Targeted drug delivery system for breast cancer brain metastases based on targeting grp94 to avoid normal brain cells and preparation method thereof

Abstract

This invention discloses a targeted drug delivery system for breast cancer brain metastases that bypasses normal brain cells based on GRP94 targeting, and its preparation method. Using a biodegradable polymer material as the base carrier, a drug delivery system with a surface-modified GRP94 antibody or targeting peptide OmpA is prepared. This system can penetrate the blood-brain barrier by targeting GRP94, specifically recognizing breast cancer brain metastases rather than normal brain cells due to the lack of GRP94 on normal brain cell membranes. Compared to other target receptors that cross the blood-brain barrier for brain-targeted drug delivery, targeting GRP94 more specifically targets brain metastases. GRP94 is expressed only on the surface of blood-brain barrier endothelial cells and breast cancer brain metastases, without leading to drug accumulation or neurotoxicity in normal intracranial regions, making GRP94 a novel targeted drug delivery target for brain metastases.

Description

Technical Field

[0001] This invention belongs to the field of nanobiomedicine technology, specifically relating to a targeted drug delivery system for breast cancer brain metastases based on targeting GRP94 to avoid normal brain cells and its preparation method. Background Technology

[0002] Brain metastases are among the most common intracranial tumors, affecting approximately 20% of cancer patients. Despite advancements in clinical treatments including surgery, radiotherapy, chemotherapy, targeted therapy, and immunotherapy, the prognosis remains poor. Currently, surgery, radiotherapy, and chemotherapy are the preferred treatment options. However, due to the blood-brain barrier (BBB) ​​and the small, invasive nature of brain metastases, they cannot effectively eradicate the tumor, and these treatments have significant side effects. Therefore, developing efficient drug delivery systems targeting brain metastases has become an urgent social issue. Currently, most preclinical and clinical work focuses on overcoming the BBB barrier to improve drug penetration into the brain. Current research indicates that BBB receptors that can mediate brain entry include transferrin receptor, low-density lipoprotein receptor-associated protein 1 (LRP1), N-methyl-D-aspartate receptor (NMDAR), and nicotinic acetylcholine receptor. However, there are certain risks associated with drug delivery to the brain via these receptors. These receptors, which can mediate BBB penetration, are also expressed in normal brain parenchyma regions, such as normal neurons, astrocytes, and pericytes, where LRP1 is expressed. Normal intracranial expression of these receptors can lead to unnecessary intracranial drug accumulation and neurotoxicity. Therefore, there is an urgent need to discover novel drug delivery targets expressed only on the blood-brain barrier and the cell membranes of diseased cells, capable of penetrating the blood-brain barrier, effectively avoiding normal brain cells, and reaching the diseased areas within the brain parenchyma. Summary of the Invention

[0003] The purpose of this invention is to provide a targeted drug delivery system for breast cancer brain metastases that targets GRP94 and avoids normal brain cells, as well as its preparation method.

[0004] The technical solution of this invention is:

[0005] A targeted drug delivery system for breast cancer brain metastases that targets GRP94 and avoids normal brain cells utilizes biodegradable polymeric materials to prepare drug-loadable nanoparticles as the basic carrier. The biodegradable polymeric material is polylactic acid-glycolic acid, and the surface of the nanoparticles is modified with GRP94 antibody or the targeting peptide OmpA.

[0006] Another technical solution of the present invention is:

[0007] A method for preparing a targeted drug delivery system for breast cancer brain metastases based on GRP94 targeting to avoid normal brain cells, the method comprising:

[0008] (1) Weigh out the polymer material and dissolve it in an organic solvent to form an oil phase;

[0009] (2) Dissolve the drug doxorubicin and probe Dir in the oil phase to form part of the oil phase, and the aqueous phase is water itself, or dissolve the drug in water to form the aqueous phase;

[0010] (3) The aqueous phase is added dropwise to the swirling oil phase and ultrasonically emulsified to form a water-in-oil emulsion;

[0011] (4) The emulsion is then added drop by drop to the external aqueous phase of the vortex and ultrasonically emulsified to form a water-in-oil-in-water complex emulsion;

[0012] (5) Quickly pour the double emulsion into the volatile aqueous phase, stir overnight to evaporate, and form a nanoparticle suspension;

[0013] (6) Centrifuge the nanoparticle suspension at low speed and collect the supernatant;

[0014] (7) The supernatant is purified by high-speed centrifugation to obtain purified and unmodified nanoparticle precipitate;

[0015] (8) The nanoparticles were ultrasonically dispersed in a buffer solution, and the surface was modified with functional linking molecules. After reacting at room temperature for 1-2 hours, the excess functional linking molecules were removed by high-speed centrifugation, and the precipitate was obtained to obtain nanoparticles with surface-modified functional linking molecules.

[0016] (9) The nanoparticles of the surface-modified functional linker molecule are ultrasonically dispersed in a buffer solution, and the surface is modified with GRP94 antibody or targeting peptide OmpA by the functional linker molecule. After reacting for 1-2 hours, the nanoparticles of the surface-modified GRP94 antibody or targeting peptide OmpA are obtained by high-speed centrifugation.

[0017] (10) The surface-modified GRP94 antibody or targeting peptide OmpA nanoparticles were ultrasonically dispersed in water and centrifuged at high speed to obtain a precipitate, thereby obtaining a targeted drug delivery system for breast cancer brain metastases based on targeting GRP94 to avoid normal brain cells.

[0018] (11) Disperse the targeted drug delivery system for breast cancer brain metastases based on targeting GRP94 to avoid normal brain cells in ultrapure water.

[0019] Furthermore, in step (1), the polymer material is polylactic acid-hydroxyacetic acid, and the organic solvent is ethyl acetate.

[0020] Furthermore, in step (4), the external aqueous phase is polyvinyl alcohol with a mass fraction of 2.5%.

[0021] Furthermore, in step (5), the volatile aqueous phase is polyvinyl alcohol with a mass fraction of 0.3%.

[0022] Furthermore, in step (6), the low-speed centrifugation speed is 1000-1500 r, and the time is 10 min.

[0023] Furthermore, in step (7), the high-speed centrifugation speed is 35000 r and the time is 20 min.

[0024] Furthermore, in step (8), the buffer solution is a phosphate buffer with a pH of 7.4, the functional linker is PEG, and the high-speed centrifugation speed is 35000 r for 20 min.

[0025] Furthermore, in step (9), the buffer solution is a phosphate buffer with a pH of 7.4, and the high-speed centrifugation speed is 35000 r for 20 min.

[0026] Furthermore, in step (10), the high-speed centrifugation speed is 35000 r and the time is 20 min.

[0027] This invention provides a targeted drug delivery system for breast cancer brain metastases based on GRP94 targeting, bypassing normal brain cells, and its preparation method. The beneficial effects are: a novel drug target is discovered, expressed only on the cell membranes of diseased cells that penetrate the blood-brain barrier. After penetrating the blood-brain barrier, it effectively avoids normal brain cells and reaches the lesion area within the brain parenchyma, without causing unnecessary intracranial drug accumulation or neurotoxicity. Biodegradable polymeric materials, specifically polylactic-co-glycolic acid (PLGA), are used to prepare drug-loadable nanoparticles as the basic carrier. The nanoparticle surface is modified with GRP94 antibody or the targeting peptide OmpA. This novel target exhibits strong specificity for tumor targeting and low neurotoxicity. Furthermore, the preparation method is simple, the raw materials are readily available, and the operation is convenient, demonstrating considerable economic benefits and promising translational potential. Attached Figure Description

[0028] Figure 1 This is a schematic diagram illustrating the construction and in vivo behavior of the targeted drug delivery system for breast cancer brain metastases based on targeting GRP94 to avoid normal brain cells, as described in this invention.

[0029] Figure 2 The image shows the uptake of doxorubicin-loaded brain-targeted drug delivery systems (unmodified nanoparticles, Angiopep 2 modified nanoparticles, OmpA-modified nanoparticles, and GRP94 antibody-modified nanoparticles) on brain microvascular endothelial cells, with a doxorubicin concentration of 5 μg / mL and an incubation time of 3 h.

[0030] Figure 3 This is a schematic diagram of GRP94 protein expression on the cell membrane of endothelial cells bEnd.3 as characterized by confocal microscopy.

[0031] Figure 4 This is a schematic diagram of Western blot characterization of GRP94 expression in various cells of the brain environment. In this diagram, A shows the expression of GRP94 in endothelial cells bEnd.3, pericytes MBVP, neurons HT22, and human breast cancer brain metastases 231Br. B shows the expression of LRP1 in endothelial cells bEnd.3, pericytes MBVP, neurons HT22, and human breast cancer brain metastases 231Br. A and B are from the same sample. C and D are quantitative plots of the characterization results of A and B, respectively.

[0032] Figure 5 The image shows the colocalization of GRP94 and LRP1 with brain cells in a mouse model of breast cancer brain metastasis using confocal microscopy. In the image, A shows the colocalization of GRP94 and LRP1 with astrocytes, neurons, and tumor cells, respectively; B shows the colocalization of GRP94 and LRP1 with tumor cells surrounding astrocytes, respectively; and C is a quantitative graph of the colocalization coefficients related to the characterization results in A.

[0033] Figure 6 Figure 1 shows the expression of GRP94 and LRP1 in endothelial cells bEnd.3, pericytes MBVP, neurons HT22, and mouse breast cancer brain metastases 4TBr by Western blot.

[0034] Figure 7 Figure 1 shows the expression of GRP94 and LRP1 in endothelial cells bEnd.3, human breast cancer cells 231, human breast cancer brain metastases 231Br, mouse breast cancer cells 4T1, and mouse breast cancer brain metastases 4T1Br by Western blot.

[0035] Figure 8 Western blot was used to characterize the expression of NMDAR2B in endothelial cells bEnd.3, pericytes MBVP, neurons HT22, human breast cancer brain metastases 231Br, and mouse breast cancer brain metastases 4TBr, and to quantify the grayscale values ​​of the characterization results.

[0036] Figure 9 To provide a quantitative graph of the expression of GRP94 on the cell membranes of neurons HT22, human breast cancer brain metastases 231Br and mouse breast cancer brain metastases 4T1Br, and the proportion of GRP94 on different cell membranes in the total cell characterization results.

[0037] Figure 10The image shows the uptake of the doxorubicin-loaded brain-targeted drug delivery system (targeting peptide OmpA modified nanoparticles, GRP94 antibody modified nanoparticles, and Angiopep 2 modified nanoparticles) of the present invention on neuron HT22, which is used to quantitatively characterize the doxorubicin concentration of 5 μg / ml and the incubation time of 3 h.

[0038] Figure 11 The image shows the uptake of the doxorubicin-loaded brain-targeted drug delivery system (targeting peptide OmpA modified nanoparticles, GRP94 antibody modified nanoparticles, and Angiopep 2 modified nanoparticles) described in this invention on human breast cancer brain metastases 231Br, which is quantitatively characterized. The doxorubicin concentration was 5 μg / ml and the incubation time was 3 h.

[0039] Figure 12 The image shows the uptake of the doxorubicin-loaded brain-targeted drug delivery system (targeting peptide OmpA modified nanoparticles, GRP94 antibody modified nanoparticles, and Angiopep 2 modified nanoparticles) described in this invention on mouse breast cancer brain metastases 4T1Br, which is quantitatively characterized. The doxorubicin concentration was 5 μg / ml and the incubation time was 3 h.

[0040] Figure 13 To quantitatively characterize the uptake ratio of the OmpA-modified nanoparticles of the target peptide described in this invention compared with the Angiopep 2-modified nanoparticles in neurons HT22, human breast cancer brain metastases 231Br, and mouse breast cancer 4T1Br, the concentration of doxorubicin was 5 μg / ml and the incubation time was 3 h.

[0041] Figure 14 To quantitatively characterize the uptake ratio of the GRP94 antibody-modified nanoparticles described in this invention compared with the Angiopep2-modified nanoparticles in neuronal HT22, human breast cancer brain metastases 231Br, and mouse breast cancer 4T1Br, the concentration of doxorubicin was 5 μg / ml and the incubation time was 3 h.

[0042] Figure 15 The distribution of the doxorubicin-loaded, targetable GRP94 biomimetic nanosystem in different brain regions of normal mice, characterized by confocal microscopy, is shown in the image. The doxorubicin dose was 5 mg / kg in mice, administered twice at 12-hour intervals, with perfusion occurring 12 hours after the second injection.

[0043] Figure 16 The image shows the distribution of doxorubicin-loaded Angiopep 2 modified nanoparticles in different brain regions of normal mice, characterized by confocal microscopy. The doxorubicin dose was 5 mg / kg mice, injected twice with an interval of 12 h. Perfusion was performed 12 h after the second injection.

[0044] Figure 17 The uptake map of the Dir-loaded nanosystem (ordinary nanoparticles, erythrocyte membrane biomimetic nanoparticles, and GRP94-targeting biomimetic nanoparticles) described in this invention on human breast cancer brain metastases 231Br is shown for quantitative characterization. The concentration of Dir at the administered dose is 2 μg / ml, and the administration time is 1.5 h.

[0045] Figure 18 The image shows the uptake of a Dir-loaded GRP94 biomimetic nanosystem on human breast cancer brain metastases 231Br after treatment with antibodies targeting the OmpA peptide of different volumes for 2 hours (Dir concentration was 2 μg / ml, administration time was 1.5 h). Detailed Implementation

[0046] The novel target GRP94 is highly expressed on the cell membrane of brain metastases, but its expression is low and predominantly intracellular in other normal cells in the brain parenchyma. Therefore, this invention aims to construct a targeted drug delivery system for brain metastases that can penetrate the blood-brain barrier and bypass normal cells based on GRP94 targeting, along with its preparation method. Glucose-regulated protein GRP94 is normally located in the endoplasmic reticulum of normal cells, but it is overexpressed and translocated to the cell membrane in breast cancer brain metastases. This invention provides a targeted drug delivery system for breast cancer brain metastases that bypasses normal brain cells based on GRP94 targeting. Using biodegradable polymer materials as the base carrier, a drug delivery system with surface-modified GRP94 antibodies or the targeting peptide OmpA is prepared. This system can penetrate the blood-brain barrier by targeting GRP94, specifically recognizing breast cancer brain metastases rather than normal brain cells due to the lack of GRP94 on normal brain cell membranes. Compared to other target receptors that cross the blood-brain barrier for brain-targeted drug delivery, targeting GRP94 more specifically targets brain metastases. GRP94 is expressed only on the surface of blood-brain barrier endothelial cells and breast cancer brain metastases, and does not lead to drug accumulation or neurotoxicity in normal intracranial areas, making GRP94 a novel target for targeted drug delivery to brain metastases.

[0047] The preparation method of the above-mentioned targeted drug delivery system for brain metastases based on GRP94 that can bypass normal brain cells includes:

[0048] Step 1: Weigh the polymer material and dissolve it in an organic solvent to form an oil phase;

[0049] Step 2: Dissolve the drug doxorubicin and the fluorescent probe Dir in the oil phase to form part of the oil phase. The aqueous phase is water itself, or the drug can be dissolved in water to form the aqueous phase.

[0050] Step 3: Add the aqueous phase dropwise to the swirling oil phase and ultrasonically emulsify to form a water-in-oil emulsion;

[0051] Step 4: Add the emulsion dropwise to the external aqueous phase of the vortex and ultrasonically emulsify to form a water-in-oil-in-water complex emulsion;

[0052] Step 5: Quickly pour the emulsion into the evaporating aqueous phase, stir overnight to evaporate, and form a nanoparticle suspension;

[0053] Step 6: Centrifuge the nanoparticle suspension at low speed and collect the supernatant;

[0054] Step 7: The supernatant is purified by high-speed centrifugation to obtain purified and unmodified nanoparticle precipitate;

[0055] Step 8: The nanoparticle precipitate is ultrasonically dispersed in a buffer solution, and the surface is modified with functional linking molecules. After reacting at room temperature for 1-2 hours, excess functional linking molecules are removed by high-speed centrifugation, and the precipitate is obtained to obtain nanoparticles with surface-modified functional linking molecules.

[0056] Step 9: Disperse the nanoparticles with surface-modified functional linker molecules in a buffer solution using ultrasonication. Modify the surface with GRP94 antibody or targeting peptide OmpA through the functional linker molecules. After reacting for 1-2 hours, centrifuge at high speed to obtain surface-modified GRP94 antibody or targeting peptide OmpA nanoparticles.

[0057] Step 10: The surface-modified GRP94 antibody or targeting peptide OmpA nanoparticles are ultrasonically dispersed in water and centrifuged at high speed to obtain a precipitate, thereby obtaining a targeted drug delivery system for breast cancer brain metastases based on targeting GRP94 to avoid normal brain cells.

[0058] Step 11: Disperse the targeted drug delivery system for breast cancer brain metastases based on targeting GRP94 to avoid normal brain cells in ultrapure water.

[0059] Please refer to the above-prepared targeted drug delivery system for breast cancer brain metastases based on GRP94 that avoids normal brain cells. Figure 1 , Figure 1 This is a schematic diagram illustrating the construction and in vivo behavior of the targeted drug delivery system for breast cancer brain metastases based on GRP94 targeting to avoid normal brain cells, as described in this invention. Figure 1 As shown, the brain metastasis targeted drug delivery system of this invention first penetrates the blood-brain barrier through the target GRP94, enters the brain parenchyma, avoids normal brain cells, and specifically targets breast cancer brain metastases without causing unnecessary neurotoxicity. The aforementioned breast cancer brain metastasis drug delivery system based on GRP94, which avoids normal brain cells, can be used to develop drug formulations that penetrate the blood-brain barrier, target brain metastases, and bypass normal brain parenchyma cells.

[0060] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, the technical solutions of the present invention are further described below with reference to embodiments. However, the present invention is not limited to the listed embodiments, but should also include any other known modifications within the scope of the claims of the present invention.

[0061] The term "an embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the invention. The phrase "in one embodiment" appearing in different places throughout this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0062] Example 1

[0063] This implementation case demonstrates the preparation method of a targeted drug delivery system for breast cancer brain metastases based on GRP94 targeting to avoid normal brain cells, as follows:

[0064] Weigh out the polymer material and dissolve it in an organic solvent to form an oil phase; if a drug is loaded, dissolve the drug in the oil phase to become part of the oil phase, and the aqueous phase is water itself, or the drug is dissolved in water to form an aqueous phase; add the aqueous phase dropwise to the vortexed oil phase and ultrasonically emulsify to form a water-in-oil emulsion; then add the emulsion dropwise to the outer aqueous phase of the vortex and ultrasonically emulsify to form a water-in-oil-in-water complex emulsion; quickly pour the complex emulsion into the volatile aqueous phase, stir overnight to evaporate, and form a nanoparticle suspension;

[0065] The above nanoparticle suspension was centrifuged at low speed to collect the supernatant; the centrifugation speed was 1000-1500 r and the centrifugation time was 10 min; the nanoparticle suspension was purified by high-speed centrifugation at a speed of 35000 r and a centrifugation time of 20 min to obtain purified and unmodified nanoparticle precipitate.

[0066] The above nanoparticles were ultrasonically dispersed in a phosphate buffer solution with a pH of 7.4, and surface-modified with functional linker molecules (PEG). After reacting at room temperature for 1-2 hours, excess functional linker molecules were removed by high-speed centrifugation, and the precipitate was obtained to obtain nanoparticles with surface-modified functional linker molecules.

[0067] Nanoparticles with the aforementioned surface-modified functional linker molecules were ultrasonically dispersed in phosphate buffer at pH 7.4. The surface-modified targeting peptide OmpA and GRP94 antibody were then reacted with the functional linker molecules at room temperature for 1-2 hours. Excess fusion peptide was removed by rapid centrifugation, and the precipitate was obtained, yielding nanoparticles with surface-modified GRP94 antibody or targeting peptide OmpA. Finally, the nanoparticles were ultrasonically dispersed in water, centrifuged at high speed, and precipitated to obtain the final nanoparticles, i.e., the brain metastasis targeted drug delivery system based on GRP9 targeting and penetrating the blood-brain barrier to avoid normal brain cells. The final precipitated nanoparticle drug delivery system was dispersed in ultrapure water.

[0068] Example 2

[0069] To investigate the penetration of the blood-brain barrier by the GRP94-targeted nanosystem, the uptake of doxorubicin-loaded nanoparticles on brain microvascular endothelial cells was quantitatively assessed. Mouse brain endothelial cells (bEND.3) were seeded in 6-well plates, and nanoparticles modified with functional linker molecules, Angiopep 2, OmpA-targeting peptides, and GRP94 antibody were added, respectively. The concentration of doxorubicin was 5 μg / mL. Cell uptake was detected by flow cytometry using the FL2 channel. The conclusions of this example can be found in [link to relevant documentation]. Figure 2 , Figure 2 This image shows the uptake of doxorubicin-loaded brain-targeted drug delivery systems (unmodified nanoparticles, Angiopep 2-modified nanoparticles, OmpA-modified nanoparticles, and GRP94 antibody-modified nanoparticles) on brain microvascular endothelial cells, as quantitatively characterized. Figure 2 As shown, compared with unmodified nanocarriers, Angiopep 2 modified nanoparticles, OmpA-modified nanoparticles targeting peptides, and GRP94 antibody-modified nanoparticles were taken up by bEND.3 cells more, with GRP94 antibody-modified nanoparticles being taken up more, indicating that they are more likely to accumulate in endothelial cells.

[0070] Example 3

[0071] To investigate the expression of GRP94 on the cell membrane of endothelial cells (bEnd.3), the proportion of GRP94 protein on the cell membrane relative to the total cell mass was examined using confocal microscopy. Please refer to [link to relevant documentation]. Figure 3 , Figure 3 This is a schematic diagram illustrating the expression of GRP94 protein on the cell membrane of endothelial cells bEnd.3 as characterized by confocal microscopy. Figure 3 As shown, confocal microscopy revealed the expression of GRP94 protein on the endothelial cell membrane, demonstrating the feasibility of penetrating the blood-brain barrier by targeting GRP94.

[0072] Example 4

[0073] To investigate the expression of GRP94 and LRP1 proteins in different cell lysates, Western blot was used to characterize the differences in GRP94 and LRP1 protein expression in different cell lysates. (Please refer to [link to relevant documentation]). Figure 4 , Figure 4 This is a schematic diagram illustrating the expression of GRP94 in various cells of the brain environment using Western blot. (Example) Figure 4As shown, Western blot analysis revealed that GRP94 expression was significantly higher in brain metastases than in normal brain cells, while LRP1, which can also mediate crossing of the blood-brain barrier, was significantly higher in normal brain cells than in breast cancer brain metastases. This demonstrates that targeting GRP94 can specifically target breast cancer brain metastases, bypassing normal brain cells.

[0074] Example 5

[0075] To investigate the colocalization of GRP94 and LRP1 proteins with different cells in mice, confocal microscopy was used to examine the colocalization of GRP94 and LRP1 proteins with different cells. Please refer to [link / reference]. Figure 5 , Figure 5 This image shows the colocalization of GRP94 and LRP1 with brain cells in a mouse model of breast cancer brain metastasis using confocal microscopy. Figure 5 As shown in the confocal characterization map, GRP94 protein has a higher co-localization coefficient with brain metastases compared to LRP1, while it is largely unco-localized with normal cells in the brain parenchyma. LRP1, on the other hand, co-localizes with astrocytes and neurons in the brain parenchyma, but is largely unco-localized with breast cancer brain metastases. This demonstrates that targeting GRP94 specifically targets breast cancer brain metastases, avoiding normal brain cells.

[0076] Example 6

[0077] To investigate the expression of GRP94 and LRP1 proteins in different cell lysates, Western blot was used to characterize the differences in GRP94 and LRP1 protein expression in different cell lysates. (Please refer to [link to relevant documentation]). Figure 6 , Figure 6 The image shows the expression of GRP94 and LRP1 in endothelial cells (bEnd.3), pericytes (MBVP), neurons (HT22), and murine breast cancer brain metastases (4TBr) as characterized by Western blot. Figure 6 As shown, Western blot analysis revealed that GRP94 expression was slightly higher in murine brain metastases than in normal brain cells, while LRP1, which can also mediate crossing of the blood-brain barrier, was significantly higher in neurons than in breast cancer brain metastases. This demonstrates that targeting GRP94 specifically targets breast cancer brain metastases, bypassing normal brain cells.

[0078] Example 7

[0079] To investigate the expression of GRP94 and LRP1 proteins in different cell lysates, Western blot was used to characterize the differences in GRP94 and LRP1 protein expression in different cell lysates. (Please refer to [link to relevant documentation]). Figure 7 , Figure 7The figure shows the expression of GRP94 and LRP1 in endothelial cells (bEnd.3), human breast cancer cells (231), human breast cancer brain metastases (231Br), mouse breast cancer cells (4T1), and mouse breast cancer brain metastases (4T1Br) as characterized by Western blot. Figure 7 As shown, Western blot analysis revealed that GRP94 was most highly expressed in human brain metastases, demonstrating the potential of targeting breast cancer brain metastases specifically with respect to GRP94, bypassing normal brain cells, in therapeutic drug formulations.

[0080] Example 8

[0081] To investigate the expression of NMDAR2B protein in different cell lysates, Western blot was used to characterize the differences in NMDAR2B protein expression in different cell lysates. Please refer to [link to relevant documentation]. Figure 8 , Figure 8 This study used Western blot to characterize the expression of NMDAR2B in endothelial cells (bEnd.3), pericytes (MBVP), neurons (HT22), human breast cancer brain metastases (231Br), and mouse breast cancer brain metastases (4TBr), and generated a quantitative graph of the characterization results using grayscale values. (See attached graph.) Figure 8 As shown, western blot analysis revealed that NMDAR2B can mediate crossing of the blood-brain barrier, but its expression in normal cells of the brain parenchyma is higher than that in tumor cells.

[0082] Example 9

[0083] To investigate GRP94 expression in brain parenchyma, the proportion of GRP94 protein on the cell membranes of various cell types was examined using confocal microscopy. (See also: [link to relevant documentation]). Figure 9 , Figure 9 This image provides a quantitative representation of the expression of GRP94 on the cell membranes of neurons HT22, human breast cancer brain metastases 231Br, and mouse breast cancer brain metastases 4T1Br using confocal microscopy, as well as the proportion of GRP94 on different cell membranes in the total cell count. Figure 9 As shown, confocal microscopy revealed that the vast majority of GRP94 was localized on the cell membranes of breast cancer brain metastases, but it was not co-localized on the cell membranes of neurons. This demonstrates the potential of targeting breast cancer brain metastases specifically with respect to GRP94, avoiding normal brain cells, in therapeutic drug formulations.

[0084] Example 10

[0085] To investigate the targeting effect of the GRP94-targeting nanosystem on neurons, the uptake of doxorubicin-loaded nanodelivery system on neurons was quantified. Mouse neurons (HT22) were seeded in 6-well plates, and functional linker-modified nanoparticles, Angiopep 2-modified nanoparticles, OmpA-targeting peptide-modified nanoparticles, and GRP94 antibody-modified nanoparticles were added, respectively. The concentration of doxorubicin was 5 μg / mL. Cell uptake was detected by flow cytometry using the FL2 channel. The conclusions of this example can be found in [link to relevant documentation]. Figure 10 , Figure 10 The image shows the uptake of the doxorubicin-loaded brain-targeted drug delivery system (targeting peptide OmpA modified nanoparticles, GRP94 antibody modified nanoparticles, and Angiopep 2 modified nanoparticles) on neuron HT22, as quantitatively characterized by the present invention. Figure 10 As shown, compared with functional linker nanoparticles, Angiopep 2 modified nanoparticles were taken up more by neuronal cells. There was no significant difference in the uptake of targeted peptide OmpA modified nanoparticles and GRP94 antibody modified nanoparticles compared with functional linker nanoparticles. This proves that by targeting GRP94 and avoiding normal brain cells, unnecessary neurotoxicity will not be caused.

[0086] Example 11

[0087] To investigate the targeting efficacy of the GRP94-targeted nanosystem on human breast cancer brain metastases, the uptake of doxorubicin-loaded nanoparticles on these cells was quantitatively assessed. Human breast cancer brain metastases (231Br) were seeded in 6-well plates, and nanoparticles modified with functional linker molecules, Angiopep 2, OmpA-targeting peptides, and GRP94 antibody were added, respectively. The concentration of doxorubicin was 5 μg / mL. Cell uptake was detected using the FL2 channel of flow cytometry. The conclusions of this example can be found in [link to relevant documentation]. Figure 11 , Figure 11 The image shows the uptake of the doxorubicin-loaded brain-targeted drug delivery system (targeting peptide OmpA modified nanoparticles, GRP94 antibody modified nanoparticles, and Angiopep 2 modified nanoparticles) described in this invention on human breast cancer brain metastases 231Br, for quantitative characterization. Figure 11 As shown, compared with functional linker nanoparticles, OmpA-modified nanoparticles and GRP94 antibody-modified nanoparticles were taken up more by 231Br cells, while Angiopep 2-modified nanoparticles showed no significant difference in uptake compared with functional linker nanoparticles. This demonstrates the potential of targeting breast cancer brain metastases specifically with respect to GRP94 as a target, avoiding normal brain cells, in therapeutic drug formulations.

[0088] Example 12

[0089] To investigate the targeting efficacy of the GRP94-targeted nanosystem on murine breast cancer brain metastases, the uptake of doxorubicin-loaded nanoparticles on human breast cancer brain metastases was quantitatively assessed. Murine breast cancer brain metastases (4T1Br) were seeded in 6-well plates, and nanoparticles modified with functional linker molecules, Angiopep 2, OmpA-targeting peptides, and GRP94 antibody were added, respectively. The concentration of doxorubicin was 5 μg / mL. Cell uptake was detected using the FL2 channel of flow cytometry. The conclusions of this example can be found in [link to relevant documentation]. Figure 12 , Figure 12 The image shows the uptake of the doxorubicin-loaded brain-targeted drug delivery system (targeting peptide OmpA modified nanoparticles, GRP94 antibody modified nanoparticles, and Angiopep 2 modified nanoparticles) described in this invention on mouse breast cancer brain metastases 4T1Br, for quantitative characterization. Figure 12 As shown, compared to functional linker nanoparticles, OmpA-modified nanoparticles and GRP94 antibody-modified nanoparticles were taken up more by 4T1Br cells, while Angiopep 2-modified nanoparticles showed no significant difference in uptake compared to functional linker nanoparticles. Figure 13 To quantitatively characterize the uptake ratio of the OmpA-modified nanoparticles of the present invention compared to the Angiopep 2-modified nanoparticles in neurons HT22, human breast cancer brain metastases 231Br, and mouse breast cancer 4T1Br, a graph was created. Figure 14 To quantitatively characterize the uptake ratios of the GRP94 antibody-modified nanoparticles described in this invention compared to Angiopep 2-modified nanoparticles in neurons HT22, human breast cancer brain metastases 231Br, and mouse breast cancer 4T1Br, as shown in the graph... Figure 13 and 14 As shown, this demonstrates the potential of therapeutic drug formulations that specifically target breast cancer brain metastases by targeting GRP94, bypassing normal brain cells.

[0090] Example 13

[0091] To investigate the co-localization of the doxorubicin-targeted GRP94 nanosystem in different regions and cells of mice, the co-localization was characterized using confocal microscopy. Normal mice were injected intravenously via the tail vein twice, 12 hours apart. Twelve hours after the second injection, the mice were perfused, and brain tissue was harvested, dehydrated with sucrose, and frozen sections were prepared. The distribution of the nanoparticles in different brain regions was observed using confocal microscopy. The dose of doxorubicin was 5 mg / kg of mouse per injection. The conclusions of this example can be found in the [link to previous section]. Figure 15-16 , Figure 15 This image shows the distribution of a doxorubicin-loaded, targetable GRP94 biomimetic nanosystem in different cells of different brain regions in a normal mouse brain, as characterized by confocal microscopy. Figure 16 This image shows the distribution of doxorubicin-loaded Angiopep 2-modified nanoparticles in different cells of different brain regions in normal mice, as characterized by confocal microscopy. Figure 15 As shown, the co-localization of the GRP94-targeted nanosystem with breast cancer brain metastases was significantly stronger in different regions than with normal brain parenchyma cells, such as... Figure 16 As shown, Angiopep 2-modified nanoparticles did not exhibit significant co-localization with breast cancer brain metastases in different regions, but showed some co-localization with neurons. In this embodiment, based on the target GRP94, avoiding normal brain cells, this specific targeting of breast cancer brain metastases demonstrates promise in therapeutic drug formulations while avoiding unnecessary neurotoxicity.

[0092] Example 14

[0093] To investigate the targeting efficacy of the GRP94-targeted nanosystem on human breast cancer brain metastases, the uptake of the Dir nanoparticle drug delivery system on these cells was quantitatively assessed. Human breast cancer brain metastases (231Br) were seeded in 6-well plates, and ordinary nanoparticles, erythrocyte membrane-inspired nanoparticles, and GRP94-targeted biomimetic nanoparticles were added, respectively. The concentration of Dir was 2 μg / ml. Cell uptake was detected using the FL4 channel of a flow cytometer. The conclusions of this example can be found in [link to relevant documentation]. Figure 17 , Figure 17 The image shows the uptake of the Dir-loaded nanosystems (ordinary nanoparticles, erythrocyte membrane biomimetic nanoparticles, and GRP94-targeted biomimetic nanoparticles) described in this invention on human breast cancer brain metastases 231Br, for quantitative characterization. Figure 17 As shown, compared to ordinary nanoparticles and erythrocyte membrane biomimetic nanoparticles, GRP94-targeted biomimetic nanoparticles are taken up more by 231Br cells. In this embodiment, the targeted biomimetic nanoparticles are based on the target GRP94 and specifically target breast cancer brain metastases.

[0094] Example 15

[0095] To investigate the uptake of the GRP94-targeting biomimetic nanosystem by human breast cancer brain metastases after treatment with OmpA antibody, human breast cancer brain metastases (231Br) were seeded in 6-well plates. Pretreatment with OmpA antibody-free, 7.5 μl, and 15 μl OmpA antibody-targeting nanosystems, respectively, for 1.5 h was performed. Then, the GRP94-targeting biomimetic nanosystem loaded with Dir (2 μg / ml) was added simultaneously and co-incubated with the aforementioned different doses of exogenous outer membrane proteins for 1.5 h. Cell uptake was detected using the FL4 channel of flow cytometry. The conclusions of this example can be found in [link to relevant documentation]. Figure 18 , Figure 18This is a graph showing the quantitative uptake of a Dir-loaded GRP94 biomimetic nanosystem on human breast cancer brain metastases 231Br after treatment with antibodies targeting the OmpA peptide of different volumes for 2 hours (Dir concentration: 2 μg / ml, administration time: 1.5 h). Figure 18 As shown, the OmpA antibody can inhibit the uptake of the GRP94-targeting biomimetic nanosystem by human breast cancer brain metastases, up to 34%, confirming that the OmpA-targeting peptide participates in the uptake of the GRP94-targeting biomimetic nanosystem on human breast cancer brain metastases. In this embodiment, the targeted biomimetic nanoparticles are based on the target GRP94 and specifically target breast cancer brain metastases.

[0096] Compared with existing technologies, the beneficial effects of this invention are as follows: The targeted drug delivery system for breast cancer brain metastases based on GRP94 targeting to avoid normal brain cells, and its preparation method, as described in this invention, target brain metastases more specifically with GRP94 than other target receptors that cross the blood-brain barrier for targeted drug delivery. GRP94 is expressed only on the surface of blood-brain barrier endothelial cells and breast cancer brain metastases, and does not lead to drug accumulation or neurotoxicity in normal intracranial areas.

[0097] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A targeted drug delivery system for breast cancer brain metastases based on targeting GRP94 to avoid normal brain cells, characterized in that: Drug-loadable nanoparticles were prepared using biodegradable polymeric materials, specifically polylactic acid-glycolic acid (PLA), as a basic carrier. The nanoparticles were surface-modified with GRP94 antibody or the targeting peptide OmpA. Its preparation methods include: (1) Weigh out the polymer material and dissolve it in an organic solvent to form an oil phase; (2) The drug doxorubicin and the fluorescent probe Dir are dissolved in the oil phase to become part of the oil phase, and the aqueous phase is water itself, or the drug is dissolved in water to form an aqueous phase; (3) The aqueous phase is added dropwise to the swirling oil phase and ultrasonically emulsified to form a water-in-oil emulsion; (4) The emulsion is then added drop by drop to the external aqueous phase of the vortex and ultrasonically emulsified to form a water-in-oil-in-water complex emulsion; (5) Quickly pour the double emulsion into the volatile aqueous phase, stir overnight to evaporate, and form a nanoparticle suspension; (6) Centrifuge the nanoparticle suspension at low speed and collect the supernatant; (7) The supernatant is purified by high-speed centrifugation to obtain purified and unmodified nanoparticle precipitate; (8) The nanoparticles were ultrasonically dispersed in a buffer solution, and the surface was modified with functional linking molecules. After reacting at room temperature for 1-2 hours, the excess functional linking molecules were removed by high-speed centrifugation, and the precipitate was obtained to obtain nanoparticles with surface-modified functional linking molecules. (9) The nanoparticles of the surface-modified functional linker molecule are ultrasonically dispersed in a buffer solution, and the surface is modified with GRP94 antibody or targeting peptide OmpA by the functional linker molecule. After reacting for 1-2 hours, the nanoparticles of the surface-modified GRP94 antibody or targeting peptide OmpA are obtained by high-speed centrifugation. (10) The surface-modified GRP94 antibody or targeting peptide OmpA nanoparticles were ultrasonically dispersed in water and centrifuged at high speed to obtain a precipitate, thereby obtaining a targeted drug delivery system for breast cancer brain metastases based on targeting GRP94 to avoid normal brain cells. (11) Disperse the targeted drug delivery system for breast cancer brain metastases based on targeting GRP94 to avoid normal brain cells in ultrapure water.

2. The targeted drug delivery system for breast cancer brain metastases based on targeting GRP94 to avoid normal brain cells, as described in claim 1, is characterized in that: In step (1), the polymer material is polylactic acid-hydroxyacetic acid, and the organic solvent is ethyl acetate.

3. The targeted drug delivery system for breast cancer brain metastases based on targeting GRP94 to avoid normal brain cells, as described in claim 1, is characterized in that: In step (4), the external aqueous phase is polyvinyl alcohol with a mass fraction of 2.5%.

4. The targeted drug delivery system for breast cancer brain metastases based on targeting GRP94 to avoid normal brain cells, as described in claim 1, is characterized in that: In step (5), the volatile aqueous phase is polyvinyl alcohol with a mass fraction of 0.3%.

5. The targeted drug delivery system for breast cancer brain metastases based on targeting GRP94 to avoid normal brain cells according to claim 1, characterized in that: In step (6), the low-speed centrifugation speed is 1000-1500 r and the time is 10 min.

6. The targeted drug delivery system for breast cancer brain metastases based on targeting GRP94 to avoid normal brain cells, as described in claim 1, is characterized in that: In step (7), the high-speed centrifugation speed is 35000 r and the time is 20 min.

7. The targeted drug delivery system for breast cancer brain metastases based on targeting GRP94 to avoid normal brain cells according to claim 1, characterized in that: In step (8), the buffer solution is a phosphate buffer with a pH of 7.4, the functional linker is PEG, the high-speed centrifugation speed is 35000 r, and the time is 20 min.

8. The targeted drug delivery system for breast cancer brain metastases based on targeting GRP94 to avoid normal brain cells according to claim 1, characterized in that: In step (9), the buffer solution is a phosphate buffer solution with a pH of 7.4, and the high-speed centrifugation speed is 35000 r for 20 min.

9. The targeted drug delivery system for breast cancer brain metastases based on targeting GRP94 to avoid normal brain cells according to claim 1, characterized in that: In step (10), the high-speed centrifugation speed is 35000 r and the time is 20 min.

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