WO2018098705A1 - 葡聚糖磁性铁奈米颗粒、制备及在治疗癌症与造影的用途 - Google Patents

葡聚糖磁性铁奈米颗粒、制备及在治疗癌症与造影的用途 Download PDF

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WO2018098705A1
WO2018098705A1 PCT/CN2016/108073 CN2016108073W WO2018098705A1 WO 2018098705 A1 WO2018098705 A1 WO 2018098705A1 CN 2016108073 W CN2016108073 W CN 2016108073W WO 2018098705 A1 WO2018098705 A1 WO 2018098705A1
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cancer
dextran
magnetic iron
fedc
nanoparticle
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PCT/CN2016/108073
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French (fr)
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陈震宇
亚米德·阿提夫亚米德·阿里
许斐婷
黄旭山
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硕英生医股份有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
    • A61K49/10—Organic compounds
    • A61K49/12—Macromolecular compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to the use of a dextran magnetic iron nanoparticle conjugated to a small molecule active drug for the treatment of cancer and contrast.
  • Epidermal growth factor receptor is a transmembrane glycoprotein with tyrosine kinase activity, and EGFR affects cancer cell growth, apoptosis, angiogenesis, invasiveness and invasiveness. A variety of key signaling pathways. EGFR is overexpressed in a variety of solid tumors, including lung, rectal cancer, breast cancer, ovarian cancer, and head and neck cancer. EGFR performance is associated with adverse drug treatment (References 1, 2, 3, and 4).
  • erlotinib small molecule tyrosine kinase inhibitors
  • erlotinib small molecule tyrosine kinase inhibitors
  • Non-invasive magnetic resonance imaging is an important technique for the diagnosis, grading, staging, and follow-up of cancer in clinical applications.
  • MRI imaging requires the use of contrast probes such as iron oxide, manganese oxide, and gold. , silver and gadolinium nanoparticles (Ref. 10).
  • Nanoparticles contain drugs that confer target capabilities, including monoclonal antibodies, peptides, aptamers, and small molecules. Compared to other types of target molecules, small molecules are small in size, diverse in structure, and stable. Sex and low production costs make small molecules more suitable for clinical applications in the field of targeted anti-cancer nanoparticle treatment (Refs. 11, 12).
  • the object of the present invention is to provide a dextran magnetic iron nanoparticle, comprising a dextran layer, a magnetic iron nanoparticle and an active drug, wherein the dextran is coated in a thin layer on the magnetic iron nanoparticle. Additionally, the active drug is conjugated to the dextran layer by non-covalent bonding.
  • the magnetic iron nanoparticle is a single crystal layer of magnetic iron nanoparticle.
  • the thickness of the dextran layer coated outside the magnetic iron nanoparticle is less than 3 nm.
  • the dextran magnetic iron nanoparticle has a median diameter of less than 50 nm.
  • the dextran magnetic iron nanoparticle has a median diameter of less than 10 nm.
  • the active drug has an amine group.
  • the active drug is erlotinib.
  • Another object of the present invention is to provide a method for preparing dextran magnetic iron nano particles, and the preparation steps are as follows:
  • Step 1 Add ice-cold ammonia to an aqueous solution of FeCl 3 ⁇ 6H 2 O, FeCl 2 ⁇ 4H 2 O and dextran, stir on ice in an inert atmosphere, heat to 85 ° C for 1 hour, stir. To cool down, dialyzed with Milli-Q water for 3 days;
  • Step 2 The dextran coating was crosslinked with epichlorohydrin/NaOH and dialyzed against Milli-Q water for 3 days to produce a stable FeD NP;
  • Step 3 FeD NPs were treated with ammonia to produce a primary amino functional group on the surface of the nanoparticle, followed by dialysis with Milli-Q water for 3 days to produce FeDN NP;
  • Step 5 Excess active drug was added to FeDC NP, ultrasonicated, and stirred for 2 days at a controlled temperature of 25 ° C, sterilized using a 0.22 ⁇ M MCE filter and the active drug without binding was removed to produce FeDC- E NP.
  • the active drug is erlotinib.
  • a further object of the present invention is to provide a use of the above-described dextran magnetic iron nanoparticle for the preparation of a medical contrast composition.
  • the medical contrast is used for cancer imaging.
  • the cancer is a cancer that exhibits EGFR.
  • the cancer includes brain cancer, lung cancer, colorectal cancer, colorectal cancer, breast cancer, prostate cancer, liver cancer, pancreatic cancer, bladder cancer, stomach cancer, kidney cancer, salivary gland cancer, ovarian cancer, Uterine body cancer, cervical cancer, oral cancer, skin cancer, brain cancer, malignant lymphoma or leukemia.
  • the cancer is a cancer that exhibits EGFR.
  • the cancer includes brain cancer, lung cancer, colorectal cancer, colorectal cancer, breast cancer, prostate cancer, liver cancer, pancreatic cancer, bladder cancer, stomach cancer, kidney cancer, salivary gland cancer, ovarian cancer, Uterine body cancer, cervical cancer, oral cancer, skin cancer, brain cancer, malignant lymphoma or leukemia.
  • the dextran magnetic iron nanoparticle can be moved by an applied magnetic field and stagnated at the cancer site.
  • Figure 1 is a schematic diagram showing the steps of synthesizing FeDC-E NPs
  • Figure 3 is the effect of surface material changes on the zeta potential of nanoparticles
  • Figure 5 is a high resolution TEM observation of dextran coated magnetic iron nanoparticle
  • Figure 6 is a graph showing the particle size distribution of FeDC-E NP by DLS and TEM;
  • Figure 7 is the cytotoxic effect of nanoparticle on CL1-5-F4 and Jurkat;
  • Figure 8 is a Prussian blue analysis of nanoparticle into CL1-5-F4 cells
  • Figure 9 is a TEM observation of nanoparticles entering the CL1-5-F4 cells via endocytosis
  • Figure 10 shows the effect of erlotinib release on FeDC-E NP in different pH environments
  • Figure 11 is a graph showing the effect of nanoparticle on T2-weighted MRI images and T2 relaxation times of CL1-5-F4 cells;
  • Figure 12 is the effect of nanoparticle on the T2 signal intensity of CL1-5-F4 cells
  • Figure 13 is the effect of nanoparticle on the molecular signal of CL1-5-F4 cells
  • Figure 14 is a graph showing the effect of nanoparticle on the nuclear shift of NF- ⁇ B by immunochemical fluorescence imaging
  • Figure 15 is a possible mechanism by which FeDC-E NP inhibits the EGFR-ERK-NF ⁇ B pathway
  • Figure 16 is the effect of nanoparticle on the migration ability of CL1-5-F4 cells
  • Figure 17 is the effect of nanoparticle on the invasion ability of CL1-5-F4 cells.
  • Figure 18 is a graph showing the effect of nanoparticle on the tumor size of male BALB/c nude mice implanted with heterologous CL1-5-F4 cells;
  • Figure 19 is a graph showing the effect of nanoparticle on the body weight of male BALB/c nude mice implanted with heterologous CL1-5-F4 cells;
  • Figure 20 is a T2-weighted image and T2 signal intensity of male BALB/c nude mice implanted with xenogeneic CL1-5-F4 cells by nanoparticle observation by MRI.
  • Single crystal iron oxide nanoparticles are synthesized by alkaline coprecipitation (as shown in Figure 1).
  • ice-cold ammonia is added to FeCl 3 ⁇ 6H 2 O, FeCl 2 ⁇
  • the aqueous solution of 4H 2 O and dextran was stirred on ice in an inert atmosphere, heated to 85 ° C for 1 hour, stirred until cooled, and dialyzed against Milli-Q water for 3 days.
  • the dextran coating was crosslinked with epichlorohydrin/NaOH and dialyzed against Milli-Q water for 3 days to produce a stable FeD NP (dextran coated magnetic iron nano) Granules).
  • FeD NPs were treated with ammonia to produce primary ammonia functional groups on the surface of the nanoparticles, followed by dialysis with Milli-Q water for 3 days to produce FeDN NP (dextran coated magnetic iron nanoparticles with amino groups on the surface) .
  • erlotinib Excess erlotinib (erlotinib) was added to FeDC NP, ultrasonicated and stirred for 2 days at a controlled temperature of 25 °C, sterilized using a 0.22 ⁇ M MCE filter and the unconjugated erlotinib was removed, resulting in FeDC-E NP (Glucan-coated magnetic iron nanoparticle with erlotinib on the surface).
  • any amino-containing drug such as aspirin, steroids, etc.
  • Qn is the cumulative amount of erlotinib ( ⁇ g/mL) per unit time (minutes)
  • Cn is the drug concentration in the medium at each monitoring time point
  • Ci is the drug concentration of the sample at the i-th monitoring time point
  • V0 For the volume of the dissolved medium
  • Vi is the volume of the sample.
  • dextran shows a water OH stretching peak and a HOH bending peak near 3300 and 1600 cm-1, and a CH stretching peak near 2900 cm-1 and a strong CO peak at 1000 cm-1, indicating ⁇ of dextran. - alpha-glucopyranose ring.
  • the spectral peaks of FeD NP show the characteristics of dextran, especially in the fingerprint region, the intensity and characteristics of the peaks are slightly changed, indicating the coordination of dextran and magnetic iron on the surface of the nanoparticles.
  • the nanoparticle solution was placed on a strong magnet for one week to check the precipitate and aggregate of the solution to test the physical stability and aggregation of the nanoparticle.
  • It has strong physical stability, but the application of an external magnetic field can move all the nanoparticle solution in the tube.
  • a magnetic bed, a magnetic plate, a magnetic coat, a magnetic cap, or the like can be used as an external magnetic field, which is worn during the treatment to concentrate the magnetic iron nanoparticles. fixed.
  • the FeDC-E NPs solution was stored in a sealed glass tube at 25 ° C for one year. After one year of storage, visual and TEM microscopy revealed nano particles. Still completely dispersed, without particle agglomeration or precipitation, further illustrates the physical stability of this glucan coating.
  • the surface charge of the nanoparticles determined by zeta potential measurement (Fig. 3), the surface charge of FeD NPs is close to zero -0.3 ⁇ 0.1 mV, and the zeta potential of FeDN NPs is reversed to a positive value of 8.6 ⁇ 0.5 mV, FeDC NPs because The zeta potential of the carboxyl functionalization reversal was high negative -11.4 ⁇ 1.2 mV, and the zeta potential of FeDC-E NPs conjugated with erlotinib base was reduced to -2.4 ⁇ 0.4 mV, which was due to the carboxyl group of FeDC NPs. Neutralization of the N atom of erlotinib base produces a stable erlotinib-loaded nanoparticle.
  • the erlotinib content in the NP solution was 151.32 ⁇ g/mL, which was higher than the erlotinib water solubility (14.02 ⁇ g/mL). It can be known that the present invention is very effective in conjugated erlotinib to MION.
  • the nanoparticles are loaded with a large amount of erlotinib (more than ten times their water solubility).
  • This example conjugates erlotinib to the nanoparticles, maintaining the biological activity of erlotinib in FeDC-E NP, avoiding the chemical structure of the drug and impeding its biological activity due to other covalent attachment methods.
  • the morphology of the synthesized nanoparticles was observed by Transmission Electron Microscopy (TEM), and the dextran-coated magnetic iron nanoparticles were aggregated into irregular shapes and sizes (Fig. 4);
  • the dextran-coated FeD NP is dispersed and not aggregated, and the shape of the FeD NP is a regular spherical shape and is smaller in size than the nanoparticle having no dextran coating.
  • HRTEM High resolution TEM
  • FeDC-E NPs are magnetic iron core plus monodisperse isotopic-shaped particles with an average diameter of 4.28 ⁇ 1.1 nm and a polydispersity index (PDI) of 0.07.
  • the FeDC-E NP particle size measured by dynamic light scattering (DLS) was 6.06 ⁇ 0.9 nm (narrow log-normal size distribution), PDI was 0.02, which was consistent with the TEM results (Fig. 6).
  • the particle size of the sugar magnetic iron nanoparticle is less than 50 nm, preferably less than 10 nm, and the median is the frequency of occurrence. The highest particle size.
  • the present invention utilizes high concentration dextran and reacts on ice.
  • the dextran is instantaneously adsorbed on the surface of the newly formed MION, restricting the space around the nanoparticle and further aggregation, and producing a stable and ultra-small size nanoparticle in the solution. It can be completely dispersed, and the dextran coating on the surface of the nanoparticles can prevent the aggregation generated during the synthesis of MION.
  • the Theranostic probe is characterized by targeting, therapeutic and imaging capabilities.
  • CL1-5-F4 cells overexpressing EGFR
  • EGFR epidermal growth factor receptor
  • erlotinib erlotinib
  • CL1-5-F4 cells are derived from human lung adenocarcinoma cells, overexpressing EGFR, with highly invasive and migrative cells; Jurkat cells are human lymphoid cancer cells, commonly used as representatives of EGFR cell.
  • This example tested the sensitivity of two EGFR status cells to erlotinib.
  • the results in Figure 7 show that erlotinib has toxic effects on CL1-5-F4 cells with high EGFR expression, and is concentration dependent, on the contrary. High concentrations of erlotinib did not cause toxic effects on Jurkat cells without EGFR expression.
  • the erlotinib-free FeDC NP was not cytotoxic on both CL1-5-F4 and Jurkat cells, indicating the biocompatibility of FeDC NPs nanoparticles.
  • the toxicity of erlotinib-containing FeDC-E NP on cells was similar to that of erlotinib alone.
  • FeDC-E NP significantly inhibited the survival of CL1-5-F4 cells, and the cytotoxicity against Jurkat was statistically significant. Significant differences on. The results of cell survival showed that conjugation with nanoparticle did not affect the activity of erlotinib, and FeDC NP itself was not cytotoxic. In addition, the selective cytotoxic effect of FeDC-E NPs indicated that FeDC-E NP could be used to identify overexpression. Tumor cells of EGFR.
  • this example tested the cellular uptake capacity of nanoparticles using Prussian blue staining ( Figure 8) on CL1-5-F4 cells. None of the control group and erlotinib-treated cells showed any blue staining spots and the staining reagent did not precipitate, indicating that no endogenous iron was present in the cells.
  • the cells treated with FeDC NPs had several blue spots with a staining density of 10.69% (representing the amount of FeDC NP uptake by the cells).
  • FeDC-E NPs The treated cells had a 4-fold increase in uptake of the nanoparticles, a strong blue spot observed under the microscope, and a dyeing density of 42.53%.
  • this example examined the CL1-5-F4 cells of each treatment group using TEM (Fig. 9), wherein untreated and The cells treated with erlotinib showed clear cells and nuclear membranes without any electron dense areas.
  • the cells treated by FeDC NPs have electron-intensive endocytic vesicles of nanoparticles (high electron density is due to the magnetic iron core of nanoparticles), and cells treated with FeDC-E NPs have similar dense electron endocytosis of nanoparticles. There are more endocytotic vesicles and more cells than FeDC NPs.
  • Example 6 pH affects the release of erlotinib from nanoparticles
  • FeDC-E NPs are phagocytosed by endocytotic vesicles, the pH of the humoral and extracellular environments is 7.4, and the late endosomes of the cells have a pH of about 5, this example Simulated the effects of FeDC-E NPs on the release of erlotinib in different cellular physiological environments.
  • the cumulative release of erlotinib after 76 minutes was 76.7%
  • the same 140 minutes the cumulative erlotinib release 60.8%, statistically significant (Figure 10).
  • erlotinib released from FeDC-E-NPs is low during the circulation of nanoparticles in the blood until it reaches the tumor site and is swallowed by the cells to the low pH environment of the endocytic vesicles.
  • Release of erlotinib, that is, FeDC-E NPs have a pH-adjusted intelligent release system that increases drug delivery to tumor cells and reduces systemic effects, optimizing overall treatment outcomes.
  • this embodiment treats CL1-5-F4 cells with different nanoparticles to be in vitro.
  • the MRI experiment measured the change in T2 relaxation time and the signal intensity (Fig. 11).
  • the median T2 flaccid time of untreated cells was 103.9 ms
  • the median T2 flaccid time of FeDC NPs treated cells was 92.5 ms, indicating that the amount of iron absorbed by the cells was comparable in MRI.
  • Treatment of cells with FeDC-E NPs significantly reduced the median T2 flaccid time to 84.2 ms (significantly different from FeDC NPs treated cells, Figure 11).
  • the T2 signal intensity of the three treatment groups was the most different at the echo time of 8.5 ms, wherein the median T2 signal intensity of the untreated cells was 191.4, and the median T2 signal intensity of the cells treated with FeDC NPs was 183.5.
  • the median T2 signal intensity of FeDC-E NPs treated cells was 159.8 (the statistically significant difference in T2 intensity between FeDC-E NPs treated cells and untreated and FeDC NPs treated cells, Figure 12).
  • FeDC-E NPs are sufficient for MRI tracking by uptake and accumulation in cells via cells, and therefore are suitable for use as therapeutic probes (Theranostic probe).
  • target-targeted FeDC-E NPs treated cells with and without labeling The T2 results of the target FeDC NP treated cells further confirmed the ability to confer nanoparticle targeting by conjugation of erlotinib.
  • Example 8 Nanoparticles inhibit EGFR and ERK signaling pathways
  • Erlotinib is a highly selective small molecule drug characterized by the tyrosine kinase domain of EGFR, which is effective in inhibiting the EGFR signaling pathway, and EGFR is involved in many downstream signaling pathways in cancer.
  • the extracellular-signal-regulated kinase (ERK) pathway can serve as a biomarker for the activation of EGFR inhibitors.
  • ERK-induced ERK stepwise activation involves signaling through Ras, Raf and mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK), termed the EGFR-Ras-Raf-MEK-ERK signaling pathway.
  • NF- ⁇ B transcription factor NF- ⁇ B
  • MMP-932 invasion and migration-enhanced matrix metallopeptidase 9
  • VEGF vascular endothelial growth factor
  • XIAP X-linked inhibitor of apoptosis protein
  • this example is The expression of EGFR and its phosphorylation (p-EGFR) in CL1-5-F4 cells was analyzed by Western blotting.
  • the untreated cells were found to have receptors of both EGFR and p-EGFR.
  • Erlotinib treatment did not affect EGFR performance, but inhibited EGFR phosphorylation, which is the primary mechanism of the drug as a tyrosine kinase inhibitor ( Figure 13).
  • Cells treated with FeDC-E NPs showed no altered expression of EGFR, but inhibited phosphorylation of EGFR, indicating that FeDC-E NPs can inhibit EGFR signaling.
  • FIG. 13 shows that the treatment of erlotinib and FeDC-E NPs significantly reduced the expression of p-ERK 1/2 (compared with untreated group and FeDC NPs treated group), and the ERK 1/2 expression of the four treatment groups did not change. .
  • Nanoparticles inhibit NF- ⁇ B signaling pathway and its regulated tumor promoting protein
  • the transcription factor NF- ⁇ B is a heterodimeric complex of the Rel family of proteins, and the Rel family including the RelA (p65) subunit is the most common dimeric subunit of NF- ⁇ B in cells.
  • NF- ⁇ B usually binds to I ⁇ B inhibitory protein and becomes inactive; after activation, inhibitory I ⁇ B is degraded, NF- ⁇ B is translocated to the nucleus and binds to the promoter region of DNA, and NF- ⁇ B target gene is activated. Transcription and performance.
  • this example uses Western blotting method to analyze the amount of NF- ⁇ B p65 subunit in CL1-5-F4 nuclear extract and use immunization. Cytochemical fluorescence imaging was performed to analyze the cytoplasmic translocation of NF- ⁇ B p65 subunit from CL1-5F4 cells to the nucleus. Figure 13 shows that high levels of NF- ⁇ B p65 subunits in the nuclei of untreated cells showed high activity of NF- ⁇ B, and FeDC NPs treated cells showed similar results to untreated cells, indicating drug-free nai.
  • NF- ⁇ B p65 subunits significantly accumulate in the cytoplasm, indicating that nuclear translocation of NF- ⁇ B is inhibited, and drug treatment inhibits NF- ⁇ B activity.
  • This example further analyzes the effect of nanoparticle treatment on the expression of NF- ⁇ B transcriptional regulatory proteins XIAP and MMP-9, which are involved in the progression of cancer.
  • the performance of XIAP in CL1-5-F4 cells treated with erlotinib and FeDC-E NP1 was significantly reduced (compared to untreated and FeDC NPs treated cells (Fig. 13).
  • MMP-9 enzyme was highly aggressive. And a large number of migrating cells, which are capable of degrading the cell basement membrane and extracellular matrix of type IV collagen, promoting its invasion and migration;
  • the performance of MMP-9 was significantly reduced in erlotinib and FeDC-E NP treated CL1-5-F4 cells (compared to untreated and FeDC NPs treated groups).
  • migration and invasion experiments will be performed in the following examples.
  • Figure 15 shows that FeDC-E NP produces potent anticancer activity via inhibition of the domain of tyrosine kinase of EGFR, inhibiting downstream ERK signal signaling pathway. Inhibition of ERK leads to inhibition of NF- ⁇ B activity and inhibition of NF- ⁇ B translocation from the cytoplasm to the nucleus. Negative regulation of the NF- ⁇ B target gene is associated with cancer promotion and progression, and NF- ⁇ B transcriptional activity is inhibited by XIAP (anti-apoptosis) and MMP-9 (migration and invasion) proteins that are further inhibited.
  • XIAP anti-apoptosis
  • MMP-9 migration and invasion
  • Example 11 nanoparticle inhibits migration and invasion of CL1-5-F4 cells
  • activated MMP-9 is shown to induce cell migration and invasion.
  • the previous examples found that FeDC-E NPs treatment of CL1-5-F4 cells reduced the performance of MMP-9, so this example uses a transwell to observe the effect of nanoparticle on migration and invasion, CL1-5- F4 cells migrated highly underneath the membrane of transwell, FeDC NPs treated with less cell migration than untreated cells (but not statistically significant), and erlotinib-treated cells migrated only to untreated cells. 21.75%, while FeDC-E NPs treated cells almost completely inhibited cell migration, with a mobility rate of 3.26% of untreated cells (Figure 16). Consistent with the results of MMP-9 expression in the previous examples, FeDC-E NPs potently inhibited cell migration.
  • Matrigel-coated transwell was further used to observe the effect of nanoparticle on cell invasion ability.
  • Untreated CL1-5-F4 cells showed a large number of cells invaded through Matrigel to transwell membrane, FeDC NPs-treated cells. Showing a small challenge potential (statistically no significant difference from untreated cell results), erlotinib-treated cells showed a significant reduction in invasive rate to 32.37% of untreated cells, FeDC-E NPs treated fine The invasive rate was 15.57% of untreated cells ( Figure 17), which was lower than erlotinib.
  • FeDC-E NPs significantly inhibited the invasion and migration of CL1-5F4 cells than the same concentration of erlotinib.
  • Example 12 In vivo therapeutic effect of nanoparticle
  • this embodiment uses a heterologous CL1-5-F4.
  • Cell-implanted male BALB/c nude mice were treated with Ctrl (untreated), erlotinib, FeDC NP, and FeDC-E NP, respectively, and the tumor volume of FeDC NPs-treated mice was not compared with the untreated group.
  • any statistically significant difference indicates that the nanoparticle does not have an intrinsic activity to inhibit tumor growth; erlotinib-treated mice significantly inhibited tumor growth compared to the untreated group, indicating that the tumor cells are sensitive to erlotinib, FeDC -E NP treatment significantly inhibited tumor growth in mice, indicating that FeDC-E NP has a therapeutic effect (Fig. 18).
  • FeDC-E NPs have slightly less tumor suppression than erlotinib, but there is no statistically significant difference, indicating FeDC-E NP retained the therapeutic activity of erlotinib after the formulation process.
  • Example 13 MRI contrast enhancement and targeting ability of nanoparticles tested in invasive non-invasive magnetic resonance imaging
  • this embodiment uses a heterologous CL1-5-F4 cell plant.
  • Male BALB/c nude mice were subjected to in vitro non-invasive MRI experiments.
  • Figure 20 shows tumor imaging of four treatment groups (Ctrl, Erlotinib, FeDC NP, FeDC-E NP) of the present embodiment, T2 weighted normalized MRI of regions of interest (ROI) before and after treatment.
  • the normalized T2-weighted MRI signal intensities were quantified.
  • the normalized signal intensity of the untreated and erlotinib-treated tumors did not change significantly after treatment.
  • the FeDC NPs- and FeDC-E NPs-treated groups were averaged.
  • the normalized signal intensity was reduced by approximately 1.6 and 2.8, and the normalized signal intensity of the FeDC-E NPs treated group was statistically significantly different from the other three treatment groups, indicating FeDC- compared to the non-targeted FeDC NP.
  • Targeted accumulation of E NPs in tumors is enhanced.
  • Such in vivo target MRI contrast enhancement further supports the possibility of the FeDC-E NPs of the invention being clinically useful as a Theranostic probe.
  • the present invention is the first to disclose a small molecule of erlotinib as a target agent for nanoparticle, and the nanoparticle of the present invention exhibits remarkable effects on highly invasive and migratory cancer cells. Intelligent treatment and standard nature, and can be monitored by MRI.
  • nanoparticle inhibits the expression of the EGFR-ERK-NF- ⁇ B signaling pathway and related tumor-promoting proteins MMP-9 and XIAP, and subsequently inhibits the migration and invasion of cancer cells. And confirming the nm with mice carrying xenografts Particles are targeted, therapeutic and imaging in vivo.
  • the ultra-small size of the nanoparticle formulation of the present invention makes it possible to use MRI for metastatic brain tumors.

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Abstract

一种葡聚糖磁性铁奈米颗粒，包括葡聚糖层、磁性铁奈米颗粒与活性药物，该葡聚糖为薄层包覆在该磁性铁奈米颗粒之外，该活性药物以非共价键结与该葡聚糖层缀合，该活性药物可为厄洛替尼，该葡聚糖磁性铁奈米颗粒可以用于治疗癌症与造影的用途。

Description

葡聚糖磁性铁奈米颗粒、制备及在治疗癌症与造影的用途 技术领域
本发明关于一种缀合小分子活性药物的葡聚糖磁性铁奈米颗粒用于治疗癌症与造影的用途。
背景技术
表皮生长因子受体(Epidermal growth factor receptor，EGFR)是具有酪氨酸激酶(tyrosine kinase)活性的跨膜糖蛋白，EGFR影响与癌细胞生长、凋亡、血管生成、侵袭性和侵袭性相关的多种关键信号传导途径。EGFR在多种实性瘤中过度表现，包括肺、直肠癌、乳腺癌、卵巢癌和头颈癌，EGFR表现与治疗的不良反应有关联性(参考文献1、2、3、4)。抑制EGFR的最有效的标靶策略之一是使用小分子酪氨酸激酶抑制剂，例如厄洛替尼，厄洛替尼已被证明对EGFR酪氨酸激酶具有高度选择性，会导致细胞周期停滞、抑制癌症的增殖且可以引起凋亡(参考文献4、5、6、7)。但患者对厄洛替尼治疗的反应有显著变化(参考文献8)且在治疗过程中会出现的后天的抗药性(acquire resistance)(参考文献9)，因此需要诊断工具来分类、识别肿瘤类型，并且在治疗期间定期监测治疗反应。
非侵入性核磁共振成像(magnetic resonance imaging，MRI)是临床应用中用于癌症的诊断、分级、分期和随访的一种重要技术，MRI成像需要利用对比探针，如氧化铁、氧化锰、金、银和钆(gadolinium)奈米颗粒(参考文献10)。
新兴的“诊治”奈米颗粒，具有治疗和诊断性质，具有诊断成像能力以及标靶药物递送的优势。奈米颗粒载有赋予标靶能力的药物，包括单株抗体、胜肽、适体(aptamer)和小分子等，与其他类型的标靶分子相比，小分子因为小尺寸、多样结构、稳定性和低生产成本，使小分子在标靶抗癌奈米颗粒治疗领域更适合用于临床应用(参考文献11、12)。
因此，如何设计出一种装载有小分子药物的奈米颗粒，具有治疗、标靶性质，更可以直接监测治疗反应，同时具有诊治(theranostic)的功效，即成为本发明在此欲解决的一重要课题。
发明内容
本发明的目的即在于提供一种葡聚糖磁性铁奈米颗粒，包括葡聚糖层、磁性铁奈米颗粒与活性药物，葡聚糖为薄层包覆在所述磁性铁奈米颗粒之外，所述活性药物以非共价键结与所述葡聚糖层缀合。
为达前述发明目的，所述磁性铁奈米颗粒为单晶层磁性铁奈米颗粒。
为达前述发明目的，包覆于磁性铁奈米颗粒外的所述葡聚糖层厚度小于3奈米。
为达前述发明目的，所述葡聚糖磁性铁奈米颗粒的粒径仲数小于50奈米。
为达前述发明目的，所述葡聚糖磁性铁奈米颗粒的粒径仲数小于10奈米。
为达前述发明目的，所述活性药物具有胺基。
为达前述发明目的，所述活性药物为厄洛替尼。
本发明的另一目的即在于提供一种葡聚糖磁性铁奈米颗粒的制备方法，制备步骤如下：
步骤1：将冰冷的氨加入FeCl3·6H2O、FeCl2·4H2O和葡聚糖的水溶液中，于惰性气体环境中置于冰上搅拌，再加热至85℃维持1小时，搅拌至冷却，以Milli-Q水进行透析3天；
步骤2：葡聚糖(dextran)涂层使用环氧氯丙烷/氢氧化钠(Epichlorohydrin/NaOH)交联，用Milli-Q水进行透析3天，产生稳定的FeD NP；
步骤3：FeD NPs用氨处理以在奈米颗粒的表面上产生一级氨官能基，随后用Milli-Q水进行透析3天，产生FeDN NP；
步骤4：FeDN NP与琥珀酸酐(succinic anhydride)在缓冲溶液(pH＝8.5，0.1M NaHCO3)中反应，随后用Milli-Q水进行透析3天以充分透析，然后通过加入4-吗啉乙磺酸(Morpholineethanesulfonic acid)和氯化钠缓冲溶液，产生FeDC NP；
步骤5：将过量的活性药物加入FeDC NP中，超音波处理，并在25℃的控制温度下搅拌2天，使用0.22μM MCE过滤器灭菌且移除没有接合上的活性药物，产生FeDC-E NP。
为达前述发明目的，所述活性药物为厄洛替尼。
本发明的再一目的即在于提供一种如上述葡聚糖磁性铁奈米颗粒用于制备医学造影组合物的用途。
为达前述发明目的，所述医学造影用于癌症造影。
为达前述发明目的，所述癌症为表现EGFR的癌症。
为达前述发明目的，所述癌症包括脑癌、肺癌、大肠癌、结肠直肠癌、乳癌、摄护腺癌、肝癌、胰脏癌、膀胱癌、胃癌、肾癌、唾腺癌、卵巢癌、子宫体癌、子宫颈癌、口腔癌、皮肤癌、脑癌、恶性淋巴瘤或白血病。
本发明的又一目的即在于提供一种上述葡聚糖磁性铁奈米颗粒用于制备治疗癌症医药组合物的用途。
为达前述发明目的，所述癌症为表现EGFR的癌症。
为达前述发明目的，所述癌症包括脑癌、肺癌、大肠癌、结肠直肠癌、乳癌、摄护腺癌、肝癌、胰脏癌、膀胱癌、胃癌、肾癌、唾腺癌、卵巢癌、子宫体癌、子宫颈癌、口腔癌、皮肤癌、脑癌、恶性淋巴瘤或白血病。
为达前述发明目的，所述葡聚糖磁性铁奈米颗粒可以利用外加磁场移动且停滞于所述癌症位置。
附图说明
图1为FeDC-E NPs合成步骤示意图；
图2为奈米颗粒的红外线光谱(FT-IR)分析图；
图3为奈米颗粒表面物质变化对zeta电位影响；
图4为以TEM观察奈米颗粒的影像及示意图；
图5为以高分辨率TEM观察葡聚糖涂覆的磁性铁奈米颗粒；
图6为以DLS及TEM分析FeDC-E NP粒径分布；
图7为奈米颗粒对CL1-5-F4、Jurkat细胞毒性影响；
图8为以普鲁士蓝分析奈米颗粒进入CL1-5-F4细胞；
图9为以TEM观察奈米颗粒经由胞吞进入CL1-5-F4细胞；
图10为于不同pH环境对FeDC-E NP厄洛替尼释出的影响；
图11为奈米颗粒对CL1-5-F4细胞T2加权性MRI影像及T2弛缓时间(T2relaxation times)的影响；
图12为奈米颗粒对CL1-5-F4细胞T2信号强度的影响；
图13为奈米颗粒对CL1-5-F4细胞分子信号的影响；
图14为以免疫化学荧光成像分析奈米颗粒对NF-κB核位移的影响；
图15为FeDC-E NP抑制EGFR-ERK-NFκB途径的可能机制；
图16为奈米颗粒对CL1-5-F4细胞迁移能力的影响；
图17为奈米颗粒对CL1-5-F4细胞侵袭能力的影响；
图18为奈米颗粒对携带异种CL1-5-F4细胞植入的雄性BALB/c裸鼠肿瘤尺寸的影响；
图19为奈米颗粒对携带异种CL1-5-F4细胞植入的雄性BALB/c裸鼠体重的影响；
图20为以MRI观察奈米颗粒对携带异种CL1-5-F4细胞植入的雄性BALB/c裸鼠肿瘤的T2加权性影像及T2信号强度。
具体实施方式
本发明以下面的实施例予以示范阐明，但本发明不受下述实施例所限制。
奈米颗粒合成
单晶层氧化铁奈米颗粒(monocrystalline iron oxide nanoparticles，MION)合成利用碱性共沉淀法(如图1所示)，简言之，将冰冷的氨加入FeCl3·6H2O、FeCl2·4H2O和葡聚糖的水溶液中，于惰性气体环境中置于冰上搅拌，再加热至85℃维持1小时，搅拌至冷却，以Milli-Q水进行透析3天。葡聚糖(dextran)涂层使用环氧氯丙烷/氢氧化钠(Epichlorohydrin/NaOH)交联，用Milli-Q水进行透析3天，产生稳定的FeD NP(葡聚糖涂层磁性铁奈米颗粒)。FeD NPs用氨处理以在奈米颗粒的表面上产生一级氨官能基，随后用Milli-Q水进行透析3天，产生FeDN NP(表层有氨基的葡聚糖涂层磁性铁奈米颗粒)。FeDN NP与琥珀酸酐(succinic anhydride)在缓冲溶液(pH＝8.5，0.1M NaHCO3)中反应，随后用Milli-Q水进行透析3天以充分透析，然后通过加入4-吗啉乙磺酸(Morpholineethanesulfonic acid)和氯化钠缓冲溶液，产生FeDC NPs(表层有羧基的葡聚糖涂层磁性铁奈米颗粒)。将过量的厄洛替尼(erlotinib)加入FeDC NP中，超音波处理并在25℃的控制温度下搅拌2天，使用0.22μM MCE过滤器灭菌且移除没有接合的厄洛替尼，产生FeDC-E NP(表层有厄洛替尼的葡聚糖涂层磁性铁奈米颗粒)。
除厄洛替尼外，任何含氨基药物，如阿司匹林、类固醇等，皆可以其胺基与FeDC NPs的羧基利用非共价键形式缀合。
奈米颗粒的厄洛替尼释出实验
取1毫升FeDC-E NPs溶液置于透析袋，透析袋的截留尺寸为分子量12000-14000，透析液为200毫升PBS缓冲液(pH5或pH7.4)，在37℃的控制温度下透析，定时将透析液置换为新鲜PBS缓冲液，厄洛替尼累积释放量的计算公式为：
Figure PCTCN2016108073-appb-000001
其中，Qn为单位时间(分钟)的厄洛替尼累积量(μg/mL)，Cn为每个监测时间点培养基中的药物浓度，Ci为第i个监测时间点样本的药物浓度，V0为溶解的培养基的体积，Vi为样本的体积。
实施例1、合成奈米颗粒的结构与物理、化学特性
在合成奈米颗粒时面临的主要挑战之一是保留厄洛替尼在与奈米颗粒缀合后的生物活性。如前面所述，使用碱性共沉淀法合成单晶磁性铁奈米粒子(MION)，随后在奈米粒子表面上交联上薄的葡聚糖涂层，奈米颗粒的表面修饰为羧基并与厄洛替尼缀合，合成过程的细节在方法呈现在图1。为了证实奈米颗粒结构，葡聚糖与合成的MION的配位，以红外线光谱(FT-IR)分析非葡聚糖包覆的奈米颗粒、葡聚糖和葡聚糖包覆的磁性铁奈米颗粒(FeD NP)(图2)，其中，非葡聚糖涂覆奈米颗粒的光谱显示在3300和1600cm-1附近有吸收峰，分别表示O-H伸缩和H-O-H弯曲也就是颗粒表面吸附着水，且在600cm-1处的宽吸收带，表示Fe-O振动。葡聚糖的光谱显示在3300和1600cm-1附近的水O-H伸缩峰和H-O-H弯曲峰，以及在2900cm-1附近的C-H伸缩峰和在1000cm-1处的强C-O峰，表示葡聚糖的α-吡喃葡萄糖环(α-glucopyranose ring)。FeD NP的光谱波峰显示葡聚糖的特征，特别是在指纹区域中，波峰的强度和特征具有轻微变化，显示葡聚糖与奈米颗粒表面磁性铁的配位。
将奈米颗粒溶液置于强力磁铁上一个星期检查溶液的沉淀物及聚集体来测试奈米颗粒的物理稳定性和聚集力。结果没有葡聚糖涂覆的磁性铁奈米颗粒全部都沉淀且聚集，而FeD NP没有沉淀或聚集，FeD NP完全分散且为澄清的红棕色溶液，表明由于葡聚糖涂层使奈米颗粒具有强的物理稳定性，但施加外部磁场可以移动管中的全部奈米颗粒溶液。医疗应用上可使用磁床、磁版、磁衣、磁帽等作为外加磁场，于治疗期间穿戴，使磁性铁奈米颗粒集中 固定。
为了进一步测试葡聚糖稳定于产品上的时效，将FeDC-E NPs溶液在密封的玻璃管中在25℃下储存一年，经过一年储存后，目视和TEM显微镜检查结果发现奈米颗粒仍然完全分散，没有颗粒聚集或沉淀，进一步说明了这种葡聚糖涂层的物理稳定性。
以zeta电位测量确定的奈米颗粒的表面电荷(图3)，FeD NPs的表面电荷接近零为-0.3±0.1mV，FeDN NPs的zeta电位反转到正值为8.6±0.5mV，FeDC NPs因为羧基官能化反转的zeta电位为高负值-11.4±1.2mV，缀合上厄洛替尼碱的FeDC-E NPs的zeta电位降低至-2.4±0.4mV，这是由于FeDC NPs的羧基被厄洛替尼碱的N原子中和，产生稳定的载有厄洛替尼碱的奈米颗粒。
微波电浆原子放射光谱仪(Microwave plasma-atomic emission spectrometry，MP-AES)分析FeDC-E NP溶液中铁含量为1.913mg/mL，高效液相层析仪(high performance liquid chromatography,HPLC)分析FeDC-E NP溶液中厄洛替尼含量为151.32μg/mL，较厄洛替尼的水中溶解度(14.02μg/mL)更高，可以知道本发明将厄洛替尼缀合MION的结果十分有效，可以在奈米颗粒中加载大量的厄洛替尼(超过其水溶性的十倍)。
本实施例将厄洛替尼与奈米颗粒缀合，保持厄洛替尼在FeDC-E NP的生物活性，避免因为其他共价连接方法改变药物的化学结构并阻碍其生物活性。
实施例2、合成奈米颗粒的型态分析
以穿透式电子显微镜(Transmission Electron Microscopy，TEM)观察合成奈米颗粒的型态，没有葡聚糖涂覆的磁性铁奈米颗粒聚集成不规则的形状和尺寸(图4)；相反的，葡聚糖涂覆的FeD NP成分散且没有聚集，FeD NP的形状为规则球形且尺寸小于没有葡聚糖涂覆的奈米颗粒。高分辨率TEM(HRTEM)影像显示出FeD NP的单颗粒晶格面的清楚的规则条纹，显示FeD NP是单晶层的(图5)。根据TEM结果与ImageJ软件分析，FeDC-E NPs是磁性铁核心加上单分散同位素颗粒(monodisperse isotopic-shaped particles)，其平均直径为4.28±1.1nm，多分散指数(polydispersity index，PDI)为0.07，以动态光散射(dynamic light scattering，DLS)测定的FeDC-E NP粒径为6.06±0.9nm(narrow log-normal size distribution)、PDI为0.02，与TEM结果一致(图6)所述葡聚糖磁性铁奈米颗粒的粒径仲数小于50奈米，优选小于10奈米，仲数是指出现频 率最多的颗粒粒径。
根据结果分析，MION核心外约有1-2nm的葡聚糖涂层，与Kumar等人利用碱性共沉淀法合成的奈米粒子相比，本发明利用高浓度葡聚糖、在冰上反应以减慢反应速率等有利的合成条件，使葡聚糖瞬间吸附在新形成的MION的表面上，限制奈米颗粒周围空间与进一步聚集，而产生稳定且超小尺寸的奈米颗粒，在溶液中可以完全分散，奈米颗粒表面上的葡聚糖涂层可预防MION合成时产生的聚集。
实施例3、奈米颗粒的标靶与治疗效果
诊治探针(Theranostic probe)的特色是具有标靶、治疗与成像能力，为了研究FeDC-E NPs与厄洛替尼药物的标靶能力与治疗效果，我们以WST-1分析奈米颗粒处理对于CL1-5-F4细胞(过度表现EGFR)、Jurkat细胞的存活率的影响。CL1-5-F4细胞由人类肺腺癌细胞演化而得，过度表现EGFR，具有高度侵袭(invasive)和迁移(migrative)的细胞；Jurkat细胞为人类淋巴癌细胞，常用于作为没有表现EGFR的代表细胞。
本实施例测试了两种EGFR状态细胞对厄洛替尼的敏感性，图7结果显示厄洛替尼对高度EGFR表现的CL1-5-F4细胞造成毒性作用，且具有浓度相关性，相反的，高浓度厄洛替尼对无EGFR表现的Jurkat细胞没有造成毒性作用。不含厄洛替尼的FeDC NP在CL1-5-F4、Jurkat两种细胞上都没有细胞毒性，显示FeDC NPs奈米颗粒的生物兼容性。含厄洛替尼的FeDC-E NP对细胞上的毒性，与单独厄洛替尼的效果相似，FeDC-E NP对CL1-5-F4细胞存活有明显抑制，与对Jurkat的细胞毒性有统计上的显著差异。由细胞存活结果显示与奈米颗粒缀合不影响厄洛替尼的活性，且FeDC NP本身没有细胞毒性，此外，FeDC-E NPs具有选择性细胞毒性效应表示FeDC-E NP可用于辨识过度表现EGFR的肿瘤细胞。
实施例4、以细胞摄食实验观察奈米颗粒的标靶能力
为了研究厄洛替尼对奈米颗粒标靶能力的影响，本实施例使用普鲁士蓝染色(Prussian blue staining)测试了奈米颗粒的细胞摄取的能力(图8)，于CL1-5-F4细胞，控制组与厄洛替尼处理的细胞都没有显示任何蓝色染色斑且染色试剂不沉淀，表明细胞内没有内源性铁存在。FeDC NPs处理的细胞有几个蓝色斑点，染色密度为10.69％(代表细胞摄取的FeDC NP的量)。FeDC-E NPs 处理的细胞，其奈米颗粒的摄取增加4倍，显微镜下观察到的强蓝色斑点、染色密度为42.53％。
厄洛替尼与奈米颗粒的缀合增加奈米颗粒摄取量，这可能是由于厄洛替尼对表现EGFR细胞的标靶能力。由于这种普鲁士蓝染色搭配显微镜检查不能排除奈米颗粒位于细胞表面而非经摄入而进入细胞内，因此进一步以TEM确认这些奈米颗粒是否真的进入细胞中。
实施例5、以TEM检测的奈米颗粒的细胞摄入
为了检测奈米颗粒的细胞定位以确认前述实施例中从普鲁士蓝实验获得的细胞摄取结果，本实施例利用TEM(图9)检查各个处理组的CL1-5-F4细胞，其中，未处理和厄洛替尼处理的细胞显示透明细胞和核膜而没有任何电子密集区域的。FeDC NPs处理的细胞有奈米颗粒的电子密集的内吞囊泡(高电子密集是由于奈米颗粒的磁性铁核心)，FeDC-E NPs处理的细胞有相似的奈米颗粒的电子密集内吞囊泡(endocytotic vesicles)且数量比FeDC NPs处理的细胞更多。
本实施例结果支持前述实施例普鲁士蓝染色结果，证明厄洛替尼增加奈米粒子的吞噬，这些TEM结果说明摄取的奈米颗粒位在细胞的内吞囊泡(endocytotic vesicles)内，而不是聚集于细胞外；此外，也证实厄洛替尼与奈米颗粒的缀合可以显著增加奈米颗粒的细胞摄取(以普鲁士蓝实验证明有4倍增加)，证明于厄洛替尼赋予奈米颗粒标靶能力。
实施例6、pH值影响奈米粒子的厄洛替尼释放
FeDC-E NPs被细胞吞噬到内吞囊泡(endocytotic vesicles)中，体液和细胞外的环境的pH值为7.4，细胞内晚期胞内体(late endosomes)的pH值约为5，本实施例模拟于不同细胞生理环境中，FeDC-E NPs将厄洛替尼释出的影响。于内吞囊泡模拟液(pH＝5)，140分钟后厄洛替尼累积释放76.7％，而在细胞外环境模拟液(pH＝7.4)，相同的140分钟时，累积厄洛替尼释放60.8％，统计上具有显著差异(图10)。这样的差异说明，奈米颗粒在血液中循环期间，从FeDC-E-NPs释放的厄洛替尼较低，直到其到达肿瘤部位被细胞吞噬置至内吞囊泡的低pH环境才会高速释放厄洛替尼，也就是说，FeDC-E NPs具有根据pH调整的智能释放系统，可以增加药物传递到肿瘤细胞并减少系统效应、优化整体治疗结果。
实施例7、以MRI分析研究奈米颗粒的标靶能力
为了验证本发明的奈米颗粒可以有效用于核磁共振摄影(magnetic resonance imaging，MRI)和确认其标靶能力，本实施例以不同奈米颗粒处理CL1-5-F4细胞进行体外(in vitro)MRI实验，测量T2弛缓时间(T2relaxation times)变化和的信号强度(图11)。未处理的细胞的T2弛缓时间的中间值为103.9ms，FeDC NPs处理的细胞的T2弛缓时间中间值为92.5ms，这表明细胞吸收的铁量在MRI可产生对比。用FeDC-E NPs处理细胞显著降低T2弛缓时间中间值至84.2ms(与FeDC NPs处理的细胞结果有显著差异，图11)。三个处理组别的T2信号强度在回讯时间(echo time)8.5ms时差异最大，其中未处理细胞的T2信号强度中间值为191.4，FeDC NPs处理的细胞的T2信号强度中间值为183.5，FeDC-E NPs处理细胞的T2信号中间值强度为159.8(FeDC-E NPs处理细胞与未处理和FeDC NPs处理细胞的T2强度在统计上有显著差异，图12)。
MRI图像中证明了三个处理组之间的差异。这些结果表示FeDC-E NPs经由细胞摄入和累积于细胞内而足以由MRI追踪，因此适合用作诊治探针(Theranostic probe)，此外，具标靶力的FeDC-E NPs处理细胞与不具标靶力FeDC NP处理细胞的T2结果进一步证实了通过厄洛替尼的缀合赋予奈米颗粒标靶能力。
实施例8、奈米颗粒抑制EGFR和ERK信号传导途径
厄洛替尼是一种高选择性小分子药物，其特征在于EGFR的酪氨酸激酶(tyrosine kinase)结构域，可有效抑制EGFR信号途径，EGFR与癌症发生的许多下游信号途径有关。细胞外训号调节激酶(extracellular-signal-regulated kinase，ERK)途径可作为EGFR抑制剂活化的生物标志。EGFR引起的ERK阶梯式活化涉及通过Ras、Raf和mitogen-activated protein kinase/extracellular signal-regulated kinase(MEK)的信号传导，称为EGFR-Ras-Raf-MEK-ERK信号途径。在CL1-5-F4细胞中，ERK途径的活化导致转录因子NF-κB的活化，与许多调节参与癌症进展的基因表现有关，例如侵入和迁移强化matrix metallopeptidase 9(MMP-932)，血管生成刺激血管内皮生长因子(vascular endothelial growth factor，VEGF)和X-linked inhibitor of apoptosis protein(XIAP)，可能抑制肿瘤细胞增殖。
为了研究FeDC-E NPs处理后经由这些途径的细胞信号改变，本实施例通 过西方墨点转渍法分析CL1-5-F4细胞中EGFR及其磷酸化(p-EGFR)的表现，结果发现未处理的细胞有EGFR和p-EGFR两种形式的受体。厄洛替尼处理不影响EGFR的表现，但会抑制EGFR磷酸化，这也是该药物作为酪氨酸激酶抑制剂的主要机制(图13)。FeDC-E NPs处理的细胞显示EGFR没有改变的表现，但抑制EGFR的磷酸化，表示FeDC-E NPs能够抑制EGFR信号。
在EGFR抑制后，其下游ERK信号被抑制。图13结果证明erlotinib、FeDC-E NPs处理使细胞p-ERK 1/2表现明显降低(与未处理组、FeDC NPs处理组相比)，四个处理组别的ERK 1/2表现都没有改变。
实施例9、奈米颗粒抑制NF-κB信号途径及其调节的肿瘤促进蛋白
转录因子NF-κB是Rel家族蛋白质的异二聚体复合物(heterodimeric complex)，Rel家族包括RelA(p65)次单元是在细胞中最常见的NF-κB的二聚体次单元。在细胞质中NF-κB通常与IκB抑制蛋白结合呈现无活性状态；被激活后，抑制性IκB被降解，使NF-κB移位到细胞核并结合到DNA的启动子区，启动NF-κB目标基因的转录和表现。
为了研究奈米颗粒对EGFR和ERK下游的NF-κB途径的抑制，本实施例利用西方墨点印渍法分析CL1-5-F4细胞核萃取物中NF-κB p65次单元的量，并使用免疫细胞化学荧光成像分析NF-κB p65次单元的从CL1-5F4细胞的细胞质移位到细胞核的成像。图13结果显示，未处理细胞的细胞核中有高量的NF-κB p65次单元，表现高活性NF-κB，FeDC NPs处理细胞组与未处理细胞组显示相似的结果，表示不含药物的奈米颗粒不影响NF-κB途径，厄洛替尼和FeDC-E NPs处理组都显示出核中的NF-κB p65次单元量降低，表示药物处理后NF-κB途径被抑制。图14免疫细胞化学成像结果显示，在未处理及FeDC NPs处理的CL1-5-F4细胞NF-κB p65次单元驻留于细胞核，证明NF-κB核移位途径被活化。然而，厄洛替尼以及FeDC-E NPs处理的细胞，NF-κB p65次单元在细胞质中明显聚积，显示NF-κB的核移位被抑制，药物处理抑制NF-κB活性。
本实施例进一步分析奈米颗粒处理对NF-κB转录活性调节蛋白质XIAP和MMP-9表现的影响，XIAP和MMP-9与癌症发展进度有关。厄洛替尼、FeDC-E NP1处理CL1-5-F4细胞中XIAP的表现显著降低(与未处理和FeDC NPs处理的组细胞相比(图13)。另外，MMP-9酶于高侵袭性和迁移性细胞中大量表现，而能够降解IV型胶原蛋白的细胞基底膜和细胞外基质，促进其侵入和迁移； 厄洛替尼、FeDC-E NP处理CL1-5-F4细胞中MMP-9的表现显著降低(与未处理和FeDC NPs处理的组相比)。为了了解MMP-9表现降低对CL1-5-F4细胞的迁移和侵袭能力的影响，于后面实施例将进行迁移和侵袭实验。
实施例10、奈米粒子经由分子信号途径产生抗癌活性
基于前述实施例中关于由于奈米颗粒处理而改变的分子信号途径的结果发现，图15显示，FeDC-E NP经由抑制EGFR的酪氨酸激酶的结构域而产生有效的抗癌活性，抑制下游ERK信号信号途径。抑制ERK导致NF-κB活性的抑制，抑制了NF-κB从细胞质移位至细胞核。NF-κB目标基因的负调节，与癌症促进和进展中有关，NF-κB转录活性被抑制会进一步抑制的XIAP(抗细胞凋亡)和MMP-9(迁移和侵袭)蛋白质。
实施例11、奈米颗粒抑制CL1-5-F4细胞的迁移和侵袭能力
在多种癌症中，活化的MMP-9表现诱导细胞的迁移和侵袭。先前实施例发现FeDC-E NPs处理CL1-5-F4细胞降低MMP-9的表现，因此本实施例使用通透性培养皿(transwell)观察奈米颗粒对迁移和侵袭的影响，CL1-5-F4细胞会高度迁移至transwell的膜的下面，FeDC NPs处理的细胞迁移率较未处理的细胞更少(但统计上没有显著差异)，厄洛替尼处理的细胞的迁移率只有未处理细胞的21.75％，而FeDC-E NPs处理细胞几乎完全抑制细胞的迁移能力，迁移率为未处理细胞的3.26％(图16)。与先前实施例中MMP-9表现的结果一致，FeDC-E NPs有效抑制细胞的迁移。
本实施例进一步以Matrigel包覆的transwell观察奈米颗粒对细胞侵袭能力的影响，未处理的CL1-5-F4细胞显示有大量的侵袭通过Matrigel到transwell的膜的下侧，FeDC NPs处理的细胞显示较小的袭潜力(与未处理细胞结果在统计学上没有显著差异)，厄洛替尼处理的细胞显示其侵袭率的显著降低至未处理细胞的32.37％，FeDC-E NPs处理细的侵袭率是未处理细胞的15.57％(图17)，较厄洛替尼更低。
依据本实施例结果，我们发现FeDC-E NPs比相同浓度的厄洛替尼更显著抑制CL1-5F4细胞的侵袭和迁移能力。
实施例12、奈米颗粒的In vivo治疗效果
为了证实先前实施例中in vitro实验中FeDC-E NP对CL1-5-F4细胞的毒性作用，并进一步研究其in vivo抗肿瘤活性，本实施例使用携带异种CL1-5-F4 细胞植入的雄性BALB/c裸鼠模式，分别处理Ctrl(未处理)、厄洛替尼、FeDC NP、FeDC-E NP，与未处理组相比，FeDC NPs处理的小鼠的肿瘤体积没有任何统计学显著差异，表明奈米颗粒没有抑制肿瘤生长的内在活性；与未处理组相比，厄洛替尼处理的小鼠显著抑制的肿瘤生长，表示肿瘤细胞对厄洛替尼敏感，FeDC-E NP处理显著抑制小鼠肿瘤生长，表示FeDC-E NP具有治疗效果(图18)，FeDC-E NPs对肿瘤抑制略小于厄洛替尼，但没有统计上的显著差异，表明FeDC-E NP在配制过程后保留了厄洛替尼的治疗活性。
整个实验期间，各处理组之间没有观察到小鼠体重有显著改变，显示药物处理对小鼠没有毒性迹象(图19)，表示本发明使用的奈米颗粒具有生物兼容性。
实施例13、in vivo非侵入性核磁共振成像测试的奈米颗粒的MRI对比增强和标靶能力
为了确定先前实施例中用FeDC-E NPs观察到in vitro的MRI对比增强是否可以在in vivo实行，并且确认FeDC-E NP的标靶能力，本实施例使用携带异种CL1-5-F4细胞植入的雄性BALB/c裸鼠进行in vitro非侵入性MRI实验。图20显示本实施例的4个处理组(Ctrl、Erlotinib、FeDC NP、FeDC-E NP)肿瘤成像，在处理前和处理后对肿瘤感兴趣区域(regions of interest，ROI)的T2加权标准化MRI信号强度(Normalized T2-weighted MRI signal intensities)进行定量，未处理和厄洛替尼处理组的肿瘤的标准化信号强度在处理后没有显著变化，FeDC NPs-和FeDC-E NPs-处理组分别在平均标准化信号强度有约1.6倍和2.8倍的降低，FeDC-E NPs处理组的标准化信号强度的降低较其他三个处理组有统计上的显著差异，表明与非靶向FeDC NP相比，FeDC-E NPs在肿瘤中的标靶聚积增强。
这样的in vivo标靶MRI对比增强进一步支持本发明的FeDC-E NPs于临床上作为诊治探针(Theranostic probe)的可能性。
综合上述实施例结果，本发明是第一个公开小分子厄洛替尼作为奈米颗粒的标靶药剂的研究，本发明的奈米颗粒表现出对高度侵袭性和迁移性癌细胞的显著的智能治疗和标把性质，并且可以通过MRI监测。此外，奈米颗粒抑制EGFR-ERK-NF-κB信号途径以及相关肿瘤促进蛋白MMP-9和XIAP的表现，并随后抑制癌细胞的迁移和侵袭能力。并以携带异种移植物的小鼠确认奈米 粒子在体内标靶、治疗和成像能力。最后，本发明的奈米颗粒制剂的超小尺寸使其有可能用于转移性脑肿瘤的MRI。
上列详细说明针对本发明的可行实施例的具体说明，惟该实施例并非用以限制本发明的专利范围，凡未脱离本发明技艺精神所为的等效实施或变更，均应包括于本案的专利范围中。
上述多项功效，实属充分符合新颖性及进步性的法定专利要件，爰依法提出申请，恳请贵局核准本件发明专利申请案，以励发明。
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Claims (17)

  1. 一种葡聚糖磁性铁奈米颗粒，其特征在于，包括葡聚糖层、磁性铁奈米颗粒与活性药物，葡聚糖为薄层包覆在所述磁性铁奈米颗粒之外，所述活性药物以非共价键结与所述葡聚糖层缀合。
  2. 如权利要求1所述的葡聚糖磁性铁奈米颗粒，其特征在于，所述磁性铁奈米颗粒为Milli-Q铁奈米颗粒。
  3. 如权利要求1所述的葡聚糖磁性铁奈米颗粒，其特征在于，包覆于磁性铁奈米颗粒外的所述葡聚糖层厚度小于3奈米。
  4. 如权利要求1所述的葡聚糖磁性铁奈米颗粒，其特征在于，所述葡聚糖磁性铁奈米颗粒的粒径仲数小于50奈米。
  5. 如权利要求1所述的葡聚糖磁性铁奈米颗粒，其特征在于，所述葡聚糖磁性铁奈米颗粒的粒径仲数小于10奈米。
  6. 如权利要求1所述的葡聚糖磁性铁奈米颗粒，其特征在于，所述活性药物具有胺基。
  7. 如权利要求1所述的葡聚糖磁性铁奈米颗粒，其特征在于，所述活性药物为厄洛替尼。
  8. 一种磁性铁奈米颗粒的制备方法，其特征在于，制备步骤如下：
    步骤1：将冰冷的氨加入FeCl3·6H2O、FeCl2·4H2O和葡聚糖的水溶液中，于惰性气体环境中置于冰上搅拌，再加热至85℃维持1小时，搅拌至冷却，以Milli-Q水进行透析3天；
    步骤2：葡聚糖(dextran)涂层使用环氧氯丙烷/氢氧化钠(Epichlorohydrin/NaOH)交联，用Milli-Q水进行透析3天，产生稳定的FeD NP；
    步骤3：FeD NPs用氨处理以在奈米颗粒的表面上产生一级氨官能基，随后用Milli-Q水进行透析3天，产生FeDN NP；
    步骤4：FeDN NP与琥珀酸酐(succinic anhydride)在缓冲溶液(pH＝8.5，0.1M NaHCO3)中反应，随后用Milli-Q水进行透析3天以充分透析，然后通过加入4-吗啉乙磺酸(Morpholineethanesulfonic acid)和氯化钠缓冲溶液，产生FeDC NP；
    步骤5：将过量的活性药物加入FeDC NP中，超音波处理，并在25℃的控制温度下搅拌2天，使用0.22μM MCE过滤器灭菌且移除没有接合上的活性药物，产生FeDC-E NP。
  9. 如权利要求8所述的置备方法，其特征在于，所述活性药物为厄洛替尼。
  10. 一种如权利要求1-7中任一项所述葡聚糖磁性铁奈米颗粒用于制备医学造影组合物的用途。
  11. 如权利要求10所述的用途，所述医学造影用于癌症造影。
  12. 如权利要求11所述的用途，其特征在于，所述癌症为表现EGFR的癌症。
  13. 如权利要求11所述的用途，其特征在于，所述癌症包括脑癌、肺癌、大肠癌、结肠直肠癌、乳癌、摄护腺癌、肝癌、胰脏癌、膀胱癌、胃癌、肾癌、唾腺癌、卵巢癌、子宫体癌、子宫颈癌、口腔癌、皮肤癌、脑癌、恶性淋巴瘤或白血病。
  14. 一种如权利要求1-7中任一项所述葡聚糖磁性铁奈米颗粒用于制备治疗癌症医药组合物的用途。
  15. 如权利要求14所述的用途，其特征在于，所述癌症为表现EGFR的癌症。
  16. 如权利要求15所述的用途，其特征在于，所述癌症包括脑癌、肺癌、大肠癌、结肠直肠癌、乳癌、摄护腺癌、肝癌、胰脏癌、膀胱癌、胃癌、肾癌、唾腺癌、卵巢癌、子宫体癌、子宫颈癌、口腔癌、皮肤癌、脑癌、恶性淋巴瘤或白血病。
  17. 如权利要求14所述的用途，其特征在于，所述葡聚糖磁性铁奈米颗粒可以利用外加磁场移动且停滞于所述癌症位置。
PCT/CN2016/108073 2016-11-30 2016-11-30 葡聚糖磁性铁奈米颗粒、制备及在治疗癌症与造影的用途 WO2018098705A1 (zh)

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