Fig. 2. For each class, typical two-dimensional averages were established, summarizing the main features of caged structures and representing them graphically. As shown in Figure 2b (different viewing directions, scale bar: 20nm), it can be seen that class 1 and class 2 have more complex features, such as nearly elliptical and thicker protein shells, while class 3 and class 4 show more obvious Cage-like structure, nearly round shape and thinner shell.

和

)和Glu478(键长:

)之间观察到4种氢键相互作用，这是Dox和亲水阴离子口袋之间的主要界面相互作用。Dox还被BSA的残基Phe205、Ala209、Ala212、Leu480和Val481包围，形成疏水绑定。所有这些相互作用均有助于Dox锚定在空腔结合位点。上述结构分析有力地证实了Dox的封装稳定性，为理解Dox@BMNT作为药物载体的构效关系提供了有价值的信息。In order to understand the basic structural properties of Dox@BMNT, the proposed model was obtained by MD simulation and computational modeling (based on Amber 14 program) (all MD simulations were performed on a GPU workstation based on linux (Ubuntu 14.04)), where The three-dimensional (3D) structure (PDB ID: 4F5S) of BSA was downloaded from the miv RCSB protein database (www.rcsb.org). The results of MD simulation and computational modeling are shown in ce in Fig. 2, where c in Fig. 2 shows the structural model and surface charge distribution of Dox@BMNT (FR: 3/1) under different viewing angles. It can be seen that 6 BSA particles The units are intertwined with each other to form a complex sphere and are shaped into a "torpedo" topology with magnetite and Dox molecules located in the cavity of the complex sphere. Inside this "torpedo" topology, 1 magnetite nanoparticle is combined with 72 Dox molecules (on average, each BSA protein participates in the loading of 12 Dox molecules), and is stabilized by the self-assembled "torpedo" shell of BSA protein. From the surface, a large number of hydrophilic residues are randomly exposed on the surface of the "torpedo", which greatly promotes the "torpedo"-water interaction, thereby improving the stability of the "torpedo" in aqueous solution. Figure 2d shows the internal structure of the Dox@BMNT (FR: 3/1) model. It can be seen that the Dox molecules are entangled with the magnetite particles and buried together in the hydrophilic anion pocket (ie, the cavity of the nanocage) with a diameter of about 4.2 nm. Figure 2 e shows the interface information between Dox and BSA in the cavity of the nanocage, and the interface interaction between BSA and magnetite, it can be seen that the magnetite particles and BSA Arg81, Glu82, Asp86, Asp89 The residues of Glu92 and Glu92 are tightly bound in the hydrophilic anion pocket, while the aryl group of Dox forms cationic-π interactions with the Arg208 and Lys350 residues of BSA. Importantly, between Dox and residue Arg208 (bond length:

and

) and Glu478 (key length:

), which are the main interfacial interactions between Dox and the hydrophilic anion pocket. Dox is also surrounded by residues Phe205, Ala209, Ala212, Leu480 and Val481 of BSA, forming a hydrophobic binding. All of these interactions contribute to the anchoring of Dox at the cavity binding site. The above structural analysis strongly confirmed the encapsulation stability of Dox and provided valuable information for understanding the structure-activity relationship of Dox@BMNT as a drug carrier.

Example 2: Preparation of drug-loaded albumin-magnetite nanoparticle drug delivery system and its controlled release effect in serum

This Example 2 is carried out according to the operation of preparing the drug-loaded albumin-magnetite nanoparticle drug delivery system in Example 1, the difference is only: (1) 1mg Dox is dissolved in DMSO in step 1.1; ( 2) In step 1.2, respectively control the molar ratio of Fe and BSA to 110/1, 220/1, 330/1 or 82.5/1, and control the FR to 3/1, respectively, to obtain biomimetic mineralization under different Fe/BSA conditions The synthesized Dox@BMNT were named as: Dox@BMNT (Dox@BMNT(Fe/BSA=110/1), Dox@BMNT(Fe/BSA=220/1), Dox@BMNT(Fe/BSA=330/1) 1) and Dox@BMNT (Fe/BSA=82.5/1).

Evaluation of the controlled release effect of Dox@BMNT in serum:

The Dox@BMNT (Fe/BSA=165/1) prepared in Example 1 (that is, the Dox@BMNT (FR: 3/1) prepared in Example 1), and the four Dox@BMNTs prepared in Example 2 (Dox@BMNT(Fe/BSA=110/1), Dox@BMNT(Fe/BSA=220/1), Dox@BMNT(Fe/BSA=330/1) and Dox@BMNT(Fe/BSA=82.5/1) 1)) were placed in a dialysis cup (MW cut-off 14kDa, Novagen), and serum was added to incubate with gentle stirring. After incubation for a period of time (about 240 h), the incubation solution was extracted and centrifuged at 8000 rpm for 5 min. The stability of free Dox released in different incubation times in serum was determined by high performance liquid chromatography.

The measurement results are shown in Figure 3. Compared with Dox@BMNT (Fe/BSA=330/1) and Dox@BMNT (Fe/BSA=82.5/1), there is more leakage in serum, and Dox@BMNT (Fe/BSA=82.5/1) has more leakage in serum. /BSA=110/1), Dox@BMNT(Fe/BSA=165/1) and Dox@BMNT(Fe/BSA=220/1) showed good stability in serum, and incubated in serum for 120 hours Almost non-leaky (Dox release lower than about 15%), especially Dox@BMNT (Fe/BSA=110/1) and Dox@BMNT (Fe/BSA=165/1) showed better stability in serum It is almost leak-free (Dox release is less than about 10%) after incubation in serum for 120 hours, even after incubation for 240 hours, the release of Dox is less than about 20%, which indicates that in the process of preparing Dox@BMNT, when Fe and When the molar ratio of BSA is in the range of 110/1-220/1, preferably in the range of 110/1-165/1, it is more helpful to improve the drug-loading stability of the prepared Dox@BMNT.

Example 3: Preparation of drug-loaded albumin-magnetite nanoparticle drug delivery system

This embodiment prepares a bovine serum albumin (BSA)-magnetite nanoparticle drug delivery system (named Dox Hcl@BMNT) loaded with Dox Hcl (a hydrophilic chemotherapeutic drug), and according to the embodiment It was characterized by the method described in 1. Wherein the preparation method can be carried out according to the operation of preparing the albumin-magnetite nanoparticle drug delivery system loaded with drugs in Example 1, the difference is only in: (1) in step 1.1, 1 mg/ml (in terms of Dox) Dox·Hcl is dissolved in sterile water; (2) In step 1.2, the molar ratio of BSA and Fe is controlled to be 1/165, and the FR (the molar ratio of Dox to BSA) is controlled to be 3/1, thereby obtaining Dox·Hcl@ BMNT.

(图4中F)，与磁铁矿(311)平面的标准晶格常数相对应，表明在纳米笼中形成了磁铁矿纳米颗粒；图4中G则示出了纳米笼中形成的磁铁矿纳米颗粒的粒径统计结果，可见该磁铁矿纳米颗粒的粒径分布在1.5-5nm的范围内，平均粒径约为3.07±1.27nm，表现出小尺寸和均匀性；图4中H-I则分别示出了Dox·Hcl@BMNT的Zeta-电位(图4中H)和水动力直径HD(图4中I)的检测结果。The characterization results of Dox·Hcl@BMNT are shown in AI in Figure 4, AB in Figure 4 is the uranium acetate negative-stained TEM image of Dox·Hcl@BMNT at different resolutions; C in Figure 4 shows the negative-stained TEM image The nanocage structure in Dox·Hcl@BMNT indicates that the BSA protein in Dox·Hcl@BMNT is also mineralized to form a clear cage nanostructure. The size of the nanocage structure is about 18.5 nm, and the cavity of the nanocage is The diameter is about 4-12nm; D in Figure 4 shows the 280nm absorption peak based on the SEC elution volume of about 11.7ml, indicating the formation of BSA nanocages; EF in Figure 4 is the TEM image of Dox·Hcl@BMNT and High-resolution HRTEM image (the inset in the upper left corner is the Fourier transform data), it can be seen that the lattice spacing of the core nanoparticles is about

(F in Figure 4), corresponding to the standard lattice constant of the magnetite (311) plane, shows that magnetite nanoparticles are formed in the nanocage; G in Figure 4 shows the magnetic field formed in the nanocage The statistical results of the particle size of the iron ore nanoparticles show that the particle size distribution of the magnetite nanoparticles is in the range of 1.5-5nm, and the average particle size is about 3.07±1.27nm, showing small size and uniformity; in Figure 4 HI shows the detection results of the Zeta-potential (H in Figure 4) and hydrodynamic diameter HD (I in Figure 4) of Dox·Hcl@BMNT, respectively.

Example 4: Preparation of drug-loaded albumin-magnetite nanoparticle drug delivery system

In this example, a Dox-loaded human serum albumin (HSA)-magnetite nanoparticle drug delivery system (named Dox@HMNT) was prepared and characterized according to the method described in Example 1. Wherein the preparation method can be carried out according to the operation of preparing the drug-loaded albumin-magnetite nanoparticle drug delivery system in Example 1, the difference is only: (1) 1 mg Dox is dissolved in DMSO in step 1.1; ( 2) In step 1.2, the molar ratio of HSA to Fe is controlled to be 1/165, and FR (here, the feed ratio (molar ratio) of Dox to HSA) is controlled to be 3/1, thereby obtaining Dox@HMNT.

In this example, similar to the r results in Figure 1, as the FR of Dox to BSA increases, the loading efficiency of Dox increases significantly and eventually tends to saturate when the FR is 3/1. Therefore, when preparing Dox@HMNT, it is preferable to control the feed ratio of Dox to HSA to be above 1.5/1, preferably above 3/1, which can improve the loading efficiency of Dox. In addition, similar to the results in Figure 3, when the molar ratio of Fe to HSA is in the range of 110/1-220/1, preferably in the range of 110/1-165/1, it is more helpful to improve the drug loading stability of Dox@HMNT sex.

The characterization results of Dox@HMNT are shown in A-F in Figure 5, and A-B in Figure 5 are uranium acetate negative-stained TEM images of Dox@HMNT at different resolutions, which are similar to those in the Dox@BMNT structure prepared in Example 1 above. BSA, in the Dox@HMNT structure, the HSA protein is also mineralized to form a clear cage-like nanostructure (similar to BSA, 6 HSA subunits are intertwined with each other to form a cage-like nanostructure, in which the cage-like nanostructure In the cavity, 1 magnetite nanoparticle is combined with 72 Dox molecules (on average, each HSA protein participates in the loading of 12 Dox molecules), and is stabilized by the self-assembled shell of the HSA protein); Figure 5 C shows Dox The statistical results of the particle size of the nano-cage structure of @HMNT, it can be seen that the particle size of the nano-cage structure of Dox@HMNT is in the range of 13-23nm, and the average particle size is about 17.3±1.7nm; D in Figure 5 shows Based on the 280nm absorption peak (higher peak) with an elution volume of about 11.6ml in SEC, it indicates the formation of HSA nanocages; E in Figure 5 is the TEM characterization image of Dox@HMNT; F in Figure 5 shows the Dox The statistical results of the particle size of the inorganic core particles in the cavity of the nanocage structure of @HMNT show that the particle size of the inorganic core particles is in the range of 1.5-5nm, and the average particle size is about 2.9±1.0nm, showing a small size and uniformity.

Example 5: Preparation of drug-loaded albumin-magnetite nanoparticle drug delivery system

This example prepares a human serum albumin (HSA)-magnetite nanoparticle drug delivery system loaded with Dox Hcl (named Dox Hcl@HMNT), and characterizes it according to the method described in Example 1 . Wherein the preparation method can be carried out according to the operation of preparing the drug-loaded albumin-magnetite nanoparticle drug delivery system in Example 1, the difference is only: (1) in step 1.1, 1 mg/ml Dox Hcl is dissolved into In sterile water; (2) In step 1.2, the molar ratio of HSA to Fe is controlled to be 1/165, and the FR (the molar ratio of Dox to HSA) is controlled to be 3/1, thereby obtaining Dox·Hcl@HMNT.

The characterization results of Dox·Hcl@HMNT are shown in Figure 6A-F, and Figure 6 A-B is the uranyl acetate negative-stained TEM image of Dox·Hcl@HMNT at different resolutions; Figure 6 C shows the negative-stained TEM The nanocage structure in the image shows that the HSA protein in Dox·Hcl@HMNT is also mineralized to form a clear cage nanostructure. The size distribution of the nanocage structure is measured in the range of 13-23nm, with an average size of diameter is about 17.2 ± 1.6nm (as shown in F in Figure 6), and the diameter of the cavity of the nanocage is about 4-10nm; D in Figure 6 shows the 280nm absorption based on the elution volume of SEC about 12.6ml The peak (higher peak) indicates the formation of HSA nanocages, and the peak at 480nm (lower peak) indicates the successful loading of Dox; E in Figure 6 is the TEM image of Dox·Hcl@HMNT.

Example 6: In vitro controlled release and efficacy evaluation of drug-loaded albumin-magnetite nanoparticle drug delivery system

This embodiment is for the albumin-magnetite nanoparticle drug delivery system (including Dox@BMNT (FR: 1.5/1) prepared in Example 1, Dox@BMNT (FR: 3/1) and Dox@BMNT (FR: 6/1), Dox@BMNT (Fe/BSA=110/1) and Dox@BMNT (Fe/BSA=220/1) prepared in Example 2, The Dox·Hcl@BMNT prepared in Example 3, the Dox@HMNT prepared in Example 4, and the Dox·Hcl@HMNT prepared in Example 5) were evaluated in vitro drug controlled release and drug efficacy, specifically, the drug prepared in Example 1 Dox@BMNT (FR: 3/1) describes the evaluation method and results as an example.

(1) Determination of the Dox release of Dox@BMNT in PBS buffers with different pHs: Dox-BSA, Doxil and Dox@BMNT were placed in dialysis cups (MW cut-off 14kDa, Novagen), and PBS buffers with different pHs were added solution (pH 4.5 (simulated lysosome), pH 5.5 (simulated cytoplasm), pH 6.5 (simulated tumor microenvironment) and pH 7.5 (simulated plasma)), stirred gently. After incubation for a period of time (about 25 h), the incubation solution was extracted and centrifuged at 8000 rpm for 5 min. The concentration of Dox in the supernatant was detected by a spectrofluorometer.

The measurement results are shown in a-d in Figure 7 (the curves from top to bottom represent Dox-BSA, Doxil and Dox@BMNT respectively), it can be seen that under different pH conditions, the three drug delivery systems (Dox-BSA, Doxil and Dox@BMNT) all exhibited cumulative release of Dox, in which Dox-BSA and commercial Doxil groups had significant leakage at pH 7.5 and pH 6.5, especially the Dox-BSA group, which released about 58.7% of Dox at pH 6.5 Dox, about 78% of Dox was released at pH 5.5, which may be related to the unstable non-specific interaction between Dox and BSA. Followed by Doxil, which released more than half of Dox at pH 5.5, while at pH 7.5, pH 6.5 and pH 5.5, most of Dox@BMNT remained stable, with no more than 20% of Dox released at most (Figure 7 Middle c), until the pH value dropped to 4.5, Dox@BMNT finally reached a higher release (Fig. 7 d). The statistical results of the cumulative release of Dox in the above three drug delivery systems are shown in e in Figure 7 (the columns from left to right under each pH condition represent Dox-BSA, Doxil and Dox@BMNT respectively), it can be seen that although The acidity of the solution increased (pH decreased from 7.5 to 5.5), but Dox@BMNT still showed good stability, and the leakage of Dox was significantly lower than that of Dox-BSA and Doxil groups. Finally, the Dox@BMNT group was released in a low pH environment of 4.5, which was beneficial to the circulation of drug Dox in plasma, tumor microenvironment and cytoplasm, and facilitated the delivery and release of drug Dox in lysosomes.

(2) Determination of the Dox release of Dox@BMNT in serum: Dox-BSA, Doxil and Dox@BMNT were placed in dialysis cups (MW cut-off 14kDa, Novagen), and serum was added respectively, and stirred gently. After incubation for a period of time (about 120 h), the incubation solution was extracted and centrifuged at 8000 rpm for 5 min. The stability of free Dox released in different incubation times in serum was determined by high performance liquid chromatography.

The measurement results are shown in f in Figure 7 (the curves from top to bottom represent Dox@BMNT, Doxil and Dox-BSA respectively). It can be seen that Dox@BMNT shows better stability in serum, and it can be incubated in serum for 120 hours There is almost no leakage, while Dox-BSA and Doxil have more leakage.

The results of (1) and (2) above show that, compared with Dox-BSA and Doxil, Dox@BMNT has better stability characteristics, which may be related to the synergy of many factors, such as magnetite nanoparticles and Dox The surface chelation of BSA and the interfacial binding of Dox. In addition, the effective encapsulation of BSA nanocages can also reduce the exposure and separation of Dox, which helps to improve the stability and thus prolong its half-life in blood circulation.

(3) In vitro killing effect of Dox@BMNT on tumor cells: In this study, two tumor cell lines (4T1 and SKBR3) were selected. 4T1 cells were cultured in DMEM (Dulbecco's Modified Eagle Medium) containing 10% fetal bovine serum (FBS) and 1% antibiotic solution, and SKBR3 cells were cultured in DMEM containing 20% fetal bovine serum (FBS) and 1% antibiotic solution. Adherent culture was carried out in DMEM/F12 (Ham's F-12 nutrient medium) at a temperature of 37°C and a concentration of 5% CO ₂ .

4T1 and SKBR3 cells were incubated in the medium containing Dox-BSA, Doxil and Dox@BMNT for 24h, and the Dox concentration in each medium was 10 ^-2 , 5×10 ^-2 , 0.1, 0.5, 1, 5, 10 , 50, 10 ² , 5×10 ² , 10 ³ , 5×10 ³ , 10 ⁴ , 5×10 ⁴ , 10 ⁵ μg/mL. The cell viability of each sample was then detected with a CCK-8 kit. Each test has 5 parallel samples.

After 24 hours of incubation, the exemplary results of cytotoxicity and killing effect detection on the 4T1 tumor cell line are shown in gh in Figure 7 (wherein: *p≤0.05, **p≤0.01) (in g in Figure 7 by The curves from left to right represent Dox@BMNT, Doxil and Dox-BSA), it can be seen that Dox@BMNT has strong cytotoxicity against 4T1 tumor cell line and has a strong killing effect, and its IC ₅₀ value is about 0.5 μg /mL, significantly lower than Dox-BSA (about 2.5μg/mL) and Doxil (about 1.1μg/mL). The killing effect of Dox@BMNT on SKBR3 cell line is consistent with that on 4T1 tumor cell line, indicating that it has a good killing effect on tumor cells.

(4) Effect of Dox@BMNT on normal cells: In this study, two normal cell lines (293T and HL7702) were selected, in which 293T and HL7702 cells were maintained in DMEM containing 10% fetal bovine serum (FBS) and 1% antibiotic solution. (Dulbecco's Modified Eagle Medium) for adherent culture at a temperature of 37°C and a concentration of 5% CO ₂ .

293T and HL7702 cells were incubated overnight in medium containing Dox@BMNT (Dox concentrations of 0, 0.5, 1.0, 2.0, 5.0, 10.0 μg/mL, respectively). Cell viability was measured with CCK-8 kit. Each test has 5 parallel samples.

The measurement results are shown in Figure 8 (in which the columns from left to right for each Dox concentration represent 293T and HL7702 respectively), it can be seen that the survival rates of the normal cell lines (293T and HL7702) incubated with Dox@BMNT were all greater than 90 %, indicating that Dox@BMNT exhibits low toxicity to normal cells, so it has good biosafety performance when using this Dox@BMNT to deliver drugs.

A variety of drug-loaded albumin-magnetite nanoparticle drug delivery systems prepared in Examples 1-5 above also have similar in vitro drug release effects and tumor cell The killing effect, and safe to normal cell lines, will not repeat them here.

Example 7: Evaluation of Cellular Uptake and Retention of Drug-Loaded Albumin-Magnetite Nanoparticle Delivery System

This embodiment is for the albumin-magnetite nanoparticle drug delivery system (including Dox@BMNT (FR: 1.5/1) prepared in Example 1, Dox@BMNT (FR: 3/1) and Dox@BMNT (FR: 6/1), Dox@BMNT (Fe/BSA=110/1) and Dox@BMNT (Fe/BSA=220/1) prepared in Example 2, The Dox·Hcl@BMNT prepared in Example 3, the Dox@HMNT prepared in Example 4, and the Dox·Hcl@HMNT prepared in Example 5) were evaluated for their cellular uptake and retention, specifically the Dox@Hcl@HMNT prepared in Example 1 BMNT (FR: 3/1) describes the evaluation method and results as an example.

Seed 4T1 cells into a six-well plate at a density of 10 ⁵ cells/well. When the cell confluence reaches about 70%, replace the original medium with fresh medium containing Dox@BMNT (Dox concentration: 5 μg/ml) , wherein Dox-BSA and Doxil were used as controls. After 2 hours of incubation, the medium was removed and the cells were washed 3 times with PBS. Evaluation of cell uptake: cells were incubated with DAPI and lysosome-tracker Green staining solution for 15 min, and then observed with CLSM (Zesis, Germany). Evaluation of cell retention: cells were incubated in fresh medium for 4 hours, incubated with DAPI and lysosome-tracker Green staining solution for 15 minutes, and then observed with CLSM (Zesis, Germany).

The results are shown in a-f in Figure 9, a in Figure 9 is the distribution of Dox in the cells after incubation for 2 hours, b in Figure 9 shows the distribution of Dox in the cells after removing the drug for 4 hours, in a-b in Figure 9, white arrows Indicates the nucleus, the gray arrow indicates the lysosome, c in Figure 9 indicates the average fluorescence intensity of Dox in the lysosome, d in Figure 9 indicates the average fluorescence intensity of Dox in the nucleus, and e in Figure 9 indicates the average fluorescence intensity of Dox in the lysosome and the total fluorescence intensity in the nucleus, f in Figure 9 represents the decrease of the total fluorescence intensity after removing the drug for 4 hours, where *p≤0.05, **p≤0.01. As shown by the results of a-b in Figure 9, it can be seen that most of the Dox from Dox@BMNT was captured by lysosomes, while Dox from the other two groups (Dox-BSA and Doxil) were mainly distributed in the nucleus, and after 4 hours of drug removal ( That is, after 6 hours of incubation), the results of detecting the retention concentration of Dox showed that compared with Dox-BSA and Doxil, Dox@BMNT group still expressed higher fluorescence intensity, and it was widely gathered in the nucleus to produce strong fluorescence. As shown by the quantitative brightness results of c-f in 9, after incubation for 2 h, Dox@BMNT is the first choice for lysosomes, because its mean fluorescence intensity (MFI) is 50.3 a.u, which is 2.5 times and 25 times that of Dox-BSA group and Doxil group, respectively. times. Although the uptake of Dox (MFI: 10.7a.u) by the nucleus into Dox@BMNT was comparable to that of Dox@BSA and Doxil after 2h of incubation, the amount of Dox@BMNT was significantly increased after 4h of drug removal, with an MFI of 41.4a.u. Considering that some amount of Dox is still present in lysosomes and is transported into the nucleus, high Dox levels will persist in the nucleus over time. Notably, despite the different intracellular distribution, all three groups had similar Dox uptake, but after 4 h of drug removal, only the MFI of the Dox@BMNT group was better maintained (56.8a.u), compared with The total MFIs of the other two groups (Dox-BSA MFI: 7.1a.u and Doxil MFI: 16.3a.u) were significantly lower than those of Dox@BMNT, indicating that both have poorer Dox retention. The longer the retention time of Dox@BMNT, the more favorable the accumulation of the drug in the tumor area.

A variety of drug-loaded albumin-magnetite nanoparticle drug delivery systems prepared in Examples 1-5 above also have similar cellular uptake and retention effects as Dox@BMNT (FR: 3/1), which is not shown here. Let me repeat them one by one.

Example 8: In vivo anti-tumor activity of drug-loaded albumin-magnetite nanoparticle drug delivery system

This embodiment is for the albumin-magnetite nanoparticle drug delivery system (including Dox@BMNT (FR: 1.5/1) prepared in Example 1, Dox@BMNT (FR: 3/1) and Dox@BMNT (FR: 6/1), Dox@BMNT (Fe/BSA=110/1) and Dox@BMNT (Fe/BSA=220/1) prepared in Example 2, The Dox·Hcl@BMNT prepared in Example 3, the Dox@HMNT prepared in Example 4, and the Dox·Hcl@HMNT prepared in Example 5) were evaluated for their cellular uptake and retention, specifically the Dox@Hcl@HMNT prepared in Example 1 BMNT (FR: 3/1) describes the evaluation method and results as an example.

8.1. Establishment of tumor mouse model

Female BALB/c nude mice used in this study were purchased from Nanjing Jieyao Technology Co., Ltd., China, with an average body weight of 20 g. A suspension containing 1×10 ⁶ 4T1 cells was inoculated into the right leg of the mouse to construct a tumor model mouse. The tumor volume was accurately monitored with a vernier caliper, and the tumor volume was calculated by the formula V=LW ² /2, where L and W were the length and width of the tumor, respectively. All mice were cared for according to the guidelines outlined in the Guide for the Care and Use of Laboratory Animals.

8.2 In vivo antitumor activity of Dox@BMNT

All the tumor model mice constructed in the above step 8.1 were randomly divided into 4 groups, 5 mice in each group, divided into PBS group, Dox-BSA group, Doxil group and Dox@BMNT group. When the tumor volume in the tumor model mice reached 150 mm ³ , the mice were intravenously injected with each group of drug samples (with an equivalent Dox concentration of 5 mg/kg), once every three days, for a total of three injections. Tumor size and body weight were recorded on days 3, 6, 9, 12, 15, 18, 21 after the first injection (day 0). The mice were sacrificed on the 21st day, and the Dox@BMNT group was compared with three control groups to evaluate the therapeutic effect.

8.3. Magnetic resonance imaging

In in vivo MRI, mouse bodies were placed in a 14.1T NMR microimaging system (Bruker Biospec, Karlsruhe, Germany) and MRI studies were performed at different processing times. t1-weighted MRI images were acquired using Rapid Acquisition Relaxation Enhancement (RARE) sequence with the following parameters: TR/TE = 3500/10ms, field of view (FOV) = 25 mm × 25 mm, matrix = 256 × 256, slice thickness = 0.5 mm, mean Number=2. The calculation formula of CNR in the MRI image is: CNR=(SI tumor-SI mouse)/SI noise, where SI represents the signal intensity and the noise comes from the air background.

The in vivo MRI and anti-tumor efficacy results of Dox@BMNT are shown in Figure 10 a-i, Figure 10 a is a schematic diagram of the anti-tumor treatment timeline of tumor model mice, and Figure 10 b is the model mice after intravenous injection of Dox@BMNT Dynamic t1-weighted MRI to track the distribution of drugs in the tumor area, c in Figure 10 is the CNR ratio corresponding to MRI, d-e in Figure 10 are the body weight changes and tumor growth curves of the model mice, and f in Figure 10 is the tumor of the model mice after treatment In Figure 10, g is the tumor photo of the model mouse after treatment, in Figure 10 h is the photo of the model mouse before and after treatment, and in Figure 10 i is the histological analysis result of the tumor tissue of the model mouse after treatment, wherein Scale bar: 100 μm, **p≤0.01.

From the results of b-c in Figure 10, it can be seen that the T1 signal intensity in the tumor area of the model mice was significantly improved 30 minutes after the injection of Dox@BMNT, and reached the strongest 4 hours after the injection. At this time, the CNR ratio corresponding to the MRI was about 10.9, which was the initial Nearly 9 times the noise. The continuous high-contrast effect significantly improves the resolution and sensitivity of MRI, which can accurately reflect the distribution and accumulation of Dox@BMNT after injection into the tumor area.

It can be seen from the results in d of Figure 10 that the body weight of the model mice in all treatment groups increased, and there was no significant difference. However, compared with the other three groups, the Dox@BMNT group had the best tumor treatment effect, which significantly inhibited the tumor growth of the model mice (e in Figure 10 (the curves from top to bottom represent the PBS group, Dox-BSA group, Doxil group and Dox@BMNT group)). The statistical results of the tumor volume of the model mice shown in f in Figure 10 showed that the average tumor volume of the Dox@BMNT group was 338.1mm ³ 21 days after the first administration, which was much smaller than that of the PBS group (1649.5mm ³ ), Dox- BSA group (1271.3mm ³ ) and Doxil group (893.1mm ³ ). Such a significant treatment effect can also be directly observed from gh in Fig. 10.

From the results of histological analysis shown in i in Figure 10, hematoxylin and eosin (H&E) staining showed that the density of tumor cells in the Dox@BMNT treatment group was lower than that in the other groups, and there were more apoptotic cells. In particular, tumors treated with Dox@BMNT also showed fewer Ki67-positive cells, more TUNEL-positive cells, and large-scale DNA degradation and fragmentation. Due to the long-term retention of Dox@BMNT in the tumor area, the drug is widely accumulated in the tumor area, which can more effectively inhibit cell proliferation and induce cell apoptosis, thus achieving a good tumor treatment effect.

A variety of drug-loaded albumin-magnetite nanoparticle drug delivery systems prepared in Examples 1-5 above also have similar anti-tumor effects in vivo as Dox@BMNT (FR: 3/1), which will not be repeated here. Let me repeat them one by one.

Example 9. In vivo biocompatibility of drug-loaded albumin-magnetite nanoparticle drug delivery system

This embodiment is for the albumin-magnetite nanoparticle drug delivery system (including Dox@BMNT (FR: 1.5/1) prepared in Example 1, Dox@BMNT (FR: 3/1) and Dox@BMNT (FR: 6/1), Dox@BMNT (Fe/BSA=110/1) and Dox@BMNT (Fe/BSA=220/1) prepared in Example 2, Dox·Hcl@BMNT prepared in Example 3, Dox@HMNT prepared in Example 4, and Dox·Hcl@HMNT prepared in Example 5) were evaluated for in vivo biocompatibility, specifically the Dox@Hcl@HMNT prepared in Example 1 BMNT (FR: 3/1) describes the evaluation method and results as an example.

9.1. Analysis of renal clearance rate

Collect urine samples at different times from the model mice injected with Dox@BMNT in Example 8 above to determine the renal clearance rate of Dox@BMNT, and the control group was injected with PBS. The iron concentration of each sample was determined by ICP-MS (Thermo, USA), and the nanoparticles in urine were observed by TEM (JEM 2100).

9.2 Histopathology and blood biochemical examination

The main organs (heart, liver, spleen, lung and kidney) and serum samples of the mice treated with Dox@BMNT in the above-mentioned Example 8 were collected, and the mice injected with PBS were used as controls. Serum samples were used for blood biochemical analysis, mouse organs were fixed with 4% paraformaldehyde for 24 hours, and paraffin-embedded sections. Then, paraffin-embedded sections were stained with hematoxylin and eosin (H&E) to assess the degree of damage.

The results are shown in a-m in Figure 11, where a in Figure 11 is the iron content in the urine samples collected at different times after the model mice were injected with Dox@BMNT and PBS (for each time point, the columns from left to right represent respectively Results of PBS and Dox@BMNT), b-d in Figure 11 are urine TEM images of model mice injected with Dox@BMNT 6h and 24h, e-l in Figure 11 are blood biochemical analysis results, where ALB: albumin, ALP: Alkaline phosphatase, CREA: creatinine, BUN: blood urea nitrogen, AST: aspartate aminotransferase, ALT: alanine aminotransferase, LDH: lactate dehydrogenase, TBIL: total bilirubin, m in Figure 11 is the model mouse H&E results of major organs after treatment, scale bar: 100 μm.

As shown in Figure 11a, it can be seen that after the model mice were injected with Dox@BMNT, the iron concentration in the urine of the mice increased and reached the maximum at 6h. When the urine was collected, a large number of nanoparticles could be observed, implying that Dox@BMNT was cleared by the kidney (b–c in Fig. 11). Subsequently, the concentration of iron in the urine decreased, and returned to normal levels 24 hours after the injection of Dox@BMNT, and almost no particles were seen in the urine (Fig. 11 d). The above results indicate that the Dox@BMNT prepared in the present invention can be effectively degraded and metabolized by the kidney, thus reducing the risk of toxicity and side effects. As shown in e-l in Figure 11, after the model mice were injected with Dox@BMNT and PBS, there was no significant difference in the blood biochemical indicators between the two, so it can be confirmed that Dox@BMNT has good biological safety in blood circulation. The H&E staining analysis results of the main organs shown in m in Figure 11 show that Dox@BMNT has no obvious damage to the heart, liver, spleen, lung and kidney after injection, which further proves that Dox@BMNT has no significant damage to the mice during the treatment period. Physiological functions and health conditions have no obvious side effects, so Dox@BMNT has very stable biocompatibility and biosafety.

A variety of drug-loaded albumin-magnetite nanoparticle drug delivery systems prepared in Examples 1-5 above also have similar in vivo biocompatibility and biosafety to Dox@BMNT (FR: 3/1) , which will not be repeated here.

Finally, it should be noted that: the above is only a preferred embodiment of the present invention, and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, for those skilled in the art, it can still The technical solutions recorded in the foregoing embodiments are modified, or some of the technical features are equivalently replaced. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims (14)

1. A drug-loaded albumin-magnetite nanoparticle drug delivery system, comprising: (1) nanocages with cavities formed by self-assembly of albumin; and (2) magnetite nanoparticles positioned in the cavity of the nanocage; Wherein the surface of the magnetite nano particle is coated with medicine. 2. The albumin-magnetite nanoparticle drug delivery system loaded with medicine according to claim 1, wherein: The diameter of the nanocage is 16.5-20.5nm; and/or The magnetite nanoparticles have a diameter of 2.5-4.5 nm; and/or The diameter of the cavity is 4-12 nm. 3. The drug-loaded albumin-magnetite nanoparticle drug delivery system according to claim 1, wherein in the cavity of the nanocage, the surface of the magnetite nanoparticle and the surface of the albumin residue binding; optionally, the albumin residues are selected from one or more of the following: Arg81, Glu82, Asp86, Asp89 and Glu92. 4. the albumin-magnetite nanoparticle drug delivery system loaded with medicine according to claim 1, wherein in the cavity of the nanocage, the medicine and the iron ion on the surface of the magnetite nanoparticle Chelation by chemical bonds. 5. The drug-loaded albumin-magnetite nanoparticle drug delivery system according to claim 1, wherein in the cavity of the nanocage, the group of the drug passes through a cation-π bond and/or hydrogen The bond interacts with residues of the albumin; optionally, the group of the drug is selected from aryl groups, and the residues of the albumin are selected from one or more of the following: Arg208, Lys350 and Glu478 . 6. The drug-loaded albumin-magnetite nanoparticle drug delivery system according to claim 1, wherein in the cavity of the nanocage, the drug is surrounded by residues of the albumin, forming a hydrophobic Binding; optionally, the albumin residues are selected from one or more of the following: Phe205, Ala209, Ala212, Leu480 and Val481. 7. The drug-loaded albumin-magnetite nanoparticle drug delivery system according to any one of claims 1-6, wherein the drug comprises an anticancer chemotherapy drug; optionally, the anticancer chemotherapy drug Including hydrophobic anticancer chemotherapy drugs and hydrophilic anticancer chemotherapy drugs; further optionally, the anticancer chemotherapy drugs are selected from one or more of the following: doxorubicin, doxorubicin hydrochloride, fluorouracil, silk Split mycin, actinomycin, pingyangmycin, cisplatin, carboplatin, epirubicin, methotrexate, and cytarabine. 8. The drug-loaded albumin-magnetite nanoparticle drug delivery system according to any one of claims 1-6, wherein the albumin is selected from one or more of the following: bovine serum albumin , and human serum albumin. 9. The drug-loaded albumin-magnetite nanoparticle drug delivery system according to any one of claims 1-6, wherein the drug is doxorubicin or doxorubicin hydrochloride, and the albumin is selected from One or more of the following: bovine serum albumin, and human serum albumin, and the nanocage is formed by self-assembly of 6 albumin molecules, and the surface of the magnetite nanoparticle is coated with 72 drugs molecular. 10. the preparation method of the albumin-magnetite nanoparticle delivery system of loading medicine described in any one in claim 1-9, it comprises the following steps: (S1) premixing the drug solution with the iron salt solution to obtain a premixed solution; (S2) Add the albumin solution to the premix solution obtained in step (S1), and add a slightly excess NaOH solution relative to the iron ions in the premix solution, stir, filter, and dialyze to obtain the drug-loaded albumin solution. Protein-magnetite nanoparticle drug delivery system; Wherein step (S1) and step (S2) are both carried out under the protection of inert gas. 11. The preparation method according to claim 10, wherein in step (S2), the amount of albumin solution added meets: the mol ratio of albumin to iron in the premixed solution is 1:(110- 220); optional 1:(110-165). 12. The preparation method according to claim 10 or 11, wherein in step (S2), the amount of albumin solution added satisfies: the molar ratio of the drug to the albumin is above 1.5, optionally as 3 or more. 13. The preparation method according to claim 10 or 11, wherein in step (S1), in the premixed solution, the molar ratio of the drug to iron is ≥1.5:(110-220); ≥1.5: (110-165). 14. The preparation method according to claim 10 or 11, wherein in step (S1), Fe ³⁺ /Fe ²⁺ =(1.5-2.5):1 in the iron salt solution.

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