CN115645548A

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

Info

Publication number: CN115645548A
Application number: CN202211411090.4A
Authority: CN
Inventors: 马坤; 赵享龙; 许帅; 王俊峰
Original assignee: Hefei Institutes of Physical Science of CAS
Current assignee: Hefei Institutes of Physical Science of CAS
Priority date: 2022-11-11
Filing date: 2022-11-11
Publication date: 2023-01-31

Abstract

The invention discloses a drug-loaded albumin-magnetite nanoparticle drug delivery system and a preparation method thereof, and belongs to the technical field of drug delivery. The drug-loaded albumin-magnetite nanoparticle drug delivery system provided by the invention comprises a nanocage with a cavity formed by albumin self-assembly and magnetite nanoparticles positioned in the cavity of the nanocage, and the surface of the magnetite nanoparticles is loaded with drugs, so that the advantages of an inorganic nanoparticle-based drug delivery system and a protein carrier-based drug delivery system can be effectively integrated, and further, better drug delivery and treatment effects can be realized, and the drug-loaded albumin-magnetite nanoparticle drug delivery system can be used for preparing anticancer drugs.

Description

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

Technical Field

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

Background

A safe and efficient Drug Delivery System (DDS) is expected to prolong the half-life of chemotherapeutic drugs and reduce toxic and side effects. In recent years, with the rapid development of nanotechnology, liposome chemotherapeutic drugs such as Doxil (doxorubicin liposome) and Onivyd (irinotecan liposome) have been widely used in clinical practice. However, the effect of the liposome chemotherapeutic drug in tumor treatment is only moderate, the stability is poor, the drug leaks in blood circulation, and the accumulation amount of the drug in a lesion area is low, and the potential risk of cardiotoxicity is high.

To overcome these drawbacks, two important delivery systems have been developed: one is an inorganic nanoparticle carrier (e.g., magnetite nanoparticles or silica nanoparticles) delivery system that is effective in stabilizing chemotherapeutic agents and extending their half-life in the blood circulation. In addition, the high specific surface area of the nanoparticles can also improve drug loading efficiency, and when the functionalized nanoparticles are used as carriers, the drugs can be endowed with comprehensive diagnosis and treatment capabilities. 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 the drug stability, and can reduce or eliminate immunogenicity and side effects. However, both of these delivery systems still have their own limitations, mainly manifested by higher biotoxicity of the inorganic nanoparticles and lower drug loading efficiency of the protein carriers.

Disclosure of Invention

In view of one or more of the problems of the prior art, one aspect of the present invention is to provide a drug-loaded albumin-magnetite nanoparticle delivery system, comprising:

(1) A nanocage having a cavity formed by albumin self-assembly; and

(2) Magnetite nanoparticles located in the nanocage cavity;

wherein the surface of the magnetite nanoparticles is coated with a drug.

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

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

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

In some embodiments, within the nanocage cavity, the surface of the magnetite nanoparticles is bound to the residues of the albumin; optionally, the residues of albumin are selected from one or more of: arg81, glu82, asp86, asp89 and Glu92.

In some embodiments, within the nanocage cavity, the drug is chelated by chemical bonding with iron ions on the surface of the magnetite nanoparticles.

In some embodiments, within the cavity of the nanocage, the group of the drug interacts with the residue of the albumin through cation-pi bonds and/or hydrogen bonds; optionally, the group of the drug is selected from aryl, and the residue of albumin is selected from one or more of: arg208, lys350 and Glu478.

In some embodiments, within the cavity of the nanocage, the drug is surrounded by residues of the albumin, forming a hydrophobic binding; optionally, the residue of albumin is selected from one or more of: phe205, ala209, ala212, leu480, and Val481.

In some embodiments, the drug comprises an anti-cancer chemotherapeutic drug; optionally, the anti-cancer chemotherapeutic comprises a hydrophobic anti-cancer chemotherapeutic and a hydrophilic anti-cancer chemotherapeutic; further optionally, the anti-cancer chemotherapeutic is selected from one or more of: doxorubicin, doxorubicin hydrochloride, fluorouracil, mitomycin, actinomycin, pingyangmycin, cisplatin, carboplatin, epirubicin, methotrexate, 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, and the albumin is selected from one or more of the following: bovine serum albumin and human serum albumin, and the nanometer cage is formed by 6 albumin molecules self-assembly, the surface cladding of magnetite nanoparticle has 72 medicine molecules.

In another aspect, the present invention provides a method for preparing a drug-loaded albumin-magnetite nanoparticle drug delivery system, comprising the steps of:

(S1) premixing the medicine solution and the iron salt solution to obtain a premixed solution;

(S2) adding an albumin solution into the premixed solution obtained in the step (S1), adding a NaOH solution slightly excessive relative to iron ions in the premixed solution, stirring, filtering, and dialyzing to obtain the drug-loaded albumin-magnetite nanoparticle delivery system;

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

In some embodiments, in step (S2), the amount of albumin solution added satisfies: the molar ratio of albumin to the iron in the premix solution is 1 (110-220); can be selected as 1 (110-165).

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

In some embodiments, in step (S1), the molar ratio of drug to iron in the premix solution is ≧ 1.5 (110-220); is selected to be more than or equal to 1.5 (110-165).

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

In another aspect of the present invention, there is provided the use of the drug-loaded albumin-magnetite nanoparticle delivery system described above in the preparation of an anti-cancer drug.

The embodiment proves that the drug delivery system with the structure can effectively integrate the advantages of the drug delivery system based on inorganic nanoparticles and the drug delivery system based on protein carriers in the prior art, further improve the drug loading stability of the drug delivery system, effectively reduce drug leakage in the drug delivery process, further prolong the half-life period of the drug in blood circulation, facilitate the drug release of the drug delivery system in lysosomes, permeate and accumulate the drug in tumor regions, and further realize better disease (such as cancer) treatment effect on the basis of further improving the biocompatibility and biological safety of the drug delivery system and further improving the drug loading efficiency of the drug delivery system. 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 invention has the advantage of simple operation, can improve the synthesis efficiency of the drug delivery system, and is beneficial to the application of the drug delivery system in the research and development and delivery of drugs in the field of nano medicine.

Drawings

FIG. 1 is a representation of Dox @ BMNT synthesized in example 1 under different FR conditions, (a-e) SEC analysis results; (f-j) and (k-o) are negatively stained TEM and TEM images of Dox @ BMNT synthesized under different FR conditions, respectively (with inset 50-fold magnified HRTEM image); (p) size distributions of the protein cages and magnetite from negative staining TEM and HRTEM images; (q) HD and zeta potentials for Dox @ BMNT; (r) Dox load efficiency under different FR conditions.

FIG. 2 is a model structural analysis of Dox @ BMNT, (a) negative staining TEM image of Dox @ BMNT; (b) two-dimensional structural features of 4 main classes of Dox @ BMNT; (c) Structural model and surface charge distribution of Dox @ BMNT at different viewing angles; (d) internal section of Dox @ BMNT model; (e) Interface information between Dox and BSA within the cage cavity, and interface interactions between BSA and magnetite.

FIG. 3 is a Dox release profile over time in serum of Dox @ BMNT obtained at different Fe to BSA molar ratios.

FIG. 4 is a representation of Dox Hcl @ BMNT, (A) and (B) negative TEM images of Dox Hcl @ BMNT; (C) is Dox. Hcl @ BMNT with cage-shaped structure in a negative staining electron microscope; (D) SEC analysis of column absorption peaks; (E) And (F) TEM and HRTEM respectively for Dox Hcl @ BMNT characterize the presence of magnetite; (G) statistics of inorganic core particle size for Dox Hcl @ BMNT; (H) And (I) Zeta potential and hydrated particle size DLS of Dox. Hcl @ BMNT, respectively.

FIG. 5 is a representation of Dox @ HMNT, (A) and (B) negative TEM images of Dox @ HMNT; (C) Counting the particle size of cage-like structure Dox @ HMNT in a negative staining electron microscope; (D) SEC analysis of column absorption peaks; (E) TEM characterization of dox @ hmnt; and (F) inorganic core particle size statistics for Dox @ HMNT.

FIG. 6 is a representation of Dox Hcl @ HMNT, (A) and (B) negative TEM images of Dox Hcl @ HMNT; (C) Dox. Hcl @ HMNT in cage structure in negative staining electron microscope; (D) SEC analysis of column absorption peaks; (E) TEM characterization of Dox Hcl @ HMNT; and (F) particle size statistics for Dox Hcl @ HMNT.

FIG. 7 is the evaluation of the in vitro controlled release and killing effect on tumor cells of Dox @ BMNT, (a-d) Dox @ BMNT release accumulation under different pH conditions; (e) Statistical analysis of total Dox release amount of Dox @ BMNT under different pH conditions within 24 h; (f) Dox release over time in serum of Dox @ BMNT; (g) And (h) cytotoxicity and IC of Dox-BSA, doxil and Dox @ BMNT, respectively, on 4T1 cell line ₅₀ The value is obtained.

FIG. 8 is a graph of the effect of Dox @ BMNT on the viability of 293T and HL7702 cells.

FIG. 9 is a cell uptake and retention assay for Dox-BSA, doxil and Dox @ BMNT, (a) distribution of Dox in cells after 2h of culture; (b) distribution of Dox in cells after 4h of drug removal; (c-d) mean fluorescence intensity of Dox in lysosomes and nuclei, respectively; (e) total fluorescence intensity of Dox in lysosomes and nuclei; (f) reduction in total fluorescence intensity after 4h of drug removal.

FIG. 10 is an in vivo MRI and antitumor efficacy of Dox @ BMNT, (a) time of antitumor treatment is indicated for intent; (b) Dynamic t 1-weighted MRI after dox @ bmnt intravenous injection followed tumor area drug distribution; (c) CNR ratio for MRI; (d) Mouse weight change during treatment and (e) tumor growth curve; (f) mouse survival; (g) post-treatment tumor photographs; (h) pre-treatment and post-treatment photographs of mice; (i) histological analysis of the tumor after treatment.

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

Detailed Description

Aiming at the defects of high biological toxicity of a drug delivery system based on nano-particles and low drug loading efficiency of a drug delivery system based on a protein carrier in the prior art, the inventor is inspired by natural biomineralization, biomimetic synthesis construction is carried out by utilizing a biological macromolecule template (such as protein) to obtain an albumin-magnetite nano-particle drug delivery system which is low in biological toxicity and high in drug loading efficiency, the drug delivery system integrates the advantages of the drug delivery system based on inorganic nano-particles and the drug delivery system based on the protein carrier, on one hand, the drug loading efficiency can be improved based on the high specific surface area of the inorganic nano-particles, and drugs are stably assembled on the surfaces of the nano-particles (for example, the drugs are chelated with metal ions on the surfaces of the nano-particles through chemical bonds); on the other hand, the outer layer of the drug delivery system is protein with a cage-shaped structure, has good biocompatibility and biodegradability, can reduce or eliminate immunogenicity and side effects of the drug delivery system, and on the other hand, can further coat nanoparticles coated with drugs, reduces exposure of the drugs and improves the stability of the whole drug delivery system; more importantly, the special structure of the drug delivery system (namely, the drug is loaded on the surface of the magnetite nanoparticle in the cavity of the protein nanocage) provided by the invention can effectively solve the leakage problem of the drug small molecules in the delivery process, further prolong the half life of the drug small molecules in the blood circulation, and further facilitate the drug delivery system to release the drug in lysosomes and permeate and accumulate the drug in the tumor area. The invention also provides a preparation method of the drug delivery system.

The invention is further illustrated by the following examples. It should be understood that the specific examples are intended to be illustrative of the invention and are not intended to limit the scope of the invention.

The methods used in the following examples are conventional methods unless otherwise specified. The specific steps can be seen in: a Molecular Cloning Laboratory Manual (Molecular Cloning: A Laboratory Manual, sambrook, J., russell, david W., molecular Cloning: A Laboratory Manual,3rd edition,2001, NY, cold Spring Harbor).

The various biological materials described in the examples are obtained by way of experimental acquisition for the purposes of this disclosure and should not be construed as limiting the source of the biological material of the invention. In fact, the sources of the biological materials used are wide and any biological material that can be obtained without violating the law and ethics can be used instead as suggested in the examples. The test materials used in the following examples are all conventional biochemical reagents, and are commercially available, unless otherwise specified.

The materials used in the following examples were obtained by the following route: bovine Serum Albumin (BSA), human Serum Albumin (HSA), feSO ₄ ·7H ₂ O、FeCl ₃ ·6H ₂ O, doxorubicin (Dox), phosphate Buffered Saline (PBS), dimethyl sulfoxide (DMSO), and NaOH were purchased from Sigma-Aldrich (USA); cell counting kit-8 (CCK-8) was purchased from Beijing Sun Biotechnology, inc.; DAPI, lysosome-tracker Green fluorescent staining solution, hematoxylin and eosin (H)&E) And paraformaldehyde (4%) purchased from Biotechnology (Shanghai, china); doxil hydrochloride liposomes (Doxil) were purchased from Sequus pharmaceutical tiaks (USA).

The synthesis method of Dox-BSA used in the following examples was: dox (4 mg) was dissolved in DMSO, followed by the addition of BSA (10 mg), 37 deg.CStirring in the dark for 12 hours (N) ₂ Under the protection of atmosphere). The formed Dox-loaded BSA (Dox-BSA) was collected and filtered through a 0.22 μm filter and finally dialyzed against PBS.

Example 1: preparation and characterization of drug-loaded albumin-magnetite nanoparticle delivery systems

This example prepares a Dox (a hydrophobic chemotherapeutic drug) loaded Bovine Serum Albumin (BSA) -magnetite (Fe) ₃ O ₄ ) The nanoparticle delivery system (named dox @ bmnt) was characterized and includes the following steps.

1.1 dissolving Dox (0 mg, 0.25mg, 0.5mg, 1mg, or 2 mg) in DMSO in the dark with a solution of iron salt (FeSO) ₄ ·7H ₂ O and FeCl ₃ ·6H ₂ O preparation of a solution of an iron salt, in which Fe ³⁺ /Fe ²⁺ Can be (1.5-2.5): 1, provided that the main part of the nanoparticles resulting from this final (after addition of the NaOH solution in step 1.2) is Fe ₃ O ₄ That is, fe is specifically used in this embodiment ³⁺ /Fe ²⁺ =2/1, nanoparticles obtained after dropping NaOH solution in the following step 1.2 were magnetite (Fe) ₃ O ₄ ) Nanoparticles) pre-mix (where the molar ratio of Dox to iron is: 0/165, 0.75/165, 1.5/165, 3/165, and 6/165) to obtain premixed solutions containing varying amounts of Dox. The process is in N ₂ The atmosphere is protected, and the reaction is carried out under the stirring of a water bath at the temperature of about 37 ℃ (for example, 37 +/-2 ℃).

1.2, bovine Serum Albumin (BSA) solution is added to the different premixed solutions obtained in step 1.1 respectively at a BSA to iron molar ratio of 1/165, so that the feed ratios (molar ratio, FR) of Dox to BSA are 0/1, 0.75/1, 1.5/1, 3/1, and 6/1, respectively (corresponding to the premixed solutions containing different amounts of Dox). A NaOH solution (100 mM) in slight excess with respect to iron ions was then slowly added dropwise (in which Fe was dissolved by chemical equation 2) ³⁺ +Fe ²⁺ +8OH ^- ＝Fe ₃ O ₄ +4H ₂ And calculating the balanced addition amount of NaOH) by using O, stirring for about 3 hours, and collecting products respectively. The process is carried out at N ₂ The atmosphere is protected, and the reaction is carried out under the stirring of a water bath at the temperature of about 37 ℃ (for example, 37 +/-2 ℃).

1.3, filtering the product obtained in step 1.2 through a 0.22 μm filter membrane, and finally dialyzing the product with PBS to obtain BSA-magnetite nanoparticle delivery systems synthesized under different FR ratio conditions, which are respectively named as 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, dox @ BMNT loaded with different amounts of Dox obtained in step 1.3 above were dispersed in PBS first through a concentration tube (100K, merck Millipore, USA), and then loaded on a Superose 6 Increate 10/300GL column (GE Healthcare), with UV absorption monitoring at 280nm and 480nm, respectively. As shown in a-e in FIG. 1, the SEC analysis results of Dox @ BMNT synthesized under different FR ratio conditions, wherein 280nm is specific absorption peak (higher peak) of BSA nanocage, 480nm is specific absorption peak (lower peak) of Dox, and it can be seen that Dox @ BMNT synthesized under different FR conditions all elute 280nm absorption peak with volume of about 12.8ml, indicating formation of BSA nanocage, and with increase of Dox in the synthesis system, the absorption at 480nm increases, indicating successful Dox loading.

(2) Further characterization of Dox @ BMNTs synthesized under different FR (0/1, 0.75/1, 1.5/1, 3/1, and 6/1) conditions with transmission electron microscopy (TEM, JEM 2100) and high resolution transmission electron microscopy (HRTEM, JEM 2100) resulted in the f-o in FIG. 1, where f-j is a negatively stained TEM image and k-o is a TEM image (the inset in the TEM image is a 50-fold magnified HRTEM image), showing that BSA protein mineralized to form a well-defined cage-like nanostructure (i.e., nanocage with cavity) in Dox @ BMNTs synthesized under different FR conditions and the inorganic core particles in the nanocage cavity exhibit small size and homogeneity (k-o in FIG. 1) after negative staining with uranium acetate, which can be attributed to the spatial limitation and homogeneity of the BSA nanocage. In addition, the HRTEM image of k-o in FIG. 1 shows 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 the magnetite (311) planeIt shows that magnetite nanoparticles are formed in the nanocage cavities.

(3) The sizes of the nanocages formed by BSA protein and the magnetite nanoparticles in Dox @ BMNT synthesized under different FR conditions were calculated based on TEM images of transmission electron microscopy, and as a result, as shown by p in FIG. 1, it can be seen that although different charge ratios FR of Dox and BSA are used during the reaction process, the finally synthesized Dox @ BMNT shows 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, the Hydrodynamic Diameter (HD) and zeta potential of the synthesized Dox @ BMNT are also detected by a dynamic light scattering instrument (Malvern Zetasizer Nano ZS, UK), and as a result, as shown by q in FIG. 1, the HD and zeta of the synthesized Dox @ BMNT under different FR conditions have good consistency, especially the HD (in the range of 19-22 nm) does not change by more than 10%, and the synthesized Dox @ BMNT shows higher homogeneity and applicability. In contrast, dox-BSA showed larger size (on average about 35.3 nm) and poorer uniformity (varying over the interval of about 15-70 nm), mainly due to the lack of steric confinement of the nanocages. This shows that in the synthesized Dox @ BMNT, dox does not participate in forming the BSA nano cage, but is combined with Fe ions and loaded into the cavity of the nano cage through magnetite mineralization, and the bionic synthesis method for regulating biomineralization based on the BSA nano cage has universal applicability. From the results shown by q in FIG. 1, it is also understood that the zeta potential of Dox @ BMNT synthesized under different FR conditions is about-27 mV (about-24 to-31 mV) (whereas the zeta potential of Dox-BSA is about-12.8 mV), and it is considered that Dox @ BMNT has sufficient charge repulsion in aqueous solution to be stable, and thus it contributes to the improvement of the biosafety and stability of Dox @ BMNT.

(5) For Dox @ BMNTs synthesized under different FR conditions, the Dox loading efficiency was also measured (for Dox @ BMNTs synthesized under different FR conditions, based on SEC analysis results (absorption peaks at 280nm and 480 nm) shown in a-e in FIG. 1, the concentration of Dox was measured with a fluorescence spectrophotometer and the concentration of BSA was measured with a BCA protein assay kit (Thermo, USA), respectively, thereby calculating the Dox loading efficiency), and as shown by r in FIG. 1, it was found that the Dox loading efficiency significantly increased with the increase of FR of BSA by Dox and finally became saturated when FR was 3/1. The fitted curve shows that when the loaded Dox reaches saturation, the molar ratio of Dox to BSA in the synthesized Dox @ bmnt is about 12, i.e. the loading of 12 Dox molecules per BSA protein on average is involved. Therefore, when Dox @ BMNT is prepared, the feeding ratio of Dox to BSA is preferably controlled to be more than 1.5/1, preferably more than 3/1, so that the loading efficiency of Dox can be improved; for other drugs (e.g., anti-cancer chemotherapeutic drugs such as doxorubicin hydrochloride, fluorouracil, mitomycin, actinomycin, pingyangmycin, cisplatin, carboplatin, epirubicin, methotrexate, and cytarabine) and proteins (e.g., human Serum Albumin (HSA)), one skilled in the art can determine this by the method of this example.

1.5 structural analysis of Dox @ BMNT:

to determine the structure of Dox @ BMNT, dox @ BMNT (FR: 3/1) after SEC purification was negatively stained with uranium acetate and studied under TEM as shown in FIG. 2 a. 10000 particles were then selected from a set of micrographs and an iterative reference-free two-dimensional alignment and classification procedure (van Heel, et al. A new generation of the image processing system.j.struct.biol.116,17-24 (1996)) was performed using IMAGIC, where the alignment and classification process was iterated 15 times to converge to the final class average. For each representative class mean, the particles are randomly assigned to two equal-sized sets, the class mean is calculated, and the Fourier-loop correlation between the two class means is determined. The final 4 main classes are visualized and divided by overall size and shape, as shown by b in fig. 2. For each class, a typical two-dimensional average is established, summarizing the main features of the cage structure and graphically representing them. As shown in FIG. 2 b (different viewing directions, scale: 20 nm), it can be seen that class 1 and class 2 have more complex features, such as near-elliptical and thicker protein shells, while class 3 and class 4 exhibit more pronounced cage-like structures, near-circular and thinner shells.

To understand the basic structural properties of Dox @ BMNT, the proposed model was derived by MD simulation and computational modeling (based on the Amber 14 program) (all MD simulations were performed on a linux (Ubuntu 14.04) based GPU workstation) with three-dimensional (3D) knots of BSAConstructs (PDB ID:4F 5S) were downloaded from the miv RCSB protein database (www.rcsb.org). The results of MD simulation and computational modeling are shown in FIG. 2 c-e, where C in FIG. 2 shows the structure model and surface charge distribution of Dox @ BMNT (FR: 3/1) at different viewing angles, and it can be seen that 6 BSA subunits are interwoven with each other to form a complex sphere and are modeled with magnetite and Dox molecules located in the cavity of the complex sphere to form a "torpedo" topology. Inside this "torpedo" topology, 1 magnetite nanoparticle is bound to 72 Dox molecules (on average each BSA protein is involved in the loading of 12 Dox molecules) and is stabilized by a "torpedo" shell that is self-assembled by BSA proteins. On the surface, a large number of hydrophilic residues are randomly exposed on the surface of the torpedo, so that the interaction of the torpedo and water is greatly promoted, and the stability of the torpedo in an aqueous solution is improved. The structure inside the Dox @ BMNT (FR: 3/1) model is shown as d in FIG. 2. It can be seen that the Dox molecules are entangled with the magnetite particles and are co-buried in hydrophilic anion pockets (i.e. the cavities of the nanocages) with a diameter of about 4.2 nm. The interfacial information between Dox and BSA within the nanocage cavity, and the interfacial interactions between BSA and magnetite, are shown in fig. 2 at e, where it can be seen that the magnetite particles are tightly bound in the hydrophilic anionic pockets with the residues Arg81, glu82, asp86, asp89 and Glu92 of BSA, while the aryl groups of Dox form cation-pi interactions with the Arg208 and Lys350 residues of BSA. Importantly, in Dox and residue Arg208 (bond length:

and

) And Glu478 (bond length:

) 4 hydrogen bonding interactions were observed, which is the main interfacial interaction 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 these interactions contribute to the anchoring of Dox at the cavity binding site. The above structural analysis strongly provesThe encapsulation stability of Dox provides 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 controlled release effect of drug-loaded albumin-magnetite nanoparticle drug delivery system in serum

This example 2 was conducted in accordance with the procedure for preparing the drug-loaded albumin-magnetite nanoparticle drug delivery system of example 1, except that: (1) 1mg of Dox was dissolved in DMSO in step 1.1; (2) In step 1.2, the molar ratio of Fe to BSA is controlled to be 110/1, 220/1, 330/1 or 82.5/1 respectively, FR is controlled to be 3/1, and Dox @ BMNT biomimetically mineralized and synthesized under different Fe/BSA conditions are obtained and named as: 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).

Evaluation of controlled release effect of dox @ bmnt in serum:

dox @ BMNT (Fe/BSA = 165/1) prepared in example 1 (i.e., dox @ BMNT (FR: 3/1) prepared in example 1), and 4 kinds of Dox @ BMNTs (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)) prepared in example 2 were placed in dialysis cups (MW cut-off 14kDa, novagen), and serum was added thereto for incubation, respectively, and gently stirred. After a period of incubation (about 240 h), the incubation was extracted and centrifuged at 8000rpm for 5min. The stability of free Dox released at different incubation times in serum was determined by high performance liquid chromatography.

As shown in fig. 3, it can be seen that, compared to the greater leakage in serum of Dox @ bmnt (Fe/BSA = 330/1) and Dox @ bmnt (Fe/BSA = 82.5/1), dox @ bmnt (Fe/BSA = 110/1), dox @ bmnt (Fe/BSA = 165/1) and Dox @ bmnt (Fe/BSA = 220/1) showed better stability in serum, almost no leakage was observed for 120 hours in serum (Dox release is less than about 15%), especially Dox @ bmnt (Fe/BSA = 110/1) and Dox @ bmnt (Fe/BSA = 165/1) showed better stability in serum, almost no leakage was observed for 120 hours in serum (Dox release is less than about 10%), even if Dox release was 240 hours, dox release was less than about 20%, indicating that in the preparation of Dox bmnt, when the molar ratio of Fe to BSA is 110/165/1, the drug loading ratio of Dox @ bmnt is increased within the range of 110/1, preferably 220/1.

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

This example a Bovine Serum Albumin (BSA) -magnetite nanoparticle delivery system (named Dox Hcl @ bmnt) loaded with Dox Hcl, a hydrophilic chemotherapeutic, was prepared and characterized as described in example 1. 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, except that: (1) 1mg/ml (in Dox) Dox Hcl was dissolved in sterile water in step 1.1; (2) In step 1.2, the molar ratio of BSA to Fe was controlled to 1/165, and FR (molar ratio of Dox to BSA) was controlled to 3/1, thereby obtaining Dox. Hcl @ BMNT.

The characterization results of the Dox-Hcl @ BMNT are shown in A-I in FIG. 4, and A-B in FIG. 4 are uranium acetate negative staining TEM images of the Dox-Hcl @ BMNT at different resolutions; c in FIG. 4 shows nanocage structures in negatively stained TEM images, indicating that the BSA protein in Dox. Hcl @ BMNT also mineralizes to form well-defined caged nanostructures, which were measured to have a size of about 18.5nm and a diameter of the nanocage cavity of about 4-12nm; in FIG. 4D shows the 280nm absorption peak at an elution volume of about 11.7ml based on SEC, indicating the formation of BSA nanocages; in FIG. 4, TEM image and high resolution HRTEM image (Fourier transform data in the upper left-hand insert) of Dox Hcl @ BMNT with E-F as shown, it can be seen that the lattice spacing of the core nanoparticles is about

(F in fig. 4), corresponding to the standard lattice constant of the magnetite (311) plane, indicating the formation of magnetite nanoparticles in the nanocages; the statistical results of the particle size of the magnetite nanoparticles formed in the nanocages are shown as G in fig. 4, and it can be seen that the magnetite nanoparticles have a particle size distribution in the range of 1.5 to 5nm, an average particle size of about 3.07 ± 1.27nm, exhibit small size and uniformity; H-I in FIG. 4 shows the Zeta potential (H in FIG. 4) and hydrodynamic diameter HD (H in FIG. 4) of Dox. Hcl @ BMNT, respectivelyI) The detection result of (1).

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

This example a Dox-loaded Human Serum Albumin (HSA) -magnetite nanoparticle delivery system (named Dox @ HMNT) was prepared and characterized as described in example 1. 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, except that: (1) 1mg of Dox was dissolved in DMSO in step 1.1; (2) In step 1.2, dox @ HMNT is obtained by controlling the molar ratio of HSA to Fe to 1/165 and FR (herein, the charge ratio (molar ratio) of Dox to HSA) to 3/1.

In this example, similar to the r results in fig. 1, as Dox increases for FR of BSA, the loading efficiency of Dox increases significantly and eventually saturates when FR is 3/1. Therefore, in the preparation of Dox @ HMNT, the charge ratio of Dox to HSA is preferably controlled to be 1.5/1 or more, preferably 3/1 or more, so that the Dox loading efficiency can be improved. In addition, similar to the results of FIG. 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.

Characterization of Dox @ hmnt results are shown in fig. 5 a-F, and fig. 5 a-B are uranium acetate negative TEM images of Dox @ hmnt at different resolutions, and it can be seen that similarly to BSA in the Dox @ bmnt structure prepared in example 1 above, HSA protein is also mineralized to form a definite cage-like nanostructure (similar to BSA, 6 HSA subunits are interwoven with each other to form a cage-like nanostructure, in the cavity of which 1 magnetite nanoparticle is bound to 72 Dox molecules (each HSA protein participates in the loading of 12 Dox molecules on average) and the shell self-assembled by HSA protein is stable); c in FIG. 5 shows the statistical results of the particle size of the nanocage structure of Dox @ HMNT, which indicates that the particle size of the nanocage structure of Dox @ HMNT is in the range of 13-23nm and the average particle size is about 17.3 + -1.7 nm; in FIG. 5D shows the 280nm absorption peak (higher peak) based on SEC with an elution volume of about 11.6ml, indicating the formation of HSA nanocages; e in FIG. 5 is a TEM representation of Dox @ HMNT; FIG. 5, F shows the statistics of the particle size of the inorganic core particles in the cavities of the nanocage structure of Dox @ HMNT, and it can be seen that the particle size of the inorganic core particles is in the range of 1.5-5nm, the average particle size is about 2.9 + -1.0 nm, and small size and uniformity are exhibited.

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

This example a Human Serum Albumin (HSA) -magnetite nanoparticle delivery system loaded with Dox Hcl (named Dox Hcl @ hmnt) was prepared and characterized as described in example 1. 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, except that: (1) 1mg/ml Dox · Hcl was dissolved in sterile water in step 1.1; (2) In step 1.2, the molar ratio of HSA to Fe is controlled to 1/165, and FR (molar ratio of Dox to HSA) is controlled to 3/1, thereby obtaining Dox Hcl @ HMNT.

The characterization results of Dox Hcl @ HMNT are shown in 6A-F in the figure, and A-B in the figure 6 are uranium acetate negative dye TEM images of Dox Hcl @ HMNT under different resolutions; c in FIG. 6 shows nanocage structures in negatively stained TEM images, indicating that HSA protein in Dox. Hcl @ HMNT is also mineralized to form well-defined caged nanostructures, which were measured to have a size distribution in the range of 13-23nm, an average particle size of about 17.2 + -1.6 nm (as shown by F in FIG. 6), and a diameter of the nanocage cavity of about 4-10nm; FIG. 6, panel D shows the 280nm absorption peak (upper peak) at an SEC-based elution volume of about 12.6ml, indicating formation of HSA nanocages, and the peak at 480nm (lower peak) indicating successful Dox loading; in FIG. 6, E is a TEM image of Dox Hcl @ HMNT.

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

This example evaluates the in vitro drug controlled release and drug efficacy of various drug-loaded albumin-magnetite nanoparticle delivery systems prepared in examples 1-5 above (including Dox @ bmnt (FR: 1.5/1), dox @ bmnt (FR: 3/1) and Dox @ bmnt (FR: 6/1) prepared in example 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, dox @hmntprepared in example 5, dox · hcl @ hmnt prepared in example 5), and specifically describes the evaluation methods and results with Dox @ bmnt (FR: 3/1) prepared in example 1 as an example.

(1) Determination of Dox @ BMNT Dox release in PBS buffer at various pH: dox-BSA, doxil and Dox @ BMNT were placed in dialysis cups (MW cut-off 14kDa, novagen) and PBS buffers of different pH (pH 4.5 (mimicking lysosomes), pH 5.5 (mimicking cytoplasms), pH 6.5 (mimicking tumor microenvironment) and pH 7.5 (mimicking plasma)) were added, respectively, and gently stirred. After a period of incubation (about 25 h), the incubation was extracted and centrifuged at 8000rpm for 5min. The concentration of Dox in the supernatant was measured with a fluorescence spectrophotometer.

The results of the assay are shown in a-d in FIG. 7 (the top-down curves represent Dox-BSA, doxil and Dox @ BMNT, respectively), and it can be seen that the three delivery systems tested (Dox-BSA, doxil and Dox @ BMNT) all exhibited cumulative release of Dox at different pH conditions, where the Dox-BSA and commercial Doxil groups were significantly leaky at pH 7.5 and pH 6.5, especially the Dox-BSA group, which released about 58.7% of Dox at pH 6.5 and about 78% of Dox at pH 5.5, which may be associated with unstable non-specific interaction between Dox and BSA. The next is Doxil, which releases more than half of the Dox at pH 5.5, whereas at pH 7.5, pH 6.5 and pH 5.5 most of Dox @ BMNT remain stable with at most only no more than 20% Dox release (c in FIG. 7), and do @ BMNT does not finally reach a higher release (d in FIG. 7) until the pH drops to 4.5. The results of statistics of the cumulative release of Dox in the above three delivery systems are shown as e in fig. 7 (bars from left to right at each pH represent Dox-BSA, doxil and Dox @ bmnt, respectively), and it can be seen that Dox @ bmnt exhibits good stability despite the increased acidity of the solution (pH decreased from 7.5 to 5.5) and the Dox leakage is significantly lower than the Dox-BSA, doxil group. Finally, the Dox @ bmnt group is released in a low pH environment of 4.5, thus facilitating the circulating presence of the drug Dox in plasma, tumor microenvironment and cytoplasm, and facilitating the delivery and release of the drug Dox in lysosomes.

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

The results are shown in f in FIG. 7 (the curves from top to bottom represent Dox @ BMNT, doxil and Dox-BSA, respectively), and it can be seen that Dox @ BMNT shows better stability in serum, and almost no leakage is observed after 120 hours of incubation in serum, while Dox-BSA and Doxil show more leakage.

The results of (1) and (2) above indicate that Dox @ bmnt has more excellent stability characteristics relative to Dox-BSA and Doxil, which may be associated with synergy of various factors, such as surface chelation of magnetite nanoparticles with Dox, and interfacial binding of BSA and Dox. In addition, effective encapsulation of the BSA nanocages may also reduce Dox exposure and dissociation, helping to improve stability and thus extend their half-life in blood circulation.

(3) In vitro killing effect of dox @ bmnt on tumor cells: 2 tumor cell lines (4T 1 and SKBR 3) were selected in this study. Wherein 4T1 cells are cultured in DMEM (Dulbecco's modified Eagle Medium) containing 10% Fetal Bovine Serum (FBS) and 1% antibiotic solution in adherent manner, SKBR3 cells are cultured in DMEM/F12 (Ham's F-12 nutrient Medium) containing 20% Fetal Bovine Serum (FBS) and 1% antibiotic solution in adherent manner, the temperature is 37 ℃, and the concentration is 5% CO ₂ 。

4T1 and SKBR3 cells were incubated for 24h in medium containing Dox-BSA, doxil and Dox @ BMNT at a concentration of 10 Dox in each medium ^-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 μ g/mL. The cell viability of each sample was then tested using the CCK-8 kit. There were 5 parallel samples for each test.

Exemplary results of the cytotoxicity and killing effect assays performed on the 4T1 tumor cell lines after 24 hours of incubation are shown in g-h in FIG. 7 (where: p. Ltoreq.0.05, p. Ltoreq.0.01) (in FIG. 7, the curves from left to right in g respectively represent Dox @ BMNT, doxil and Dox-BSA), it can be seen that Dox @ BMNT exhibits strong cytotoxicity against 4T1 tumor cell line, has strong killing effect, and its IC ₅₀ The value was about 0.5. Mu.g/mL, significantly lower than Dox-BSA (about 2.5. Mu.g/mL), doxil (about 1.1. Mu.g/mL). The killing result of the Dox @ BMNT on the SKBR3 cell line is consistent with that of the 4T1 tumor cell line, and the killing effect on the tumor cells is very good.

(4) Effects of dox @ bmnt on normal cells: in this study, 2 normal cell lines (293T and HL 7702) were selected, wherein the 293T and HL7702 cells were cultured in DMEM (Dulbecco's Modified Eagle Medium) containing 10% Fetal Bovine Serum (FBS) and 1% antibiotic solution, adherent thereto, at 37 deg.C and 5% CO ₂ 。

293T and HL7702 cells were incubated overnight in medium containing Dox @ BMNT (Dox concentrations 0, 0.5, 1.0, 2.0, 5.0, 10.0. Mu.g/mL, respectively). Cell activity was measured using the CCK-8 kit. There were 5 parallel samples for each test.

The results of the assay are shown in fig. 8 (where the left-to-right bars for each Dox concentration indicate 293T and HL7702, respectively), it can be seen that the survival rates of normal cell lines (293T and HL 7702) treated with Dox @ bmnt incubation were all greater than 90%, indicating that Dox @ bmnt exhibits low toxicity to normal cells and thus has good biosafety performance when using the Dox @ bmnt to deliver drugs.

The drug-loaded albumin-magnetite nanoparticle delivery systems prepared in examples 1-5 above all also have in vitro controlled drug release and tumor cell killing effects similar to Dox @ BMNT (FR: 3/1), and are safe for normal cell lines, and will not be described in detail.

Example 7: cellular uptake and retention evaluation of drug-loaded albumin-magnetite nanoparticle drug delivery systems

This example evaluates the cellular uptake and retention of various drug-loaded albumin-magnetite nanoparticle delivery systems prepared in examples 1-5 above (including Dox @ BMNT (FR: 1.5/1), dox @ BMNT (FR: 3/1) and Dox @ BMNT (FR: 6/1) prepared in example 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, dox @ HMNT prepared in example 5, and Dox Hcl @ HMNT prepared in example 1, and specifically describes the evaluation methods and results with Dox @ BMNT (FR: 3/1) prepared in example 1 as an example.

The 4T1 cells were treated with 10 ⁵ The density of individual cells/well was seeded in six-well plates, and when the degree of cell confluence reached around 70%, the original medium was replaced with fresh medium containing Dox @ BMNT (Dox concentration 5. Mu.g/ml) with Dox-BSA and Doxil as controls. After 2 hours of incubation, the culture medium was removed and the cells were washed 3 times with PBS. Evaluation of cellular uptake: cells were incubated with DAPI and lysosome-tracker Green staining solution for 15min and then visualized with CLSM (Zesis, germany). Cell retention evaluation: cells were incubated in fresh medium for 4h, incubated with DAPI and lysosome-tracker Green stain for 15min, and observed with CLSM (Zesis, germany).

The results are shown in FIGS. 9 a-f, where a in FIG. 9 is the distribution of Dox in the cells after 2h incubation, b in FIG. 9 is the distribution of Dox in the cells after 4h removal of the drug, a-b in FIG. 9, white arrows indicate the nucleus, gray arrows indicate the lysosome, c in FIG. 9 indicates the mean fluorescence intensity of Dox in the lysosome, d in FIG. 9 indicates the mean fluorescence intensity of Dox in the nucleus, e in FIG. 9 indicates the total fluorescence intensity of Dox in the lysosome and the nucleus, and f in FIG. 9 indicates the decrease in the total fluorescence intensity after 4h removal of the drug, where p.ltoreq.0.05, and p.ltoreq.0.01. As shown by the results of a-b in FIG. 9, it can be seen that most of Dox from Dox @ BMNT is captured by lysosomes, while Dox from the other two groups (Dox-BSA and Doxil) is mainly distributed in the nucleus, and the results of detecting the retained concentration of Dox 4h after removal of the drug (i.e. after 6h of initial incubation) show that: compared with Dox-BSA and Doxil, the Dox @ BMNT group still expressed higher fluorescence intensity and accumulated extensively in the nucleus, producing intense fluorescence. As shown by the results of quantitative brightness of c-f in 9, after 2h incubation, the lysosome was most preferred to Dox @ BMNT because its Mean Fluorescence Intensity (MFI) was 50.3a.u, which is 2.5-fold and 25-fold higher than that of Dox-BSA and Doxil groups, respectively. Although the uptake of Dox (MFI: 10.7a.u) by the nuclei of cells after 2h incubation was comparable to that of Dox @ BSA and Doxil, dox @ BMNT was significantly increased after 4h drug removal with an MFI of 41.4a.u. Given that a certain amount of Dox is still present in lysosomes and transported into the nucleus, high Dox levels will persist in the nucleus over time. It is noteworthy that despite the different intracellular distribution, the Dox uptake was very similar in all three groups, but the MFI was better maintained (56.8a.u) only in the Dox @ bmnt group after 4h drug removal, compared to the overall MFI (Dox-BSA MFI:7.1a.u and Doxil MFI: 16.3a.u) in the other two groups, which was significantly lower than the Dox @ bmnt, indicating that both groups had poor Dox retention. The longer Dox @ BMNT retention time is, the more favorable the drug accumulation in the tumor area.

The drug-loaded albumin-magnetite nanoparticle delivery systems prepared in examples 1-5 above also all had similar cellular uptake and retention effects as Dox @ BMNT (FR: 3/1), and are not described in detail herein.

Example 8: in vivo anti-tumor activity of drug-loaded albumin-magnetite nanoparticle delivery systems

8.1 establishment of tumor mouse model

The female BALB/c nude mice used in this study were purchased from Nanjing Jieke technology, inc., china, and had an average body weight of 20g. Will contain 1X 10 ⁶ A suspension of 4T1 cells was inoculated into the right leg of the mouse to construct a tumor model mouse. Accurately monitoring the tumor volume by using a vernier caliper, wherein the tumor volume is calculated by the formula V = LW ² L and W are tumor length and width, respectively. All mice were cared for according to the guidelines outlined in the laboratory animal Care and use guide.

8.2 in vivo antitumor Activity of Dox @ BMNT

All tumor model mice constructed in the above step 8.1 were randomly divided into 4 groups of 5 mice each, into PBS group, dox-BSA group, doxil group and Dox @ BMNT group. When the tumor volume in the tumor model mouse reaches 150mm ³ In time, mice were injected intravenously with groups of drug samples (5 mg/kg equivalent Dox concentration) three times every three days. Tumor size and body weight were recorded on

days

3, 6, 9, 12, 15, 18, 21 after the first injection (day 0). Mice were sacrificed on day 21 and treatment effect was evaluated by comparing the dox @ bmnt group with 3 control groups.

8.3 magnetic resonance imaging

In vivo MRI, mouse bodies were placed in a 14.1T NMR micro imaging system (Bruker Biospec, karlsruhe, germany) and MRI studies were performed at different treatment times. t 1-weighted MRI images were acquired using a Rapid Acquisition Relaxation Enhancement (RARE) sequence with the following parameters: TR/TE =3500/10ms, field of view (FOV) =25mm × 25mm, matrix =256 × 256, slice thickness =0.5mm, average number =2. The CNR calculation formula in MRI images is: CNR = (SI tumour-SI mouse)/SI noise, where SI represents signal intensity and noise is from air background.

In vivo MRI and anti-tumor efficacy results of Dox @ BMNT are shown as a-i in FIG. 10, a in FIG. 10 is a schematic time line of anti-tumor treatment of tumor model mice, b in FIG. 10 is dynamic t1 weighted MRI after Dox @ BMNT is intravenously injected to model mice, drug distribution in tumor regions is tracked, c in FIG. 10 is CNR ratio corresponding to MRI, d-e in FIG. 10 are weight change and tumor growth curve of model mice, respectively, f in FIG. 10 is mean volume of tumor after treatment of model mice, g in FIG. 10 is photograph of tumor after treatment of model mice, h in FIG. 10 is photograph before and after treatment of model mice, i in FIG. 10 is histological analysis result of tumor tissue after treatment of model mice, wherein the scale: 100 μm, p is less than or equal to 0.01.

As shown in the results b-c in FIG. 10, the T1 signal intensity in the tumor region of the model mouse was significantly improved 30min after the injection of the drug Dox @ BMNT, and reached the maximum level 4h after the injection, at which time the CNR ratio corresponding to MRI was about 10.9, which is about 9 times of the initial noise. The continuous high contrast effect obviously improves the resolution and sensitivity of magnetic resonance imaging, and can accurately reflect the distribution and accumulation of Dox @ BMNT after being injected into the tumor region.

From the results d in FIG. 10, it can be seen that the body weight of the model mice was increased in all the treatment groups, and there was no significant difference. However, compared with the other three groups, the tumor treatment effect was the best in the Dox @ BMNT group, which significantly inhibited the tumor growth in the model mice (e in FIG. 10 (where the curves from top to bottom represent the PBS group, the Dox-BSA group, the Doxil group, and the Dox @ BMNT group, in that order)). Statistics of tumor volumes for model mice shown as f in FIG. 10 show that the mean tumor volume of the Dox @ BMNT group 21 days after the first dose was 338.1mm ³ Much smaller than the PBS group (1649.5 mm) ³ ) Dox-BSA group (1271.3 mm) ³ ) And Doxil group (893.1 mm) ³ ). Such significant therapeutic effect can also be directly observed from g-h in fig. 10.

From the histological analysis results shown in i in FIG. 10, hematoxylin and eosin (H & E) staining revealed that the tumor cell density was lower and more apoptotic cells were present in the Dox @ BMNT treated group than in the other groups. In particular, tumors treated with dox @ bmnt also showed fewer Ki 67-positive cells, more TUNEL-positive cells, and extensive DNA degradation and fragmentation. As Dox @ BMNT is preserved in the tumor area for a long time, the medicine is widely accumulated in the tumor area, and can effectively inhibit cell proliferation and induce cell apoptosis, thereby obtaining good tumor treatment effect.

The drug-loaded albumin-magnetite nanoparticle delivery systems prepared in examples 1-5 above also all had similar in vivo anti-tumor effects as Dox @ BMNT (FR: 3/1), and are not further described herein.

Example 9 in vivo biocompatibility of drug-loaded Albumin-Magnetite nanoparticle delivery System

This example evaluates the in vivo biocompatibility of the various drug-loaded albumin-magnetite nanoparticle delivery systems prepared in examples 1-5 above (including Dox @ BMNT (FR: 1.5/1), dox @ BMNT (FR: 3/1) and Dox @ BMNT (FR: 6/1) prepared in example 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, dox @ HMNT prepared in example 5, and Dox Hcl @ HMNT (FR: 3/1) prepared in example 1, and specifically describes the evaluation methods and results with the Dox @ BMNT (FR: 3/1) prepared in example 1 as an example.

9.1 renal clearance analysis

Urine samples of model mice injected with Dox @ BMNT in example 8 above were collected at various times to determine the renal clearance of Dox @ BMNT, and the control group was PBS injection. In which the iron concentration of each sample was measured by ICP-MS (Thermo, USA) and nanoparticles in urine were observed by TEM (JEM 2100).

9.2 histopathological and blood Biochemical examinations

Major organ (heart, liver, spleen, lung and kidney) and serum samples of mice treated with dox @ bmnt in example 8 above were collected, and PBS-injected mice were used as controls. Wherein the serum specimen is used for blood biochemical analysis, the mouse organ is fixed by 4% paraformaldehyde for 24h, and the section is embedded by paraffin. Paraffin-embedded sections were then stained with hematoxylin and eosin (H & E) to assess the extent of injury.

The results are shown in a-m in FIG. 11, where a in FIG. 11 is the iron content in urine samples collected at different times after the injection of Dox @ BMNT and PBS in model mice (where the left-to-right bars represent the results for PBS and Dox @ BMNT, respectively, for each time point), b-d in FIG. 11 are urine TEM images of 6h and 24h after the injection of Dox @ BMNT in model mice, and e-l in FIG. 11 are the results of biochemical blood analysis, 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 fig. 11 is the H & E results of the major organs of the model mice after treatment, scale bar: 100 μm.

As shown in FIG. 11 a, it can be seen that the concentration of iron in the urine of the mouse increased after the injection of Dox @ BMNT in the model mouse, reaching the maximum value at 6 h. When urine was collected, a large number of nanoparticles could be observed, which means that Dox @ BMNT was cleared by the kidney (b-c in FIG. 11). The concentration of iron in the urine then dropped to normal levels 24h after dox @ bmnt injection, with almost no particles visible in the urine (d in fig. 11). The above results indicate that dox @ bmnt prepared by the present invention can be effectively degraded and metabolized by the kidney, thus reducing the risk of toxicity and side effects. As shown by e-l in FIG. 11, the blood biochemical indicators of Dox @ BMNT and PBS injected into the model mice were not significantly different, and thus it was confirmed that Dox @ BMNT had good biosafety in blood circulation. As shown in m in FIG. 11, H & E staining analysis results of major organs show that after Dox @ BMNT is injected, no significant damage is caused to organs such as heart, liver, spleen, lung and kidney, and further, dox @ BMNT has no significant side effect on physiological functions and health conditions of mice during treatment, so that Dox @ BMNT has very stable biocompatibility and biosafety.

The drug-loaded albumin-magnetite nanoparticle delivery systems prepared in examples 1-5 above also all have in vivo biocompatibility and biosafety similar to Dox @ BMNT (FR: 3/1), and are not described in detail herein.

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art will understand that various changes, modifications and substitutions can be made without departing from the spirit and scope of the invention as defined by the appended claims. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A drug-loaded albumin-magnetite nanoparticle delivery system, comprising:

(1) A nanocage having a cavity formed by albumin self-assembly; and

(2) Magnetite nanoparticles located in the nanocage cavity;

wherein the surface of the magnetite nanoparticles is coated with a drug.

2. The drug-loaded albumin-magnetite nanoparticle delivery system according to claim 1, wherein:

the diameter of the nano cage is 16.5-20.5nm; and/or

The diameter of the magnetite nano-particles is 2.5-4.5nm; and/or

The diameter of the cavity is 4-12nm.

3. The drug-loaded albumin-magnetite nanoparticle delivery system according to claim 1, wherein within the nanocage cavity, the magnetite nanoparticles have a surface bound to the residues of albumin; optionally, the residues of albumin are selected from one or more of: arg81, glu82, asp86, asp89 and Glu92.

4. The drug-loaded albumin-magnetite nanoparticle delivery system according to claim 1, wherein within the nanocage cavity, the drug is chemically chelated to iron ions on the surface of the magnetite nanoparticles.

5. The drug-loaded albumin-magnetite nanoparticle delivery system according to claim 1, wherein within the nanocage cavity, the drug groups interact with the albumin residues through cation-pi bonds and/or hydrogen bonds; optionally, the group of the drug is selected from aryl, and the residue of albumin is selected from one or more of: arg208, lys350 and Glu478.

6. The drug-loaded albumin-magnetite nanoparticle delivery system according to claim 1, wherein within the nanocage cavity, the drug is surrounded by residues of the albumin forming a hydrophobic binding; optionally, the residues of albumin are selected from one or more of: phe205, ala209, ala212, leu480, and Val481.

7. The drug-loaded albumin-magnetite nanoparticle delivery system according to any one of claims 1-6, wherein the drug comprises an anti-cancer chemotherapeutic drug; optionally, the anti-cancer chemotherapeutic comprises a hydrophobic anti-cancer chemotherapeutic and a hydrophilic anti-cancer chemotherapeutic; further optionally, the anti-cancer chemotherapeutic is selected from one or more of: doxorubicin, doxorubicin hydrochloride, fluorouracil, mitomycin, actinomycin, pingyangmycin, cisplatin, carboplatin, epirubicin, methotrexate, and cytarabine.

8. The drug-loaded albumin-magnetite nanoparticle 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 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 nanometer cage is formed by 6 albumin molecules self-assembly, the surface cladding of magnetite nanoparticle has 72 medicine molecules.

10. A method of preparing a drug-loaded albumin-magnetite nanoparticle delivery system according to any one of claims 1 to 9, comprising the steps of:

11. The production method according to claim 10, wherein in step (S2), the amount of the albumin solution added satisfies: the molar ratio of albumin to the iron in the premix solution is 1 (110-220); can be selected as 1 (110-165).

12. The production method according to claim 10 or 11, wherein in step (S2), the amount of the albumin solution added satisfies: the molar ratio of the drug to the albumin is 1.5 or more, optionally 3 or more.

13. The production method according to claim 10 or 11, wherein in step (S1), the molar ratio of the drug to iron in the premix solution is not less than 1.5 ≥ 1.5 (110-220); is selected to be more than or equal to 1.5 (110-165).

14. The production method according to claim 10 or 11, wherein in step (S1), fe is contained in the iron salt solution ³⁺ /Fe ²⁺ ＝(1.5-2.5)：1。