CN114344263B - Nano protein micelle for targeting macrophages to enhance tumor treatment effect and preparation method and application thereof - Google Patents
Nano protein micelle for targeting macrophages to enhance tumor treatment effect and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a nano protein micelle for targeting macrophages to enhance tumor treatment effect, a preparation method and application thereof, belonging to the technical field of biological medicine; hb-DOXM can spontaneously bind endogenous plasma Hp to achieve specific targeting of the M2-type TAMs. Under the acidic and anoxic environment of tumors, hb-DOXM micelle can release DOX to directly kill cancer cells and release O 2 To reduce the aggregation of TAMs to the tumor and subsequent polarization to form M2. The targeting and synergy of Hb in Hb-DOXM@Cel micelles reprograms TAMs, and Hb-DOXM@Cel remodels TME into an immunostimulatory microenvironment, enhancing the antitumor effect of cytotoxic T lymphocytes.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a nano protein micelle for targeting macrophages to enhance tumor treatment effect, and a preparation method and application thereof.
Background
Stromal cells in the Tumor Microenvironment (TME), including fibroblasts, immune cells, inflammatory cells, glial cells, and the like, play an important role in the development, progression, and metastasis of tumors. Tumor-associated macrophages (TAMs) are one of the most abundant infiltrating cells in the tumor microenvironment and play an important role in the development, progression and metastasis of tumors, and therefore, TAMs are considered as potential biomarkers for cancer diagnosis and prognosis, and TAMs targeted therapy is a potential cancer treatment regimen.
Currently, there are three main therapeutic strategies that offer the possibility of treating TAMs-associated tumors: 1) Directly interfere with the survival of the M2-type TAMs or inhibit their signaling cascade; 2) Inhibiting the aggregation of TAMs to tumors by regulating complement, component chemokines, vascular endothelial growth factors and other mediators; 3) The repolarization of tumor-promoting M2-type TAMs is an anti-tumor M1 phenotype. However, the formation and development of TAMs is a complex, dynamic process, and single methods do not allow for comprehensive and continuous regulation of TAMs. Meanwhile, macrophages are an important component of innate immunity and widely distributed throughout the body, and TAMs-related therapeutic strategies have the safety problem of non-targeted therapy.
Hemoglobin (Hb) receptor CD163 is expressed only on M2-type macrophages, while CD163 is also an important specific receptor for M2-type TAMs. Hb is a natural protein in erythrocytes and delivers O to tissue 2 Is a biosafety oxygen carrier. Also, hypoxia is one of the important features of TME, which is the primary driving force for tumor angiogenesis, which promotes differentiation of aggregated monocytes into TAMs.
Targeting TAMs delivery systems via Hb drug loading is therefore a potential tumor treatment.
Disclosure of Invention
The invention aims to provide a nano protein micelle for targeting macrophages to enhance the tumor treatment effect, a preparation method and application thereof, so as to solve the problems in the prior art, and the nano protein micelle cooperates with a drug to realize targeted treatment of M2 TAMs, release the drug in an acidic and low-oxygen environment, reduce the enrichment of the TAMs in tumors and promote the polarization to M1 phenotype transformation, thereby enhancing the cancer treatment effect.
In order to achieve the above object, the present invention provides the following solutions:
the invention provides a preparation method of a nano protein micelle for targeting macrophages to enhance tumor treatment effect, which comprises the following steps:
preparation of DOXM
S101, dissolving oxidized glucose in dimethyl sulfoxide under a water bath condition, and naming the glucose as a system A;
s102, dissolving doxorubicin in DMSO to obtain a DMSO solution of the doxorubicin; adding the DMSO solution of the doxorubicin into the system A to obtain a system B;
s103, adding triethylamine into the system B, and stirring the obtained mixture in a dark place to obtain a system C;
s104, dissolving maleimide polyethylene glycol-amide in DMSO to obtain a DMSO solution of the maleimide polyethylene glycol-amide; adding the DMSO solution of maleimide polyethylene glycol-amide into the system C, and stirring to obtain a system D;
s105, adding phosphate buffer solution into the system D, continuously stirring, dialyzing, and drying to obtain a product DOXM;
preparation of Hb-DOXM
S201, dissolving DOXM in phosphate buffer solution to obtain a system E;
s202, dissolving hemoglobin into water to obtain a hemoglobin solution; adding the hemoglobin solution into the system E, reacting under the condition of carbon monoxide, centrifuging the reaction product to remove unreacted hemoglobin, and drying to obtain Hb-DOXM.
Further, the hemoglobin is human hemoglobin or bovine hemoglobin.
Further, in step S101, the ratio of the oxidized glucose to the dimethyl sulfoxide is 80mg:2mL, wherein the water bath condition is 50 ℃;
in step S102, the concentration of the DMSO solution of the doxorubicin is 24mg/mL;
in step S103, the light-shielding stirring is light-shielding stirring for 4 hours;
in step S104, the concentration of the DMSO solution of maleimide polyethylene glycol-amide is 20mg/mL.
Further, the ratio of the amount of the DMSO solution of doxorubicin, triethylamine and maleimide polyethylene glycol-amide to the amount of the system a was 0.5 μl:50 μl:1mL:2mL.
Further, in step S105, the dialysis is specifically: the solution was dialyzed against a 1:9 volume ratio of DMSO and PBS mixture for 24h, followed by deionized water for 48h.
Further, in step S201, 100mg of DOXM is dissolved in 5mL of phosphate buffer to obtain a system E;
in step S202, the concentration of the hemoglobin solution is 40mg/mL, and the ratio of the amount of the hemoglobin solution to the amount of DOXM is 200mg:100mg.
Further, the preparation of the Hb-DOXM@Cel further comprises the steps of:
s301, dissolving DOXM in dimethylformamide to obtain a system F;
s302, adding celecoxib into the system F under the condition of heating in a water bath, and stirring to obtain a system G;
s303, dropwise adding deionized water into the system G under the ultrasonic condition, dialyzing, and drying to obtain DOXM@Cel;
s304, dissolving the DOXM@Cel in phosphate buffer solution, adding the hemoglobin solution, reacting under the condition of carbon monoxide, centrifuging the reaction product to remove unreacted hemoglobin, and drying to obtain the Hb-DOXM@Cel.
Further, the mass ratio of celecoxib to DOXM is 3:20; the ratio of the amount of hemoglobin solution to the amount of DOXM was 200mg:100mg.
The invention also provides a nano protein micelle for targeting macrophages to enhance the tumor treatment effect, which is prepared by adopting the preparation method of the nano protein micelle for targeting macrophages to enhance the tumor treatment effect.
The invention also provides an application of the nano protein micelle for targeting macrophages to enhance the tumor treatment effect in preparing tumor treatment medicines.
The invention discloses the following technical effects:
the invention discloses a nano protein micelle for targeting macrophages to enhance the tumor treatment effect, which is used for targeting TAMs to enhance the cancer treatment effect. Hemoglobin (Hb) and the chemotherapeutic drug Doxorubicin (DOX) drug-loaded micelles (Hb-DOXM) can spontaneously bind to endogenous plasma Hp to achieve specific targeting of the M2 type TAMs (6-fold accumulation compared to the M1 type TAMs). Under the acidic and anoxic environment of the tumor, the micelle can release DOX to directly kill cancer cells and release O 2 To reduce TAMs aggregates to the tumor and subsequently polarizes to form M2. To fully reprogram the TAMs, the TAMs modulating drug celecoxib was further encapsulated in Hb protein micelles to make Hb-doxm@cel. Targeting and synergy of Hb allows TAMs to reprogram, hb-doxm@cel remodels TME into an immunostimulatory microenvironment, enhancing the antitumor effect of Cytotoxic T Lymphocytes (CTLs). Therefore, hb-DOXM@Cel strongly enhances DOX-based chemotherapy, remarkably inhibits tumor growth and metastasis, and has high safety. In conclusion, hb-DOXM@Cel protein micelles provide a therapeutic tool for targeting and coordinating TAMs for enhancing cancer treatment.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of a nano-protein micelle of the present invention;
FIG. 2 is a TEM image of DOXM (A), hb-DOXM (B) and Hb-DOXM@Cel (C);
FIG. 3 is a graph showing particle size distribution of DOXM and Hb-DOXM;
FIG. 4 is a particle size distribution plot of Hb-DOXM@Cel;
FIG. 5 is the particle size and PDI index of Hb-DOXM;
FIG. 6 is a graph showing the relative release rates of DOX and celecoxib over time, A being DOX and B being celecoxib;
FIG. 7 is an in vitro organ fluorescence imaging of DOX, DOXM and Hb-DOXM;
FIG. 8 shows drug concentrations in the main viscera 24h after DOX, DOXM and Hb-DOXM administration;
FIG. 9 shows DOX uptake in cells;
FIG. 10 shows the residual DOX (green fluorescence) and CD163 (red fluorescence) immunofluorescence staining in tumor sections;
FIG. 11 is a graph showing H22 tumor growth curves following various treatments;
FIG. 12 is H & E and TUNEL staining of isolated tumor tissue;
fig. 13 shows the different treatments (dox=3 mg kg) -1 ) The following 4T1 tumor growth curve, A is tumor volume change, B is average tumor weight, C is tumor photograph, and D is survival rate of mice.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
Example 1
Preparation of Hb-DOXM and Hb-DOXM@Cel (schematic representation shown in FIG. 1).
1. Preparation of DOXM
1) 80mg of oxidized glucose (ODex) was dissolved in 2mL of dimethyl sulfoxide (DMSO) at 50℃in a water bath.
2) 0.5mL of DMSO solution containing 24mg/mL Doxorubicin (DOX) was added.
3) 50. Mu.L of triethylamine was added, and the mixture was stirred in a dark room for 4 hours.
4) 20mg of maleimide polyethylene glycol-amide (MAL-PEG-NH) 2 ) Dissolved in 1mL DMSO, added to the mixture and stirred for 8h.
5) 6mL of phosphate buffer (PBS, pH 7.4) was added thereto, and the mixture was stirred at room temperature for 1 hour.
6) A mixture of DMSO and PBS (pH 7.4) was used (1: 9, v/v) the above-obtained mixed system was subjected to dialysis (MWCO 7000 Da) for 24 hours.
7) And (5) dialyzing with deionized water for 48 hours.
8) Freeze drying to obtain red product DOXM.
2. Preparation of Hb-DOXM
1) 100mg DOXM was dissolved in 5mL PBS (phosphate buffer).
2) 5mL of 50mg/mL aqueous Hb was added and reacted under CO for 8h.
3) The mixture was ultracentrifuged (100 kDa) 4 times to remove unreacted Hb.
4) Freeze-drying preparation, and storing the obtained Hb-DOXM in 4 ℃ environment filled with CO.
3. Preparation of Hb-DOXM@Cel
1) 100mg DOXM was dissolved in 5mL dimethylformamide.
2) After heating to 50℃in a water bath, 15mg of Celecoxib (Celecoxib) was added and stirred for 10min.
3) Under the action of ultrasonic wave, 10mL of deionized water is added dropwise, and ultrasonic is continued for 30min.
4) The mixture was dialyzed against deionized water.
5) Freeze-drying to obtain DOXM@Cel.
6) 100mg of DOXM@Cel was dissolved in 5mL of PBS.
7) 5mL of 50mg/mL Hb solution was added and reacted under CO for 8h.
3) The mixture was ultracentrifuged (100 kDa) 4 times to remove unreacted Hb.
4) Freeze-drying preparation, and storing the obtained Hb-DOXM@Cel in a CO-filled environment at 4 ℃.
Example 2
1. Characterization of DOXM and Hb-DOXM
1) TEM observation
After incubating the polymer nanoparticles prepared in example 1 in PBS for 24 hours, transmission electron microscopy was performed. The results are shown in FIG. 2. The DOXM micelle is spherical and is uniformly dispersed. PEG-NH further modified with maleimide 2 (MAL-PEG-NH 2 ) Hb-DOXM was prepared by Michael addition reaction. Hb-DOXM micelles maintain morphology and uniformity of DOXM, but have an increased particle size compared to DOXM. In the process, a proper amount of celecoxib is added to prepare the Hb-DOXM@Cel, and as can be seen from the figure, the celecoxib does not influence the morphology of the micelle and the uniformity of the system.
2) Particle size distribution
The polymer particles prepared in example 1 were measured for particle size distribution by a Dynamic Light Scattering (DLS) nanosize analyzer (NanoBrook 90Plus Zeta), and the results are shown in fig. 3 and 4, which show that the obtained DOXM micelle diameter was about 86nm and the DOX content was 8.67% according to the calibration curve. PEG-NH further modified with maleimide 2 (MAL-PEG-NH 2 ) Hb-DOXM was prepared by Michael addition reaction. Hb-DOXM micelles remained spherical with slightly larger diameters (-97 nm). Hb-DOXM@Cel micelles remain spherical with a relatively large diameter (-110 nm).
3) Stability of
The stability of the polymer particles prepared in example 1 was determined by dynamic light scattering. The particle size and PDI index (polymer dispersibility index, which describes the molecular weight distribution of a polymer, the larger the PDI, the wider the molecular weight distribution, and the smaller the PDI, the more uniform the molecular weight distribution) of Hb-DOXM in the presence of PBS (10%FBS,pH 7.4,0.01M) at 37℃are shown in FIG. 5.
2. Drug sustained release
Sample treatment: DOX release profile of Hb-DOXM in PBS at different pH values (7.4, 6.5 or 5.0).
Dissolved in PBS (0.01M, 1 mL) at different pH (7.4, 6.5, 5.0), packed in dialysis tubing (mwco=3500). The tube was then immersed in 20mL of PBS (pH 7.4, 6.5 or 5.0) and continuously shaken at 80 rpm. 1mL of dialysate was withdrawn at different time intervals and replaced with fresh PBS solution.
The detection method comprises the following steps: the content of DOX and celecoxib is determined by ultraviolet-visible spectrophotometry and high performance liquid chromatography. The relative release rates of DOX and celecoxib were calculated as a function of time.
Experimental results: hb-DOXM showed high stability in physiological environment (pH 7.4,0.01M,10% fetal bovine serum, 37 ℃) and only 27% DOX was released within 48 hours. At lower pH6.5 (tumor microenvironment pH) and pH5.0 (intracellular/lysosomal pH), DOX released 50% and 80% or more, respectively, in 48h (FIG. 6A)
Hb-DOXM@Cel showed high stability in the physiological environment (pH 7.4,0.01M,10% fetal bovine serum, 37 ℃), releasing only 20% of celecoxib within 48 hours. At lower pH6.5 (tumor microenvironment pH), celecoxib was released more than 50% in 48h (fig. 6B).
3. Efficacy detection
1) The method comprises the following steps: balb/c mice were taken 1h and 24h after intravenous injection and in vitro multi-wavelength fluorescence imaging of DOX was observed by in vitro organ fluorescence imaging using an in vivo animal imaging FX Pro Spectrum imaging system (Bruker, USA).
Results: the Hb-DOXM group accumulated more and the residence time was longer in each tissue site (He heart, li liver, sp spleen, lu lung, ki kidney and Tu tumor sites in this order from left to right in each experimental chart below) than in the DOX group and the DOXM group (FIG. 7).
2) Concentration of drug in main viscera 24h after administration
The method comprises the following steps: the concentration of DOX in the tissue was determined by fluorescence spectroscopy.
The fluorescence intensity of DOX in the supernatant was measured with a fluorescence spectrometer at an excitation wavelength of 480nm and an emission wavelength of 590 nm. The concentration of DOX was determined according to a standard curve established by adding an amount of DOX to the corresponding tissue and plasma collected from untreated mice.
Results: the results of 24h administration showed that Hb-DOXM had higher drug accumulation in Tumor (Tumor) and Liver (Liver) (fig. 8).
Tumor (12.21.+ -. 3.24% μg DOX g) -1 ) And liver (11.96.+ -. 4.21% DOX g) -1 ) Whereas the concentration of single DOX drug in the tissue was relatively low, tumor (4.12.+ -. 1.20% μg DOX g) -1 ) And liver (4.22.+ -. 1.25% μg DOX g) -1 ) The drug concentration of DOXM group tended to be between the former two, tumor (7.12.+ -. 2.34% μg DOX g -1 ) And liver (9.21.+ -. 1.66% μg DOX g) -1 )。
3) Comparison of DOX uptake by M1 and M2 TAMs (primary cells) 24h after administration
The method comprises the following steps: flow cytometry measures the uptake of DOX (fluorescence intensity) in cells.
1. Tumor tissue was excised, transferred to a petri dish, and cut into small pieces (less than 1mm 3 ). The fragments were suspended in 1mL of digestion solution (400. Mu.g/mL of type I collagenase, 100. Mu.g/mL of type IV collagenase in RPMI1640 medium) and stirred at 37℃for 0.5h. Cells were then collected by centrifugation at 1500rpm for 5 min.
2. Filtered through a 200 mesh screen. The cells were digested with erythrocyte lysate for 2min, collected in a 5ml flow tube, and centrifuged at 350g for 5min to remove the supernatant.
3. 200. Mu.L of precooled PBS was added to resuspend the cells, and the cells were shaken well and centrifuged again at 350g for 5min to remove the supernatant;
4. then 200. Mu.L of PBS was added to resuspend the cells, and the cells were immediately checked on the machine.
5. The mean fluorescence intensity MFI at excitation wavelength 480nm and emission wavelength 590nm was determined.
Results: the MFI of DOX in M2-type TAMs is only 1.5-2.0 times that of M1-type TAMs after administration of free DOX and DOXM. By comparison, after Hb-DOXM administration, the MFI of TAMS form M2 to DOX was about 6.05 times that of TAMS form M1, indicating that Hb-DOXM has specific targeting to TAMS form M2 in vivo (fig. 9).
Compared with the uptake of DOX from the M2 type TAMs cells, the Hb-DOXM group uptake is significantly higher than that of DOXM group and DOX group; compared with the uptake of DOX from the M1 type TAMs cells, the Hb-DOXM group uptake was similar to that of DOXM group and DOX group, and the uptake of DOX was significantly reduced relative to that of the M2 type TAMs cells.
4) DOX residue (green fluorescence) and CD163 (red fluorescence) immunofluorescence staining in tumor sections 24h after dosing
The method comprises the following steps: tumor specimens were collected and frozen sections of 6mm thickness were prepared using a cryometer. The sections were air dried for at least 1h, then fixed in acetone at 20℃for 10min, blocked with 20% mouse serum, incubated overnight at 4℃with anti-CD 163 monoclonal antibodies, respectively, and then with secondary antibodies for 1h. After 10min of DAPI staining, the cells were washed twice with PBS and observed under a confocal laser microscope.
Results: tumor tissues were taken 24h after intravenous injection and immunofluorescent stained. The Hb-DOXM injected group further showed greater accumulation of DOX compared to the free DOX and DOXM groups (fig. 10).
5) Different treatment methods (dox=5 mg kg) -1 ) Post H22 tumor growth curve
The method comprises the following steps: in vivo tumor targeting of Hb-DOXM was verified with a mouse liver cancer model. H22 cells were subcutaneously injected on the left side of each female Balb/c mouse (10 7 And (3) one/mL) of 100. Mu.L Phosphate Buffer (PBS) was inoculated. When the tumor grows to 150mm 3 In this case, an equivalent amount of 5mg/kg body weight of free DOX, DOXM or Hb-DOXM treatment, respectively, was administered. Tumor tissue was taken at a certain time after the operation.
To obtain quantitative information on the concentration of DOX in each tissue, the tissue and plasma were weighed, suspended in 70% ethanol with 0.3M hydrochloric acid, and homogenized. After further centrifugation, the fluorescence intensity of DOX in the supernatant was measured at excitation wavelength of 480nm and emission wavelength of 590nm by a fluorescence spectrometer. Standard curves are the same as 2) preparation method.
Results: after 12 days of treatment, hb-DOXM effectively inhibited tumor growth, and tumor volume was reduced by 78.5% compared to saline. The tumor volumes of the free DOX group and DOXM group were reduced by 46.5% and 59.5%, respectively, compared to the saline group, showing moderate antitumor effect (fig. 11).
6) Tumor tissue was isolated for HE and TUNEL detection at the end of treatment
The method comprises the following steps: HE and TUNEL assays were performed on the treated tumor tissue as in 5) above.
Results: the results of histopathological analysis of resected tumors further showed that mice treated with Hb-DOXM exhibited significant necrotic and apoptotic cells. The results show that Hb-DOXM can be combined with endogenous Hp to effectively target the CD163 receptor on the surface of M2 type TAMs, so that higher drug accumulation is obtained at a tumor site, and the anti-tumor effect is effectively improved (figure 12).
7) Different treatment methods (dox=3 mg kg) -1 ) Post 4T1 tumor growth curve
It was evaluated whether DOX-based therapy could be enhanced by Hb-DOXM@Cel.
The method comprises the following steps: a BALB/c mouse 4T1 tumor model was established. Every 3 days at 3mg kg -1 Different formulations were intravenously injected (Saline: physiological Saline, celecoxib: physiological Saline containing an equivalent amount of doxycycline) in three injections (9 days).
Results: figure 13A shows that very weak tumor inhibition was observed in mice treated with free celecoxib, and moderate antitumor effect was also observed in the free DOX and DOXM groups. After 18 days of treatment, doxm@cel showed better anti-tumor effect, with tumor volume reduced to 32.5% (relative to saline group), indicating that celecoxib enhanced doxycycline-based chemotherapy in combination. In addition, hb-doxm@cel showed the most potent tumor suppression effect (reduced to 20.3%), which is attributed to targeting of M2-type TAMs to deliver drugs and oxygen. The same trend was also observed in average tumor weight (fig. 13B), tumor photograph (fig. 13C), and mouse survival (fig. 13D).
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Claims (10)
1. The preparation method of the nano protein micelle for targeting macrophages to enhance the tumor treatment effect is characterized by comprising the following steps of:
preparation of DOXM
S101, dissolving oxidized glucose in dimethyl sulfoxide under a water bath condition, and naming the glucose as a system A;
s102, dissolving doxorubicin in DMSO to obtain a DMSO solution of the doxorubicin; adding the DMSO solution of the doxorubicin into the system A to obtain a system B;
s103, adding triethylamine into the system B, and stirring the obtained mixture in a dark place to obtain a system C;
s104, dissolving maleimide polyethylene glycol-amide in DMSO to obtain a DMSO solution of the maleimide polyethylene glycol-amide; adding the DMSO solution of maleimide polyethylene glycol-amide into the system C, and stirring to obtain a system D;
s105, adding phosphate buffer solution into the system D, continuously stirring, dialyzing, and drying to obtain a product DOXM;
preparation of Hb-DOXM
S201, dissolving DOXM in phosphate buffer solution to obtain a system E;
s202, dissolving hemoglobin into water to obtain a hemoglobin solution; adding the hemoglobin solution into the system E, reacting under the condition of carbon monoxide, centrifuging the reaction product to remove unreacted hemoglobin, and drying to obtain Hb-DOXM.
2. The method of claim 1, wherein the hemoglobin is human hemoglobin or bovine hemoglobin.
3. The method according to claim 1, wherein in step S101, the ratio of glucose oxide to dimethyl sulfoxide is 80mg:2mL, wherein the water bath condition is 50 ℃;
in step S102, the concentration of the DMSO solution of the doxorubicin is 24mg/mL;
in step S103, the light-shielding stirring is light-shielding stirring for 4 hours;
in step S104, the concentration of the DMSO solution of maleimide polyethylene glycol-amide is 20mg/mL.
4. A method of preparation according to claim 3, wherein the ratio of the amount of DMSO solution of doxorubicin, triethylamine and maleimide polyethylene glycol-amide to the amount of system a is 0.5 μl:50 μl:1mL:2mL.
5. The method according to claim 1, wherein in step S105, the dialysis is specifically: the solution was dialyzed against a 1:9 volume ratio of DMSO and PBS mixture for 24h, followed by deionized water for 48h.
6. The preparation method according to claim 1, wherein in step S201, 100mg of DOXM is dissolved in 5mL of phosphate buffer to obtain system E;
in step S202, the concentration of the hemoglobin solution is 40mg/mL, and the ratio of the amount of the hemoglobin solution to the amount of DOXM is 200mg:100mg.
7. The preparation method of the nano protein micelle for targeting macrophages to enhance the tumor treatment effect is characterized by comprising the following steps of:
preparation of DOXM
S101, dissolving oxidized glucose in dimethyl sulfoxide under a water bath condition, and naming the glucose as a system A;
s102, dissolving doxorubicin in DMSO to obtain a DMSO solution of the doxorubicin; adding the DMSO solution of the doxorubicin into the system A to obtain a system B;
s103, adding triethylamine into the system B, and stirring the obtained mixture in a dark place to obtain a system C;
s104, dissolving maleimide polyethylene glycol-amide in DMSO to obtain a DMSO solution of the maleimide polyethylene glycol-amide; adding the DMSO solution of maleimide polyethylene glycol-amide into the system C, and stirring to obtain a system D;
s105, adding phosphate buffer solution into the system D, continuously stirring, dialyzing, and drying to obtain a product DOXM;
preparation of Hb-DOXM@Cel:
s201, dissolving DOXM in dimethylformamide to obtain a system F;
s202, adding celecoxib into the system F under the condition of heating in a water bath, and stirring to obtain a system G;
s203, dropwise adding deionized water into the system G under the ultrasonic condition, dialyzing, and drying to obtain DOXM@Cel;
s204, dissolving the DOXM@Cel in a phosphate buffer solution, adding a hemoglobin solution, reacting under the condition of carbon monoxide, centrifuging a reaction product to remove unreacted hemoglobin, and drying to obtain Hb-DOXM@Cel;
wherein the hemoglobin solution is obtained by dissolving hemoglobin in water.
8. The preparation method according to claim 7, wherein the mass ratio of celecoxib to DOXM is 3:20; the ratio of the amount of hemoglobin solution to the amount of DOXM was 200mg:100mg.
9. A nano-protein micelle for targeting macrophages to enhance the tumor treatment effect, which is characterized by being prepared by adopting the preparation method of the nano-protein micelle for targeting macrophages to enhance the tumor treatment effect according to any one of claims 1-8.
10. Use of the nano-protein micelle for targeting macrophages to enhance the therapeutic effect of tumors according to claim 9 in the preparation of a tumor therapeutic drug.
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| Title |
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| Dextran-Based Doxorubicin Nanocarriers with Improved Tumor Penetration;Sharon M. Sagnella等;《Biomacromolecules》;第15卷;第262-275页 * |
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| Publication number | Publication date |
|---|---|
| CN114344263A (en) | 2022-04-15 |
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