An acidic oligopeptide modified bone targeting liposome and a preparation method thereof

Abstract

The application discloses an acid oligopeptide modified bone targeting liposome and a preparation method thereof. The surface of the bone targeting liposome is modified with acid oligopeptide, and the modification density n is 1-5, which is expressed in mol%. The bone targeting liposome is prepared by modifying the liposome surface with D-glutamic acid hexapeptide which has bone targeting effect, and optimizing the modification density of the D-glutamic acid hexapeptide on the liposome surface, so that the liposome preparation with the strongest bone targeting activity is screened, and the bone tissue distribution of the liposome preparation is increased. With the increase of the modification density of the acid oligopeptide, the combination ability of the liposome and hydroxyapatite is enhanced. However, the high negative charge generated by the modification stimulates the phagocytosis of the mononuclear phagocyte system, and the blood retention of the liposome is reduced. However, the high-density acid oligopeptide modification (5 mol%) endows the liposome with outstanding HA combination activity, and the deficiency of the weak blood retention of the liposome is completely compensated, so that the highest bone targeting delivery is realized.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of pharmaceutical preparations, and particularly relates to an acid oligopeptide modified bone targeting liposome and a preparation method thereof. BACKGROUND

[0002] Due to the gradual social aging, bone diseases (such as osteoporosis and bone tumors) constitute a major public health problem. For the treatment of bone diseases, a large dose of drugs needs to be taken orally and injected to reach the bone tissue site at an effective concentration. However, such a high concentration of drugs in the blood circulation can cause serious adverse effects on other normal tissues. Nanocarriers can target drug delivery to bone tissue sites and maximize the reduction of systemic toxicity to improve the treatment effect of bone diseases, thereby attracting widespread attention.

[0003] Therefore, it is of great significance to develop a nanodrug delivery system with the function of actively targeting bone tissue. SUMMARY

[0004] In view of the deficiencies of the prior art, the present application provides an acid oligopeptide modified bone targeting liposome and a preparation method thereof.

[0005] The purpose of the present application is achieved by the following technical solutions:

[0006] In the first aspect, the present application provides an acid oligopeptide modified bone targeting liposome, wherein the surface of the bone targeting liposome is modified with an acid oligopeptide, and the modification density n is 1-5, in terms of molar ratio mol%.

[0007] Preferably, the acid oligopeptide is D-type Glu6.

[0008] In the technical solution of the present application, the acid oligopeptide with bone targeting function, such as the acid oligopeptide represented by D-type glutamic hexapeptide (Glu6), is modified on the surface of the liposome, and the density of the modification on the surface of the liposome is optimized to screen out the liposome preparation with the strongest bone targeting activity, so as to increase the distribution in bone tissue.

[0009] It should be noted that the liposome modified with the acid oligopeptide ligand shows ideal bone targeting ability. However, with the increase of the modification density of the acid oligopeptide, the liposome may exhibit stronger hydroxyapatite (HA) binding activity, but the blood pharmacokinetics is poor, which has an opposite effect on the distribution of the liposome in the bone tissue. Therefore, simply increasing the modification density of the acid oligopeptide cannot well obtain the ideal liposome with bone targeting activity, and few studies have systematically investigated how the modification density of the acid oligopeptide affects the bone targeting activity of the liposome.

[0010] Based on this, the inventors constructed a liposome library with the same lipid composition and comparable particle size, but with different Glu6 modification densities, thereby obtaining the optimal range of Glu6 modification density. However, as described above, although high-density Glu6 modification can endow the liposomes with stronger HA binding activity, it can also make the liposomes highly negatively charged, thereby stimulating macrophage phagocytosis, enhancing the uptake of the liposomes in the liver and spleen, and weakening the blood retention of the liposomes. Ultimately, the biodistribution experiments showed the results of the dual factors, i.e., the Glu6-modified liposomes with a modification density of 1-5 mol% completely overcame the deficiency of blood pharmacokinetics due to their excellent HA binding activity, and produced Glu6 density-dependent bone tissue distribution in bone tissue. Among them, 5 mol% Glu6 modification caused the strongest bone tissue distribution. In addition, the hemolysis test further showed that the optimized bone-targeting liposomes (5 mol% Glu6-modified liposomes, referred to as 5% e-Lip for short) had good biocompatibility when administered by systemic injection.

[0011] In a second aspect, the present application provides a method for preparing the bone-targeting liposomes described above, comprising dissolving HSPC, cholesterol, and DSPE-PEG2000 with different feeding ratios of DSPE-PEG2000-DGlu6 in an organic solvent, shaking uniformly, forming a phospholipid film by vacuum rotary evaporation, vacuumizing overnight, adding PBS, and rotating in a water bath until the film is hydrated and falls off, to obtain a liposome suspension with different Glu6 modification densities, and then extruding with an extruder with a polycarbonate membrane to obtain bone-targeting liposomes with a particle size of 100 nm.

[0012] Preferably, the DSPE-PEG2000-DGlu6 is obtained by the following method:

[0013] DCys-DGlu6 is synthesized by Fmoc synthesis strategy;

[0014] DSPE-PEG2000-DGlu6 is obtained by thiol-maleimide addition reaction of DSPE-PEG2000-Mal and DCys-DGlu6.

[0015] Preferably, the organic solvent is a mixture of chloroform and methanol at a volume ratio of 3:1.

[0016] Preferably, HSPC, cholesterol, DSPE-PEG2000, and DSPE-PEG2000-DGlu6 are dissolved at the following molar ratio: 56.5:38.5:5-x:x, wherein x represents the molar ratio of DSPE-PEG2000-DGlu6, and x = 1, 2, 3, 4, 5.

[0017] Preferably, the water bath temperature is 37℃ and the water bath time is 30 min.

[0018] Preferably, the liposome suspension is extruded in an extruder through 100 nm and 50 nm polycarbonate membranes in sequence.

[0019] In a third aspect, the present application provides a bone-targeting liposome drug, which comprises the bone-targeting liposome as described above, and an active drug molecule wrapped in the bone-targeting liposome.

[0020] Preferably, the active drug molecule is selected from at least one of the following: drugs for treating osteoporosis, tumor bone metastasis, bone fracture healing disorder, bone fracture, bone defect repair, hyperostosis, and heterotopic ossification.

[0021] In a fourth aspect, the present application provides use of the bone-targeting liposome drug in the preparation of a drug for treating bone diseases.

[0022] Preferably, the bone disease is selected from osteoporosis, tumor bone metastasis, bone fracture healing disorder, bone fracture, bone defect repair, hyperostosis, and heterotopic ossification.

[0023] In a fifth aspect, the present application provides a pharmaceutical preparation, which comprises the bone-targeting liposome drug as described above, and the pharmaceutical preparation is a liquid preparation, and the liquid preparation is an injection.

[0024] Compared with the prior art, the present application has the following advantages and beneficial effects:

[0025] The acid oligopeptide-modified bone-targeting liposome of the present application first studies and clarifies the influence rule of the acid oligopeptide modification density on the bone-targeting activity of the liposome. That is, with the increase of the acid oligopeptide modification density, the binding ability of the liposome to HA is enhanced, the high negative charge generated by the modification stimulates the phagocytosis of macrophages in the mononuclear phagocyte system (MPS) to the liposome, and the blood retention of the liposome is reduced. However, the high-density acid oligopeptide modification (5 mol%) endows the liposome with outstanding HA binding activity, which can completely compensate for the weakness of the blood retention, so as to realize the highest bone-targeting delivery. BRIEF DESCRIPTION OF DRAWINGS

[0026] Other features, objects, and advantages of the present application will become more apparent from the following detailed description of non-limiting embodiments with reference to the following drawings.

[0027] Figure 1 The synthesis flowchart of DCys-DGlu6 in the present application is shown in the figure;

[0028] Figure 2HPLC chromatogram of DCys-DGlu6 gradient elution at 220 nm and mass spectrum of DCys-DGlu6 in the application, wherein A is HPLC chromatogram, B is mass spectrum, and the relative molecular mass is shown: 895.3;

[0029] Figure 3 Schematic diagram of the synthesis process of DSPE-PEG2000-DGlu6 in the application;

[0030] Figure 4 MALDI-TOF-MS spectrum of DSPE-PEG2000-Mal and DSPE-PEG2000-DGlu6 in the application, wherein A is DSPE-PEG2000-Mal, and B is DSPE-PEG2000-DGlu6;

[0031] Figure 5 Preparation and characterization of liposomes with different Glu6 modification densities in the application, wherein A shows a schematic diagram of the preparation process of DiD-labeled liposomes with different Glu6 modification densities, B shows the particle size of the liposomes, and C shows the Zeta potential;

[0032] Figure 6 Binding capacity of liposomes with different Glu6 modification densities to HA in the application, wherein A shows the fluorescence images of the binding of liposomes with different Glu6 modification densities to HA, and B shows the quantitative analysis;

[0033] Figure 7 Uptake of liposomes with different Glu6 modification densities by macrophage Raw264.7 in the application, wherein A shows representative flow cytometry histograms of DiD-labeled liposomes with different Glu6 modification densities after incubation with Raw264.7 for 4 h, B shows the semi-quantitative results of the mean fluorescence intensity (MFI) from figure A, and C shows representative confocal images of Raw264.7 cells after the same treatment as in figure A, scale bar: 10 μm;

[0034] Figure 8 Absorbance (570 nm) change of Lip and nP-Lip after incubation with 50% FBS for 48 h in the application;

[0035] Figure 9 Leakage of DiD in Lip in 100% FBS in the application;

[0036] Figure 10Blood pharmacokinetic behavior of liposomes with different Glu6 modification density in the present application. Among them, A shows the plasma DiD concentration-time curve of each liposome prescription labeled with DiD after intravenous injection into SD rats, B and C are the pharmacokinetic parameters half-life (t1 / 2) and area under the curve (AUC) calculated by non-compartment model, respectively;

[0037] Figure 11 In vivo biodistribution of liposomes with different Glu6 modification density in the present application, wherein A is the biodistribution of liposomes with different Glu6 modification density in C57BL / 6 mice after intravenous injection, and the biodistribution in each tissue is quantitatively determined 24h later, and the measurement result is expressed as μg DiD / g tissue, and the enlarged histogram of the femur and tibia biodistribution is shown in the upper right corner; B is the fluorescence imaging of the distribution of nP-Lip, Lip and 5% e-Lip in the main tissues of C57BL / 6 mice after intravenous injection 24h later;

[0038] Figure 12 Uptake of 5% e-Lip by resident macrophages in liver and spleen in the present application, DiD-labeled non-modified Glu6 liposomes (Lip), 5% e-Lip and non-PEGylated liposomes (nP-Lip) are injected into C57BL / 6 mice, and the distribution of DiD fluorescence in the liver and spleen is observed 24h later. Among them, A shows the distribution of DiD-labeled liposomes and F4 / 80 antibody-labeled macrophages in the liver, B shows the Pearson (colocalization) correlation coefficient calculated by ImageJ software for A, C shows the distribution of DiD-labeled liposomes and F4 / 80 antibody-labeled macrophages in the spleen, and D shows the Pearson (colocalization) correlation coefficient calculated by ImageJ software for C;

[0039] Figure 13 Hemolysis experiment of 5% e-Lip in the present application, red blood cells were incubated with ultrapure water (H2O), PBS, Lip, 5% e-Lip (L) and 5% e-Lip (H) at 37℃ for 2h, after centrifugation, the supernatant suspension (A) and red blood cell precipitate (C) were photographed by digital camera and microscope, respectively, and the hemolysis rate (B) of Lip, 5% e-Lip (L) and 5% e-Lip (H) was calculated. PBS and H2O were used as negative and positive controls, respectively, the scale was 10μm. 5% e-Lip (L): low concentration of 5% e-Lip at 0.6mg / mL; 5% e-Lip (H): high concentration of 5% e-Lip at 1.8mg / mL. DETAILED DESCRIPTION

[0040] The application will be described in detail below with specific examples. The following examples will help those skilled in the art to further understand the application, but do not limit the application in any form. It should be pointed out that for those skilled in the art, without departing from the concept of the application, a number of modifications and improvements can be made. These all belong to the protection scope of the application.

[0041] In nanoparticles, liposomes are bilayer vesicles similar to biological membrane structures. The bilayer structure of liposomes is formed by dispersing phospholipid components into water, and the hydrophobic tails tend to aggregate to avoid contact with water, and the hydrophilic heads are exposed to the water side. This special bilayer structure allows liposomes to load hydrophilic drugs in the internal water phase and hydrophobic drugs in the phospholipid bilayer. Liposomes are the most clinically acceptable drug delivery carriers, with good biocompatibility and biodegradability, and their surfaces are easy to modify with targeting ligands, can encapsulate drugs, vaccines, genes and imaging agents, and have the advantages of non-toxicity and non-immunogenicity, etc., and are the most common nanocarriers for targeted drug delivery.

[0042] It should be noted that the main innovation of the present case is to optimize the best modification density on the surface of the nanocarrier. The modification of Glu6 is beneficial to its bone targeting, but the modification of Glu6 makes the liposome negatively charged, which reduces blood retention and is not conducive to its bone targeting. Therefore, in order to balance the contradiction between pharmacokinetics and bone targeting, a best Glu6 modification density is optimized to achieve the maximum bone targeting ability.

[0043] HA accounts for more than 70% of the crystal component of bone, so it is an ideal target for bone targeting. Anionic ligands such as bisphosphonates (BPs), acidic oligopeptides and tetracyclines have bone homing properties due to their high binding capacity with calcium ions on HA, and have been widely used as bone targeting ligands. Previous studies have shown that BPs and tetracycline-modified liposomes exhibit excellent bone targeting. However, BPs can remain in the bone for a long time after binding to the bone, leading to osteonecrosis of the jaw, while tetracycline can inhibit the growth of children's bones. In contrast, acidic oligopeptides have no long-term toxic side effects, are easy to synthesize and chemically modify, and can add response sequences and other targeting ligands to their amino acid sequences.

[0044] However, the use of acidic oligopeptide ligands to modify liposomes for bone targeting activity is a double-edged sword. On the one hand, high-density acidic oligopeptide modification promotes the binding of liposomes to HA, promoting the distribution of liposomes in bone tissue. On the other hand, the high negative charge on the surface of liposomes caused by high-density acidic oligopeptide modification also stimulates the phagocytosis of macrophages in the MPS, leading to rapid elimination of liposomes from the blood, thereby restricting the distribution of liposomes in bone tissue. Therefore, the modification density of acidic oligopeptides needs to be optimized to achieve optimal liposome bone targeting delivery.

[0045] To optimize the density of the acid oligopeptide modification on the surface of the liposome, the inventors constructed a library of liposomes with the same lipid composition and comparable particle size, but with different densities of the acid oligopeptide modification. In this paper, the liposome formulation is HSPC / cholesterol / DSPE-PEG2000 (56.5:38.5:5, mol / mol), which has long-circulating properties and thus helps to achieve bone-targeted delivery. The surface of the liposome is modified with the bone-targeting ligand Glu6, and five modification densities are selected, i.e., 1 mol%, 2 mol%, 3 mol%, 4 mol%, and 5 mol%, to optimize the best bone-targeting modification density (5 mol% in the end). Glu6 is used as a representative of acid oligopeptides and is modified on the surface of the liposome. The amino terminus of DGlu6 is covalently linked to DCys to introduce a free thiol group (-SH), generating DCys-DGlu6, so that DCys-DGlu6 can be connected to DSPE-PEG2000-Mal through a thiol-maleimide addition reaction to synthesize DSPE-PEG2000-DGlu6. The liposomes with different Glu6 modification densities are prepared by self-assembly of HSPC, cholesterol, and different feeding ratios of DSPE-PEG2000 and DSPE-PEG2000-DGlu6. DiD (1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindodicarbocyanine, 4-Chlorobenzenesulfonate Salt) has two octadecyl "hydrophobic tails" and can be incorporated into the lipid bilayer, and is commonly used in liposome research. Therefore, the inventors encapsulate DiD into the liposomes with adjustable Glu6 modification density to track their delivery in vitro and in vivo.

[0046] The application will be further described in conjunction with specific examples. In the following examples, the experimental methods used are conventional methods unless otherwise specified. In the following examples, the materials, reagents, etc. used are commercially available unless otherwise specified. The techniques not described in detail are performed according to the standard methods well known to those skilled in the art. The reagents mentioned in this application are commercially available or can be obtained by other means, and they are only used as examples and are not the only ones for the application. Other suitable tools or biological materials can be used instead. It should be understood that these examples only illustrate the application and are not used to limit the scope of the application.

[0047] Example 1

[0048] 1. Synthesis of DCys-DGlu6

[0049] DCys-DGlu6 was synthesized according to standard Fmoc synthesis strategy. Figure 1 ) Briefly, the amino acids of DCys-DGlu6 were covalently linked to the resin one by one, and Fmoc protection was removed using 20% piperidine / DMF. After the completion of the ligation, the resin was removed by incubation in TFA solution for 2.5 h, and the filtrate was collected and precipitated with ice-ethanol to obtain the crude peptide of DCys-DGlu6. The crude peptide was further purified using preparative high performance liquid chromatography (HPLC) to obtain pure DCys-DGlu6, and the synthesized DCys-DGlu6 was identified by HPLC and mass spectrometry (MS).

[0050] The amino terminal of DGlu6 was ligated with DCys to introduce a free thiol group (-SH) to enable DGlu6 to conjugate with DSPE-PEG2000-Mal through thiol-maleimide addition reaction to synthesize DSPE-PEG2000-DGlu6. DCys-DGlu6 was synthesized according to standard Fmoc synthesis strategy, and whether it was successfully synthesized was identified by HPLS / MS. The successful synthesis of DCys-DGlu6 was confirmed by HPLC and liquid chromatography-mass spectrometry determination. Figure 2

[0051] 2. Synthesis of DSPE-PEG2000-DGlu6

[0052] DSPE-PEG2000-DGlu6 was obtained through thiol-maleimide addition reaction between DSPE-PEG2000-Mal and DCys-DGlu6. Figure 3 Briefly, DCys-DGlu6 and DSPE-PEG2000-Mal (4:1, mol / mol) were dissolved in ACN / H2O (2:1, v / v) solution, and 0.2 M phosphate buffered saline (PBS) (pH = 7.4) and 6 M guanidine hydrochloride solution (pH = 7.0) were added under stirring. The oxygen was replaced by nitrogen gas, and the reaction was carried out at room temperature under a nitrogen atmosphere for 2 h. The reaction solution was transferred to a dialysis bag (MWCO: 1000 Da) and dialyzed in pure water to remove unreacted DCys-DGlu6 polypeptide. The reaction product DSPE-PEG2000-DGlu6 in the dialysis bag was freeze-dried to obtain the product.

[0053] The mass-to-charge ratio of DSPE-PEG200-Mal and DSPE-PEG200-DGlu6 was detected by matrix-assisted laser desorption-time-of-flight mass spectrometer. From Figure 4 ​It can be seen that the mass-to-charge ratio of DSPE-PEG2000-Mal is about 3158.3, and the mass-to-charge ratio of DSPE-PEG2000-DGlu6 is about 4053.1. The difference between the mass-to-charge ratios of the two is about 894.8, which is consistent with the relative molecular mass of DCys-DGlu6 (895.3, Figure 2 B) consistent, thereby confirming the successful synthesis of DSPE-PEG2000-DGlu6.

[0054] 3. Preparation and characterization of liposomes with different Glu6 modification densities

[0055] In the present application, liposomes with different Glu6 modification densities are prepared by the thin film hydration method. Briefly, the lipid components HSPC / cholesterol / DSPE-PEG2000 / DSPE-PEG2000-DGlu6 are dissolved in chloroform / methanol (3:1, v / v) at a molar ratio of 56.5:38.5:5-x:x (x represents the molar ratio of DSPE-PEG2000-DGlu6) in a 100 mL round-bottom flask, and placed on a rotary evaporator under vacuum for 30 min to form a phospholipid film. Remove the residual organic solvent under vacuum overnight. Add PBS solution and rotate for 30 min in a 37°C water bath to hydrate the film and remove the film, obtaining a liposome suspension. The liposome suspension is successively passed through 100 nm and 50 nm polycarbonate membranes in an extruder to reduce the particle size, until the particle size reaches about 100 nm. When the molar ratio of DSPE-PEG2000-Glu6 is 0, 1, 2, 3, 4, and 5, the liposomes are named Lip, 1%e-Lip, 2%e-Lip, 3%e-Lip, 4%e-Lip, and 5%e-Lip, respectively. Non-PEGylated liposomes (nP-Lip) with lipid components HSPC / cholesterol (56.5:38.5, mol / mol) are prepared as positive controls that can be phagocytosed by macrophages.

[0056] DiD is mixed into the lipid components at a total lipid content of 1 mol% and dissolved in chloroform / methanol (3:1, v / v), and then treated according to the above method to obtain DiD-labeled liposomes. The prepared liposomes are stored in a 4°C refrigerator.

[0057] The liposomes Lip, 1%e-Lip, 2%e-Lip, 3%e-Lip, 4%e-Lip, 5%e-Lip, and nP-Lip are dispersed in deionized water, and the light scattering particle size and zeta potential are measured using a Malvern particle size analyzer.

[0058] The liposome prescription components of the present application are as follows in Table 1:

[0059] Table 1 Liposome prescription components with different Glu6 modification densities

[0060]

[0061] The liposomes prepared in this patent have comparable particle size Figure 5 B) and the same lipid composition, thus ensuring that their pharmacokinetic behavior depends mainly on their surface properties, i.e. the Glu6 modification density. The isoelectric point of glutamic acid (Glu, e) is 3.22, which means that Glu6 is negatively charged at neutral pH. As shown in Figure C, the Zeta potential of the liposomes decreases from -6.98 to -29.13 mV as the Glu6 modification density gradually increases from 0 to 5 mol%, indicating that more negatively charged Glu6 modifications are present on the surface of the liposomes. The successful preparation of liposomes with different Glu6 modification densities is demonstrated. Figure 5

[0062] Example 2

[0063] 1. Effect of Glu6 modification density on the HA binding rate of liposomes

[0064] Take 1 mL of DiD solution with a concentration of 1.5 μg / mL of Lip, 1% e-Lip, 2% e-Lip, 3% e-Lip, 4% e-Lip and 5% e-Lip, and add 10 mg of HA respectively. Stir at 37°C for 6h, centrifuge at 5000g for 10 min, collect the precipitate, resuspend with PBS, and add it to a 48-well plate respectively. Observe the fluorescence intensity by IVIS imaging system.

[0065] Use the microplate reader to measure the fluorescence intensity (DiD, excitation / emission wavelength = 630 / 670 nm) in the collected supernatant. According to the standard curve, calculate the liposome concentration before and after binding with HA, and the percentage decrease of liposome concentration in the supernatant corresponds to the HA binding rate. The calculation formula is as follows:

[0066] HA binding rate (%) = [(C 结合前 -C 结合后 ) / C 结合前 ] x 100

[0067] Where C 结合前 represents the initial liposome concentration, and C 结合后 represents the liposome concentration in the supernatant after binding with HA.

[0068] Figure 6 The results demonstrate that the highest Glu6 modification density (5 mol%) confers the strongest HA binding activity to the liposomes.

[0069] 2. Uptake of Raw264.7 cells by liposomes with different Glu6 modification densities ​

[0070] The exponentially growing mouse macrophage line Raw264.7 was used at 2×10 5 Cells were seeded at a density of 100 cells / well in 12-well plates and incubated for 12 h. After removing the culture medium, solutions of DiD-labeled Lip, 1% e-Lip, 2% e-Lip, 3% e-Lip, 4% e-Lip, 5% e-Lip, and nP-Lip (pre-incubated with fetal bovine serum (FBS) at 37°C for 1 h) were diluted 10-fold with DMEM high-glucose medium and added to the 12-well plates, incubating for 4 h. The culture medium was removed, and the cells were washed three times with PBS. The cells were then resuspended in 1 mL of PBS. The uptake of DiD-labeled Lip, 1% e-Lip, 2% e-Lip, 3% e-Lip, 4% e-Lip, 5% e-Lip, and nP-Lip by Raw264.7 cells was measured using flow cytometry.

[0071] Raw264.7 cells in the exponential growth phase were fed a 1×10⁻⁶ dose. 5 Cells were seeded at a density of 100 cells / dish in confocal culture dishes and incubated for 12 h. The culture medium was then removed. DiD-labeled Lip, 1% e-Lip, 2% e-Lip, 3% e-Lip, 4% e-Lip, 5% e-Lip, and nP-Lip solutions, pre-incubated with FBS at 37°C for 1 h, were diluted 10-fold with DMEM high-glucose medium and added to the confocal dishes. After incubation for 4 h, the culture medium was removed, and the cells were washed three times with PBS. The cells were fixed with 4% paraformaldehyde for 30 min and washed three times with PBS. DAPI staining was performed for 2 min, followed by rinsing three times with PBS. Liposome uptake at Raw 264.7 was observed under a laser confocal fluorescence microscope.

[0072] Figure 7 The results showed that high-density Glu6 (4 or 5 mol%) modification could promote macrophage phagocytosis of liposomes by imparting a high negative charge to the liposome surface (-21.47 and -29.13 mV, respectively).

[0073] 3. Serum stability study of liposomes

[0074] The serum stability of liposomes was investigated using the methods reported in the literature. Lip and nP-Lip were dissolved in PBS solution containing 50% FBS to a final lipid concentration of 100 μg / mL, and incubated at 37°C. At preset time points, the absorbance of the suspension was measured at 560 nm using a microplate reader to assess the degree of liposome aggregation.

[0075] Figure 8 The results showed that Lip had good blood stability.

[0076] 4. Investigation of DiD leakage in serum from liposomes

[0077] To investigate the leakage of liposome-entrapped DiD in blood circulation, DiD-loaded liposomes were dissolved in 100% FBS (final concentration of liposomes was 100 μg / mL) and incubated at 37°C, protected from light. At 0, 5, 15 and 30 min, 1, 2, 4, 8, 12, 24 and 48 h, the fluorescence intensity of DiD (DiD, excitation / emission wavelength = 630 / 670 nm) was measured by a microplate reader. Since free DiD hardly fluoresces in aqueous solution, the leakage of DiD can be judged by the decrease of fluorescence intensity.

[0078] Figure 9 The results show that DiD leaks less and can be used to trace the in vivo delivery of liposomes.

[0079] 5. Investigation of pharmacokinetic behavior of liposomes with different modification densities of Glu6

[0080] To investigate the pharmacokinetic behavior of liposomes with different modification densities of Glu6, 21 SD rats (200 ± 20 g) were randomly divided into 7 groups (n = 3) and injected with DiD-labeled Lip, 1% e-Lip, 2% e-Lip, 3% e-Lip, 4% e-Lip, 5% e-Lip and nP-Lip via the tail vein. The dose of DiD was 0.3 mg / kg. At 5, 15 and 30 min, 1, 2, 4, 10 and 24 h after injection, about 100 μL of blood was taken from the rat orbital plexus using a capillary tube and added to a 1.5 mL centrifuge tube pre-washed with an anticoagulant. After centrifugation at 3500 rpm for 10 min, the supernatant plasma was collected. 20 μL of plasma was removed and added to 180 μL of isopropanol containing 0.1% Triton X-100, mixed well, and sonicated for 10 min. After centrifugation at 10000 g for 10 min, the supernatant was collected and the fluorescence intensity was measured by a microplate reader (DiD, excitation / emission wavelength = 630 / 670 nm). The concentration of DiD at each time point was calculated according to the standard curve, the concentration-time curve was drawn, and the pharmacokinetic parameters were calculated by DAS 2.0 software using a non-compartment model.

[0081] Figure 10 The results show that high-density Glu6 (4 or 5 mol%) modification weakens the blood retention of liposomes.

[0082] 6. In vivo biodistribution of liposomes with different modification densities of Glu6

[0083] To investigate the biodistribution of liposomes with different Glu6 modification densities, 21 8-week-old C57BL / 6 female mice were randomly divided into 7 groups (n = 3) and were injected with Lip, 1% e-Lip, 2% e-Lip, 3% e-Lip, 4% e-Lip, 5% e-Lip, and nP-Lip via the tail vein, respectively. Among them, the DiD dose was 0.48 mg / kg. After 24 h, the mice were euthanized, and the heart, liver, spleen, lung, kidney, and bilateral femur / tibia were collected.

[0084] The collected organs were washed with PBS to remove adhering blood, filtered with paper to remove moisture, and weighed. The bilateral femur and tibia were frozen in liquid nitrogen for 10 min, and then the heart, liver, spleen, lung, kidney, and frozen bilateral femur and tibia were placed in 1.5 mL centrifuge tubes, respectively, and magnetic beads were added, and 1 mL of isopropanol solution containing 0.1% Triton X-100 was added. Among them, the liver was placed in 2 1.5 mL centrifuge tubes due to its large volume, and a total of 1.5 mL of isopropanol solution containing 0.1% Triton X-100 was added. A tissue homogenizer was used to break up for 120 s at a power of 60 Hz, and then the samples were incubated overnight in a 4°C refrigerator. Centrifugation was performed at 10000 g for 10 min, and the supernatant was collected. The fluorescence intensity value was measured using a microplate reader (DiD, excitation / emission wavelength = 630 / 670 nm). The DiD concentration was calculated according to the standard curve, and the biodistribution of liposomes was expressed as pg DiD / g tissue.

[0085] Nine 8-week-old C57BL / 6 female mice were randomly divided into 3 groups (n = 3) and were injected with Lip, 5% e-Lip, and nP-Lip via the tail vein, respectively, with a DiD dose of 0.48 mg / kg. After 24 h of injection, the mice were euthanized, and the heart, liver, spleen, lung, kidney, and bilateral femur / tibia were collected. The fluorescence intensity of these organs was observed under the IVIS live imaging system.

[0086] Figure 11 The results showed that the distribution of bone-targeting liposomes in bone tissue increased with increasing Glu6 modification density, and 5% e-Lip had the highest bone targeting activity.

[0087] 6、5% e-Lip in the liver and spleen of resident macrophages

[0088] To evaluate the phagocytosis of 5% e-Lip by resident macrophages in liver and spleen, 9 C57BL / 6 female mice at 8 weeks of age were randomly divided into 3 groups (n=3) and injected with Lip, 5% e-Lip and nP-Lip via tail vein, respectively. The DiD dose was 0.48 mg / kg. At 24 h after injection, the mice were euthanized and the liver and spleen were removed. The liver and spleen were sectioned by a freezing microtome with a thickness of 10 pm after PBS washing to remove adherent blood, dehydration, and O.C.T. embedding.

[0089] Freezing section immunofluorescence staining procedure:

[0090] (1) Freezing section fixation: The freezing section was taken out from the refrigerator, air-dried, and fixed with 4% paraformaldehyde for 1 h. After the 4% paraformaldehyde was completely dry, the section was de-stained in PBS (pH=7.4) and washed on a shaker for 3 times, 5 min each time.

[0091] (2) Antibody repair: The tissue section was placed in citric acid antigen repair buffer (pH=6.0) and subjected to antigen repair in a microwave oven. Medium heat for 8 min, stop heating for 8 min, and low heat for 7 min. After natural cooling, the section was placed in PBS and washed on a shaker for 3 times, 5 min each time.

[0092] (3) Circle drawing: The section was air-dried and a circle was drawn around the tissue with a histological pen to prevent the loss of antibodies.

[0093] (4) Blocking: Serum was added dropwise in the circle and incubated for 30 min for blocking.

[0094] (5) Incubation of primary antibody: The blocking solution was gently shaken off, and the primary antibody was added dropwise on the section. The section was placed flat in a wet box and incubated overnight at 4°C, with a small amount of water added to the wet box to prevent evaporation of the antibody.

[0095] (6) Incubation of secondary antibody: The section was placed in PBS and washed on a shaker for 3 times, 5 min each time. The section was air-dried, and the secondary antibody of the same species as the primary antibody was added dropwise in the circle to cover the tissue. The section was incubated at room temperature for 50 min in the dark.

[0096] (7) DAPI re-staining of cell nucleus: The section was placed in PBS and washed on a shaker for 3 times, 5 min each time. After the section was slightly air-dried, DAPI staining solution was added dropwise in the circle and incubated at room temperature for 10 min in the dark.

[0097] (8) Mounting: The section was placed in PBS and washed on a shaker for 3 times, 5 min each time. The section was air-dried, and an anti-fluorescence quencher was used to mount the section.

[0098] The section was observed under a fluorescence microscope, and images were collected. The Pearson correlation coefficient was calculated using ImageJ software.

[0099] Figure 12 The results show that the 5 mol% acidic oligopeptide modification density can significantly enhance the liver and spleen uptake of liposomes.

[0100] 7. Evaluation of the biocompatibility of 5% e-Lip

[0101] The blood of 8-week-old C57BL / 6 female mice was taken and placed in an anticoagulant-washed 1.5 mL centrifuge tube; centrifuged at 3500 rpm for 10 min, and the red blood cell precipitate was collected, washed with PBS for 3 times, until the supernatant after centrifugation was colorless. 20 μL of red blood cell precipitate was taken, and 980 μL of deionized water (positive control), PBS (negative control), Lip (final lipid concentration: 0.6 mg / mL), low concentration 5% e-Lip (final lipid concentration: 0.6 mg / mL), and high concentration 5% e-Lip (final lipid concentration: 1.8 mg / mL) were added, and incubated at 37°C for 2 h. The suspension was centrifuged at 3500 rpm for 5 min, and the supernatant was collected. The absorbance value was measured at 570 nm using a microplate reader, and the hemolysis rate was calculated using the following formula:

[0102] Hemolysis rate (%) = [(ODtest-ODneg) / (ODpos-ODneg)]x100

[0103] Where ODtest, ODneg, and ODpos represent the absorbance values of the liposome sample, negative and positive controls, respectively.

[0104] The supernatant was photographed using a digital camera to determine the color change. The treated red blood cell precipitate was resuspended with PBS, and the cell morphology was observed under an inverted phase contrast microscope.

[0105] Figure 13 The results show that 5% e-Lip has good biocompatibility when administered systemically.

[0106] In summary, by studying the effects of Glu6 modification density on the HA binding capacity of liposomes, macrophage phagocytosis, pharmacokinetic behavior, and bone tissue distribution, and attempting to screen the optimal Glu6 modification density, the highest bone targeting activity of liposomes was achieved.

[0107] Although the specific embodiments of the present application are described above, those skilled in the art should understand that this is only an example, and the protection scope of the present application is defined by the appended claims. Those skilled in the art can make various changes or modifications to these embodiments without departing from the principles and essence of the present application, and such changes and modifications fall within the protection scope of the present application.

Claims

1. Acidic oligopeptide modified bone targeting liposomes, characterized in that, The surface of the bone-targeting liposome is modified with an acidic oligopeptide, and the modification density n is 5 mol%. The method for preparing the bone-targeting liposome is prepared by the following method: HSPC, cholesterol, and DSPE-PEG2000 and DSPE-PEG2000-DGlu6 with different feeding ratios are dissolved in an organic solvent, shaken uniformly, vacuum rotary evaporated to form a phospholipid film, vacuumed overnight, added with PBS, water-bathed and rotated until the film is hydrated and falls off, to obtain a liposome suspension with different Glu6 modification densities, and then extruded with an extruder with a polycarbonate membrane to obtain bone-targeting liposomes with a particle size of 100 nm. The organic solvent is a mixture of chloroform and methanol with a volume ratio of 3:

1. HSPC, cholesterol, DSPE-PEG2000, and DSPE-PEG2000-DGlu6 are dissolved in the following molar ratios: 56.5:38.5:5-x:x, wherein x represents the molar ratio of DSPE-PEG2000-DGlu6, and x=1, 2, 3, 4, 5.

2. A method of preparing the bone targeting liposome of claim 1, wherein, The method for preparing the bone-targeting liposome is prepared by the following method: The organic solvent is a mixture of chloroform and methanol with a volume ratio of 3:

1. HSPC, cholesterol, DSPE-PEG2000, and DSPE-PEG2000-DGlu6 are dissolved in the following molar ratios: 56.5:38.5:5-x:x, wherein x represents the molar ratio of DSPE-PEG2000-DGlu6, and x=1, 2, 3, 4, 5.

3. The method of claim 2, wherein, The water bath temperature is 37℃, and the water bath time is 30 min.

4. The method of claim 2, wherein, The liposome suspension is sequentially extruded through 100 nm and 50 nm polycarbonate membranes in the extruder.

5. A bone-targeting liposomal drug, characterized in that, The bone-targeting liposome drug comprises the bone-targeting liposome of claim 1, and an active drug molecule wrapped in the bone-targeting liposome.

6. The bone targeting liposomal drug of claim 5, wherein, The active drug molecule is selected from at least one of the following: drugs for treating osteoporosis, tumor bone metastasis, bone fracture, bone defect repair, hyperostosis, and heterotopic ossification.

7. The bone-targeting liposome drug of claim 5 or 6 for use in the preparation of a drug for treating bone disease.

8. Use according to claim 7, characterized in that, The bone disease is selected from osteoporosis, tumor bone metastasis, bone fracture healing disorder, bone fracture, bone defect repair, hyperostosis, and heterotopic ossification.

9. A pharmaceutical preparation, characterized by, The drug preparation contains the bone-targeting liposome drug of claim 5 or 6, and the drug preparation is a liquid preparation, which is an injection.

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