CN111973572A

CN111973572A - Manganese-based dendritic macromolecular composite nanomaterial, and preparation method and application thereof

Info

Publication number: CN111973572A
Application number: CN202010532721.2A
Authority: CN
Inventors: 胡红杰; 周晓璇; 唐建斌; 韩玉鑫
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2020-06-11
Filing date: 2020-06-11
Publication date: 2020-11-24

Abstract

The invention discloses a manganese-based dendritic macromolecular composite nanomaterial, a preparation method and application thereof. The preparation method comprises the steps of adding hypericin, manganese chloride and polyglycerol structure dendritic macromolecules with cysteine on the surface into an aqueous medium, and self-assembling the product to form the manganese-based dendritic macromolecule composite nano material. The manganese-based dendritic macromolecular composite nanomaterial with high efficiency is prepared by self-assembly, and has the advantages of high relaxation efficiency, long in-vivo circulation time, rapid kidney clearing, targeting property, high biocompatibility, small toxic and side effects and the like. The nano material prepared by the invention has higher photodynamic conversion efficiency at 595nm, can be used as a photosensitizer applied to photodynamic therapy, and monitors the position and the size of a tumor and the enrichment condition of a phototherapeutic agent in tumor tissues by means of a magnetic resonance technology, so as to evaluate the treatment effect and realize the integration of photodynamic therapy diagnosis and treatment mediated by magnetic resonance imaging.

Description

Manganese-based dendritic macromolecular composite nanomaterial, and preparation method and application thereof

Technical Field

The invention relates to the field of magnetic resonance imaging-mediated photodynamic therapy diagnosis and treatment integrated materials, in particular to a manganese-based dendritic macromolecule composite nanomaterial, a preparation method and application thereof.

Background

At present, manganese-based nanomaterials are increasingly applied to Magnetic Resonance Imaging (MRI) diagnosis, and the diversity of diagnosis and combined treatment thereof are valued by researchers. Manganese-based nanomaterials comprising MnO₂、 MnO、Mn₂O₃、Mn₃O₄And MnO_xThe nano material and the derivative thereof have wide application in the aspects of biological imaging, biological sensing, drug or gene transfer, tumor treatment and the like. The capability of the manganese-based nano material in the aspect of biological imaging is mainly due to the fact that the manganese-based nano material is combined with H⁺Hydrogen peroxide (H)₂O₂) Glutathione (GSH) and the like, so that the target site manganese ions are degraded in a tumor microenvironment to increase the concentration of the target site manganese ions, and the MRI efficiency is improved. In addition to this, nanosystems of manganese-based nanomaterials can participate in ultrasound, photothermal imaging, photoacoustic imaging, and photothermal therapy and lightKinetic therapy (PDT), and the like.

PDT has become a non-invasive, light-activated cancer treatment modality, and is becoming increasingly clinically recognized for treatment of both superficial and deep tumors (esophageal, skin, and non-small cell lung). The whole process is mainly required to realize PDT, and the whole process mainly comprises the factors of exciting light, photosensitizer, molecular oxygen and the like^[45]. Under the excitation of a light source with a specific wavelength, PS can be selectively activated to generate a series of reactive oxygen Radicals (ROS), and then cytotoxicity, vascular injury, immune regulation and the like occur, and finally cancer cell death is induced. In the PDT treatment process, the light source has time and position controllability, can avoid selective retention of normal tissues in a tumor, and accurately generates and releases ROS, so that tumor specific PDT is realized, and side effects are minimized. Since ROS can activate acute inflammatory responses, enhance tumor immunogenicity, and enhance T cell infiltration, PDT may generate strong immune responses, enhancing checkpoint blockade therapy. More importantly, ROS generated in PDT can kill cancer cells either directly by inducing apoptosis/necrosis of tumor cells or indirectly by generating tumor-specific immunity, resulting in a synergistic PDT/immunotherapy. Photosensitizers for PDT are diverse in variety, and common photosensitizers include porphyrin-related macrocyclic compounds, non-porphyrin-related phthalocyanines, hypericin, porphins, phenothiazines, and metal derivatives. Wherein, hypericin is an extract of common medicinal plant hypericum perforatum, the monomer of hypericum perforatum is highly hydrophobic, a carrier is needed when in administration, and the common carrier comprises liposome, micelle, nano-particle and the like.

Therefore, it is a problem to be solved by those skilled in the art to develop a new nano material that can be used as both a magnetic resonance imaging contrast agent and a photodynamic therapy, i.e. to satisfy the above-mentioned objective of integrating high-efficiency magnetic resonance imaging and photodynamic therapy diagnosis and treatment.

The invention patent application with the publication number of 201510043825.6 of the previous application of the applicant discloses a polyglycerol-structure dendritic macromolecule and a preparation method and application thereof, wherein the structural formula of the polyglycerol-structure dendritic macromolecule is shown as the formula (I). The polyglycerol structure dendritic macromolecule contains a large number of ether bonds in the polyglycerol structure dendritic macromolecule, has good biocompatibility and stable chemical structure under physiological conditions; the surface of the material contains a large number of reactive groups, the material can be used for bonding various medicines, molecular probes and targeting groups, and the hollow cavity structure of the core of the material can also load medicines and biological probes, and can be widely used for conveying medicines, genes, contrast agents and biological probes as a biological carrier material; the preparation method has the advantages of few synthesis steps, simple separation and purification, easy synthesis of the high molecular weight dendritic macromolecule, great improvement of the synthesis efficiency of the polyglycerol structure dendritic macromolecule, and possibility of large-scale production of the dendritic macromolecule with wide application prospect.

Disclosure of Invention

The invention provides a novel nano material, which is a manganese-based dendritic macromolecule composite nano material, has higher relaxation rate and high photodynamic conversion efficiency under the condition of illumination (595nm), can be used as a magnetic resonance contrast agent and a photodynamic therapeutic agent, and is applied to magnetic resonance imaging and photodynamic therapy to realize the diagnosis and treatment integration of tumors.

A preparation method of manganese-based dendritic macromolecular composite nanomaterial comprises adding hypericin, manganese chloride and polyglycerol dendritic macromolecules with cysteine on the surface into an aqueous medium, self-assembling the product to form the manganese-based dendritic macromolecular composite nanomaterial,

wherein, the structural formula of the polyglycerol dendritic macromolecule with cysteine on the surface is shown as formula I:

the hypericin is monomer hypericin, and the molecular formula is as follows: c₃₀H₁₆O₈Molecular weight: 504.45, CAS number: 548-04-9, the structural formula is shown as follows:

preferably, the mass ratio of the hypericin to the manganese chloride to the polyglycerol structure dendritic macromolecule with cysteine on the surface is 0.004: 5.04: 50.

Preferably, the mass-volume ratio of the polyglycerol structure dendrimer with the cysteine on the surface in the reaction system to the water medium is 50 mg: 5.4 mL.

Preferably, the manganese chloride and the polyglycerol structure dendritic macromolecule with cysteine on the surface are both aqueous solution, hypericin is dissolved in dimethyl sulfoxide,

the reaction steps are as follows: adding the dimethyl sulfoxide solution of hypericin into the aqueous solution of the dendritic macromolecules of the polyglycerol structure with cysteine on the surface, stirring for 1 hour in a dark place, dropwise adding the aqueous solution of manganese chloride into the reaction solution, adjusting the pH value of the reaction solution to 9 after dropwise adding, and stirring and reacting for 2 hours at room temperature. And (3) dialyzing the solution after the reaction in deionized water by using a dialysis membrane, removing redundant Mn ions, concentrating by using an ultrafiltration membrane centrifugal tube of 3500Da, and fixing the volume to obtain the final brown nanoparticle solution.

In the preparation method of the invention, manganese chloride is subjected to oxidation reduction reaction under alkaline condition to form manganese dioxide (MnO)₂) Particles; MnO₂The particles and hypericin are coated in the dendritic macromolecule of polyglycerol structure with cysteine on the surface to form the spherical nano-grade material.

The invention also provides the manganese-based dendritic macromolecular composite nano-material prepared by the preparation method.

The invention is realized by adding MnO₂The particles and hypericin are simultaneously connected on the dendritic macromolecule with cysteine on the surface, thereby preparing the manganese-based dendritic macromolecule based composite nano contrast agent, the particle size is 109nm, the relaxation rate is 5.8mM^-1S^-1The spherical nano particles are higher than the relaxation rate of clinical small molecular gadolinium contrast agent, have better contrast effect, and can be enriched by the EPR effect of the nano-scale macromolecule contrast agentThe magnetic resonance imaging probe is integrated in tumor tissues, improves the sensitivity of MRI, and improves the diagnosis level of early cancer.

The invention also provides application of the manganese-based dendritic macromolecular composite nanomaterial in preparation of a magnetic resonance imaging contrast agent and/or a photodynamic therapeutic agent. The nano material prepared by the invention has a certain amount of absorption at the wavelength of 595nm, and researches show that the nano material can obviously inhibit the proliferation of tumor cells when being applied to the tumor cells under the condition of illumination, so the nano material can be used as a photodynamic therapeutic agent.

The invention has the following beneficial effects:

(1) the efficient manganese-based dendritic macromolecular composite nanomaterial prepared by self-assembly has the advantages of high relaxation efficiency, long in-vivo circulation time, rapid renal clearance, targeting property, high biocompatibility, small toxic and side effects and the like.

(2) The nano material prepared by the invention has higher photodynamic conversion efficiency at 595nm, can be used as a photosensitizer applied to photodynamic therapy, and monitors the position and the size of a tumor and the enrichment condition of a phototherapeutic agent in tumor tissues by means of a magnetic resonance technology, so as to evaluate the treatment effect and realize the integration of photodynamic therapy diagnosis and treatment mediated by magnetic resonance imaging.

Drawings

FIG. 1 is a distribution diagram of particle size of nano-material DHM in water measured by dynamic light scattering instrument.

FIG. 2 is a transmission electron microscope image of the nano material DHM.

Fig. 3 shows the relaxation rate of the nano-material DHM magnetic resonance contrast agent as a function of the manganese concentration.

Fig. 4 is an in vitro magnetic resonance imaging graph of various concentrations of the nanomaterial DHM.

FIG. 5 is a graph showing the effect of DHM on proliferation of 4T1 cells (595nm, 0.25mW cm) in the presence or absence of laser irradiation^-2，5min)。

Fig. 6 is a magnetic resonance imaging chart (a) and a signal intensity quantification chart (B) of the nano-material DHM as a magnetic resonance contrast agent for enhancing the in-situ breast tumor.

Figure 7 is a coronal mri image of liver slices of mice enhanced with nanomaterial DHM as a magnetic resonance contrast agent.

FIG. 8 is a tumor growth curve diagram of the nano-material DHM in the inhibition experiment of the 4T1 breast cancer cell-bearing Balb/c mouse tumor.

FIG. 9 is a graph showing the change of body weight of Balb/c mice in the experiment process of suppressing tumors of Balb/c mice with breast cancer cells of 4T1 by using the nano-material DHM.

Detailed Description

Example 1

1. Preparation of nanomaterials

(1) The polyglycerol branched macromolecule (G1-Cys, 50mg, structural formula shown in formula I) with amino acid group on the 1 st surface is dissolved in 5mL of water, and the photodynamic drug hypericin dimethyl sulfoxide (DMSO) solution (0.2mg mL) is added^-120. mu.L), stirring for 1 hour in the dark, and then dropwise adding MnCl₂(12.6 mg mL ^-1400 μ L) was added to adjust the solution to pH 9 and stirred at room temperature for 2 hours.

(2) The reaction was continued at room temperature for 3 hours in the absence of light.

(3) The dialysis membrane (3500Da) was purified by dialysis in deionized water for 24 hours.

(4) Concentrating with ultrafiltration membrane centrifuge tube (3500Da) and fixing volume to obtain brown DHM nanoparticle solution

2. Analysis of Properties of nanomaterials

(1) The average particle size and the particle size distribution of the DHM nanoparticies (the DHM nanoparticles prepared above) were determined using a dynamic light scattering particle sizer (DLS). Take 0.5mg mL^-1A (1mL) solution of DHM nanophase contrast agent was placed in a DLS instrument for measurement, and as shown in FIG. 1, the average particle size of the DHM nanophase contrast agent was 109nm, the Polydispersity index (PDI) of the particle size was 0.15, and the Zeta potential was-7.2 mV.

(2) And (3) determining the morphology, distribution and particle size of the DHM nano contrast agent by adopting a Transmission Electron Microscope (TEM). Take 0.5mg mL^-1(1mL) is immersed in a copper mesh (400 mesh, carbon support film) for about 5 minutes, the copper mesh is gently taken out by a pair of tweezers, and then the clean filter paper is used for absorbing and removing redundant liquid and drying the liquid, the liquid is placed on a TEM instrument, the appearance and the particle size of a sample are observed, and images are stored. The size of the nanoparticles was again verified by TEM as shown in fig. 2, and it can be seen that the nano-contrast agent is in a more regular uniform spherical shape and all of the sizes are more uniform.

(3) The relaxation rate of the DHM nanophase contrast agent solution was determined using a 0.52T micro magnetic resonance imager. Determination of longitudinal relaxation time (T) of DHM nanophase contrast agent solutions (Mn ion concentration 0.05-0.20mM) at different concentrations by inversion recovery method₁) Then the obtained T is₁Plotting the reciprocal values with a slope of the longitudinal relaxation rate R of the DHM under different conditions₁Longitudinal relaxation rate (R)₁). As shown in FIG. 3, the manganese content of the nano material DHM is 4.15%, R of DHM₁Is 5.8mM^-1s^-1。

(4) In vitro MRI experiments with DHM nanoparectant: 1.5mL of deionized water and equal amounts of 4 different concentrations of DHM nanoparicise solutions (Mn ion concentration 0.05-0.20mM) were placed in 2mL centrifuge tubes for MRI, respectively. The equipment is Discovery 750w 3.0T, GE Healthcare system, and the imaging parameters are as follows: TR is 200ms, TE is 3ms, and resolution is 512 × 512. As shown in FIG. 4, pure water on the rightmost side is used as a control, the concentration of the DHM nano contrast agent solution is increased, the signal is obviously increased and has concentration dependence, and the signal is stronger when the concentration is higher.

(5) The toxicity of the DHM nano contrast agent to the 4T1 cell strain in vitro is detected by adopting an MTT method. After 4T1 cells in the logarithmic growth phase are respectively paved in a 96-well plate to be cultured overnight and attached to the wall, after DHM nano contrast agent with different concentrations and Hyp are added for 2 hours, the cells are exposed to yellow light irradiation (595nm, 0.25mW cm)^-25min), for another 24 hours, and after completion of the incubation time, 20. mu.L of MTT solution (5mg mL) was added to each well^-1) After the culture is continued for 4 hours, the cells with metabolic activity can reduce MTT into blue-purple crystal precipitation (tympany), DMSO is added after the culture medium is removed by centrifugation to completely dissolve the blue-purple crystal, and finally a 96-well plate is placed on an enzyme-linked immunosorbent assay to read the absorbance value (56)The difference between 2nm and 620 nm) indirectly reflects the toxic effect of the DHM nanoparitct on cells. As shown in FIG. 5, the DHM nano contrast agent and the illumination group and the Hyp monomer and the illumination group have strong toxicity to 4T1 cells, and the IC50 values are respectively 0.18 mu g mL^-1、0.94μg mL^-1All have higher antitumor activity. When the Hyp monomer and the DHM nano contrast agent are used independently, the anti-tumor effect is poor, and the IC50 values are respectively 4.31 mu g mL^-1、4.77μg mL^-1. The Hyp monomer is matched with photodynamic therapy to kill tumor cells with good effect, but the monomer has poor water solubility and large difficulty in vivo administration; the DHM nano contrast agent is well distributed in each medium, and has a high-efficiency anti-tumor effect by matching with illumination.

(6) The tumor diagnosis effect of the DHM nano contrast agent in tumor-bearing mice is observed through MRI. Tumor-bearing 4T1 mouse DHM nanometer contrast agent group (0.1mmol Kg) injected via tail vein^-1Mn), pictures of the cross-sectional position T1WI were taken and their signal values were determined and analyzed and plotted. As shown in FIG. 6, (A) before DHM nanoparticles, T1 signals of tumor and peripheral muscle are close, and the signals are all low. After 5 minutes of DHM nanoparticle injection, significant enhancement was observed around the tumor. Tumor signal increases with time, peaking at 30 minutes and then declining. (B) The CNR of the tumor is obviously increased after the DHM nano contrast agent is injected. The CNR of the tumors was highest at 30 min after DHM nanoparit injection, about 62. The DHM nano contrast agent has longer blood circulation time and can be accumulated in tumors through an EPR effect. Due to decomposition and oxidation-reduction of the DHM nano contrast agent in a tumor microenvironment, a large amount of Mn ions are released, so that the T1 signal of a tumor part is obviously increased, and the observable time window is longer. Therefore, the DHM nano contrast agent has excellent MRI enhancement capability in a mouse breast cancer model, and greatly improves the sensitivity of tumor detection.

(7) The distribution and metabolism of the DHM nano contrast agent in the blank mice are observed through MRI. The blank mice were injected with DHM nanophase contrast agent (0.1mmol Kg) before or after tail vein injection^-1Mn), an image of the coronal T1WI was acquired. As shown in FIG. 7, the liver was highly strengthened 3 hours after the DHM nanophase contrast agent injection and was easily associated with the surrounding tissuesSeparating; the liver signal decreased significantly 1 day after injection, suggesting that a portion of the DHM nanoparticules may be ingested by the liver, degraded and excreted from the digestive tract. The kidney is obviously strengthened 3 hours after the DHM nano contrast agent is injected, the parenchyma of the kidney is easily distinguished from the renal pelvis 6 hours after the DHM nano contrast agent is injected, and the contrast is high, mainly due to the high distribution of Mn ions in the kidney. As can be seen by MRI 1 day after injection and 3 days after injection of DHM nanophase contrast agent, the signals of kidney and liver are attenuated to be basically consistent with those before injection, which proves that DHM can be metabolically excreted in vivo.

(8) The above proves that the DHM nano contrast agent has excellent cell photodynamic killing effect in vitro, and the experiment further studies the photodynamic tumor inhibition effect in vivo. Firstly, establishing a mouse in-situ breast cancer model: 4T1 murine Breast cancer cells (5X 10)⁵200 μ L) were attached to the third pair of mammary glands in the left flank of female Balb/c mice. The average tumor volume reaches 65mm³Thereafter, the mice were randomly divided into 4 groups (n ═ 5): PBS group, Hyp single drug + light group (Hyp 10. mu.g Kg^-1) DHM nanoparticel group and DHM nanoparticel (Hyp equivalent 10 ug Kg)^-1) + illumination group (595nm, 2.5W cm ^-25 min). Only one dose was given, and then the state of the mice was closely observed, and the tumor volume and body weight were measured every two days. As shown in fig. 8, 4T1 breast cancer cell-bearing Balb/c mice were randomly divided into 4 groups (n ═ 5): mice were randomized into 4 groups (n ═ 5): PBS group, Hyp single drug + light group (Hyp equivalent 10. mu.g Kg^-1) DHM nanopartical contrast agent group and DHM nanopartical contrast agent + illumination group (595nm, 2.5W cm)^-25 min). Only one administration was followed by close observation of the mouse status, and tumor volume and body weight were measured every two days. As shown in FIG. 8, the tumors in DHM + light group completely disappeared, whereas the tumors in DHM only, Hyp + light and PBS only groups grew faster with statistical difference (p)<0.001). As shown in fig. 9, no significant weight loss occurred in the mice in the experimental group and the other three control groups. The DHM nanoparticle is shown to have good biocompatibility, and acute toxicity can not be caused in a short time after the DHM nanoparticle is injected through tail vein.

Claims

1. A preparation method of manganese-based dendritic macromolecular composite nanomaterial is characterized in that hypericin, manganese chloride and polyglycerol dendritic macromolecules with cysteine on the surface are added into an aqueous medium, the product is self-assembled to form the manganese-based dendritic macromolecular composite nanomaterial,

2. the method of claim 1, wherein said hypericin is monomeric hypericin.

3. The method according to claim 1, wherein the mass ratio of hypericin, manganese chloride and the dendritic macromolecule having a polyglycerol structure with cysteine on the surface is 0.004: 5.04: 50.

4. The process according to claim 1, wherein the mass-to-volume ratio of the polyglycerol-structured dendrimer having cysteine on the surface thereof to the aqueous medium in the reaction system is 50 mg: 5.4 mL.

5. The method according to claim 1, wherein the manganese chloride and the polyglycerol structure dendrimer having cysteine on the surface are both aqueous solutions, hypericin is dissolved in dimethyl sulfoxide,

the reaction steps are as follows: adding the dimethyl sulfoxide solution of hypericin into the aqueous solution of the dendritic macromolecules of the polyglycerol structure with cysteine on the surface, stirring for 1 hour in a dark place, dropwise adding the aqueous solution of manganese chloride into the reaction solution, adjusting the pH value of the reaction solution to 9 after dropwise adding, stirring for 2 hours at room temperature, and continuously reacting for 3 hours in a dark place at room temperature.

6. The preparation method according to claim 5, wherein the manganese-based dendrimer composite nanomaterial is prepared by performing dialysis purification in deionized water using a dialysis membrane after completion of the reaction, and then concentrating.

7. The manganese-based dendritic macromolecular composite nano-material prepared by the preparation method of any one of claims 1 to 6.

8. Use of the manganese-based dendrimer composite nanomaterial according to claim 7 for the preparation of magnetic resonance imaging contrast agents and/or photodynamic therapy agents.