CN111773392A - Human serum albumin/oligonucleotide nano-particles and preparation method and application thereof - Google Patents
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Abstract
The invention belongs to the technical field of nano-drugs, and particularly relates to human serum albumin/oligonucleotide nanoparticles as well as a preparation method and application thereof. The main raw materials of the human serum albumin/lysine-oligonucleotide nano-particles comprise human serum albumin nano empty capsules and lysine-oligonucleotide conjugates, and the lysine-oligonucleotide conjugates can be embedded in the human serum albumin nano empty capsules through electrostatic attraction. Lysine-oligonucleotide conjugates are prepared by capping lysine at the 2 'position from the 3' terminus of an oligonucleotide. The human serum albumin/lysine-oligonucleotide nano-particles have small particle size and good use effect.
Description
Technical Field
The invention relates to the technical field of nano-drugs, in particular to a human serum albumin/oligonucleotide nano-particle and a preparation method and application thereof.
Background
Because of the excellent curative effect and innovation of tumor immunotherapy, the tumor immunotherapy is judged as the most important scientific breakthrough in years by the journal of science in 2013, and an immune system regulator is the earliest means for tumor immunotherapy. CpG-ODN is one kind of immune system regulator and has been found to promote body's specific and non-specific immune response obviously. Although CpG-ODN opens new avenues for tumor therapy, it requires surface modification or package delivery to reach pharmacological targets and function in vivo. Currently, the nano-carriers for delivering CpG-ODN under investigation are liposomes, virus-like particles, and polymer nano-carriers, but the safety and efficacy of these exogenous nano-particles in vivo are still uncertain. For example, VLPs of virus-like particles have great potential for encapsulation and delivery of CpG-ODN, but are immunogenic and therefore have in vivo safety to be assessed. Therefore, the development of a nano delivery system suitable for the water-soluble immunoadjuvants such as CpG-ODN is not easy, and the system needs to have the following characteristics: 1) the size of the nano particles is between 10 and 100 nm; 2) the nanoparticles can aggregate near the tumor; 3) can maintain high blood concentration for a long time. Therefore, the characteristics of the CpG-ODN nano-drug are surrounded, endogenous materials are utilized to develop nano-carriers, and the nano-carriers have higher safety and more clinical value, such as human serum albumin.
The albumin paclitaxel (Abraxane) is lipid-soluble paclitaxel embedded by using human serum albumin as a carrier based on NAB technology, and the safety of the drug loading of the human serum albumin is proved. Human serum albumin is supported by a complete set of theories and technical systems in the aspect of fat-soluble drug delivery, and needs more exploration in the aspect of encapsulation and delivery of water-soluble drugs, and particularly, no relevant report is found for preparing 10-100 nm-sized nano-carriers for delivering CpG-ODN by using human serum albumin.
Furthermore, since human serum albumin is negatively charged, it cannot be used directly to embed and deliver CPG-ODNs that are also negatively charged. The traditional solution and the traditional preparation process of delivering water-soluble drugs by human serum albumin lead to relatively large particle size of the nanoparticles, which limits the application.
Disclosure of Invention
The invention provides a lysine-oligonucleotide conjugate, which solves the problem that the human serum albumin with negative charge in the prior art cannot be used for embedding and delivering oligonucleotide (with negative charge).
The lysine-oligonucleotide conjugate adopts the following technical scheme: a lysine-oligonucleotide conjugate prepared by capping lysine at the 2 'position from the 3' terminus of an oligonucleotide. Because lysine has positive charge, the prepared lysine-oligonucleotide conjugate also has trace positive charge, and can be used for compounding with a negatively charged nano carrier (such as a common endogenous negatively charged human serum albumin nano empty capsule) to realize delivery of the oligonucleotide; in addition, the end capping of lysine at the 2 'position of the 3' end of the oligonucleotide does not change the physicochemical properties of the oligonucleotide, and the particle size of the final nanoparticle is not affected when the lysine is compounded with a human serum albumin nanocapsule and the like.
Preferably, the lysine-oligonucleotide conjugate is prepared as follows: the preparation method of the lysine-oligonucleotide conjugate comprises the following steps: (1) synthesizing N () -trifluorooctyl-L-lysine benzyl ester 2 by taking toluenesulfonic acid monohydrate and N () -trifluorolysine as raw materials; (2) synthesizing oligonucleotides on a DNA/RNA synthesizer, and vulcanizing the oligonucleotides to obtain resin-bonded oligonucleotides; (3) transferring the resin-bound oligonucleotide prepared in the step (2) into a 5ml reaction tube of a Quest 210 manual synthesizer, adding polyvinylpyrrolidone stable palladium nanoparticles and 1, 4-cyclohexadiene as hydrogen donors, and performing phase transfer hydrogenation to realize the dissociation of a benzyl protecting group on the resin-bound oligonucleotide to obtain a debenzyl protecting group-resin-bound oligonucleotide; (4) activating the carboxyl group of the oligonucleotide prepared in the step (3) in DMF by using 1-hydroxy-1H-benzotriazole and N, N' -diisopropyl-carbodiimide; (5) putting the product obtained in the step (4), N () -trifluorooctyl-L-lysine benzyl ester 2, 1-hydroxy-1H-benzotriazole, N' -diisopropyl-carbodiimide and DMF into a reaction vessel, stirring and reacting for 15-20H, and carrying out coupling reaction; after the reaction was complete, the resin-containing product was washed with DMF and water, respectively.
Preferably, the mass ratio of the trifluorolysine to the oligonucleotide is 1: (4-16); preferably, after adding 1, 4-cyclohexadiene to the PVP-stabilised palladium suspension and filtering the solution, the filtrate obtained is then used as hydrogen donor for the cleavage of the benzyl protecting group on the resin-bound oligonucleotide; preferably, the lysine-oligonucleotide conjugates containing two and three lysines can be prepared by repeating the deprotection and coupling procedures.
The second purpose of the present invention is to provide a human serum albumin/lysine-oligonucleotide nanoparticle, wherein the human serum albumin/lysine-oligonucleotide nanoparticle of the present invention adopts the following technical scheme: the main raw materials of the human serum albumin/lysine-oligonucleotide nanoparticles comprise human serum albumin nano empty capsules and the lysine-oligonucleotide conjugates in any one of the items, wherein the lysine-oligonucleotide conjugates can be embedded in the human serum albumin nano empty capsules through electrostatic attraction. Because the lysine-oligonucleotide has stronger polarity than water molecules, and simultaneously the lysine-oligonucleotide has trace positive charges, the lysine-oligonucleotide can be attracted and embedded by the human serum albumin nano empty capsule with negative charges through electrostatic reaction. The human serum albumin/lysine-oligonucleotide nanoparticles have a particle size of less than 100nm (70-100nm), and can be released in response to reduction reaction in cells. The particle size of nano-drug carrier particles recognized in the industry should be between 20-300nm, and most of the currently approved anticancer nano-carrier particles have a particle size of between 100-200 nm. Recent studies have shown that smaller drug-loaded nanoparticles may exhibit better performance in vivo, such as higher tissue penetration or tumor suppression.
Preferably, the human serum albumin nano empty capsule is a sphere with the particle size of 70-100 nm; the preparation method of the human serum albumin nano empty capsule comprises the following steps: (1) adding glutathione into human serum albumin water solution, and adjusting pH to 3.0-4 (more preferably adjusting pH to 3.5-4); (2) dropwise adding an ethanol solution into the solution obtained in the step (1), and curing for 3 h; (3) membrane filtration, separation and purification of the mixed solution obtained in the step (2); (4) adding arginine (freeze-drying protective agent) into the filtrate obtained in the step (3), and then carrying out freeze drying to obtain human serum albumin nano empty capsules; the solidification in the step (2) is carried out under the conditions of shaking and magnetic stirring; the volume fraction of ethanol in the ethanol solution is 25%.
Preferably, the surface of the human serum albumin nano empty capsule is further modified with a targeting group RGD. The introduction of the targeting group RGD can lead the prepared human serum albumin/lysine-oligonucleotide nano-particles to have the targeting aggregation characteristic, and further can be used for targeting tumor cells. In the actual use process, the surface of the human blood albumin nano empty capsule can be modified by adopting a targeting group, and then the modified human blood albumin nano empty capsule is used for encapsulating the lysine-oligonucleotide conjugate; the lysine-oligonucleotide conjugate can also be encapsulated by human serum albumin, and then the surface of the human serum albumin nano empty capsule is modified to target the RGD group. For example, a specific method for modifying the surface of a human serum albumin nanocapsule to target the group RGD can be as follows: opening a disulfide bond by using endogenous glutathione as a cross-linking agent, adding an RGD targeting ligand to enable the RGD targeting ligand to be cross-linked with human serum albumin, and then forming RGD ligand albumin nanoparticles under the condition of long-time air exposure. The pH value, the stirring speed, the stirring method and the pretreatment speed of the solution can influence the particle size of the nano empty capsule, thereby influencing the particle size of the subsequent drug-loaded nano particles. Adjusting the pH value of the solution to 3.5-4, solidifying the human serum albumin solution for 3 hours by a composite method of oscillation (200rpm) and magnetic stirring (500rpm), and avoiding the instability of the chemical bond inside the human serum albumin caused by long-time high-speed magnetic stirring; and (3) dropwise adding the ethanol/water solution with the ratio of 1:3 into the solution, shaking the solution for 3 hours in an open mode, removing the ethanol, and purifying the solution. Arginine is adopted as a freeze-drying protective agent, so that the stability of the human serum albumin nanoparticles can be maintained.
The third objective of the present invention is to provide a method for preparing human serum albumin/lysine-oligonucleotide nanoparticles as described in any one of the above, wherein the method for preparing human serum albumin/lysine-oligonucleotide nanoparticles of the present invention adopts the following technical scheme: the method comprises the following steps: (1) adding the lysine-oligonucleotide conjugate into an aqueous solution for dissolving and adjusting the zeta potential to be 5.2; (2) dropwise adding the lysine-oligonucleotide conjugate solution prepared in the step (1) into the human serum albumin nano empty capsule, uniformly mixing, centrifuging, and storing supernatant for later use; (3) and (3) adding an acidified solution into the supernatant obtained in the step (2), and performing ultrasonic dispersion for 2s to encapsulate the lysine-oligonucleotide conjugate in the human serum albumin nano empty capsule to complete drug loading, so as to obtain the human serum albumin/lysine-oligonucleotide nanoparticles.
Preferably, the method further comprises the following steps: adding 20mM sulfo-SMCC solution into the human serum albumin/lysine-oligonucleotide, mixing and then carrying out freeze-drying treatment to obtain freeze-dried powder A; dissolving the freeze-dried powder A in a PBS solution to obtain a freeze-dried powder A-PBS solution, and dissolving RGD cyclopeptide containing sulfydryl in the PBS solution to obtain RGD cyclopeptide-PBS solution with sulfydryl; mixing the freeze-dried powder A-PBS solution with RGD cyclopeptide-PBS solution with sulfydryl, and carrying out freeze-drying treatment to obtain freeze-dried powder B, wherein the freeze-dried powder B is human serum albumin/lysine-oligonucleotide with an RGD targeting group modified on the surface; the freeze-drying protective agent is selected from arginine, albumin, mannitol, glycine and levo-glucoside.
Preferably, the mass ratio of the lysine-oligonucleotide to the HSA nano empty capsule is (0.05-25): 9.5.
The invention also provides the use of the human serum albumin/lysine-oligonucleotide nanoparticle, and the specific technical scheme is as follows: the use of the human serum albumin/lysine-oligonucleotide nanoparticle as described in any of the above in the preparation of an anti-tumor medicament.
The invention has the beneficial effects that: the lysine-oligonucleotide conjugate has positive charges, so that the lysine-oligonucleotide conjugate is conveniently encapsulated in a nano carrier with negative charges; the lysine in the lysine-oligonucleotide conjugate is modified at the 2 'position of the 3' end of the oligonucleotide, so that the physicochemical property of the oligonucleotide is maintained, and the phenomenon that the particle size of a nano-composite is enlarged after the oligonucleotide is encapsulated in a nano-carrier, and the effect of the nano-composite is further influenced, is avoided.
The human serum albumin/lysine-oligonucleotide nano-particles take human serum albumin nano empty capsules as carriers, lysine-oligonucleotide can be encapsulated inside the human serum albumin nano empty capsules by means of electrostatic acting force, and the human serum albumin is an endogenous substance, so that the safety is better.
The human serum albumin/lysine-oligonucleotide nano-particles have the particle size of 70-100nm and can show better performance in a body.
The RGD targeting group is further modified on the surface of the human serum albumin/lysine-oligonucleotide nanoparticle, so that tumor cells can be targeted, and the immunotherapy effect of the human serum albumin/lysine-oligonucleotide nanoparticle is enhanced.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 is a schematic structural diagram of RGD-HSA/Lysine-CpG-ODN according to the present invention;
FIG. 2 is a diagram showing the results of the targeting experiment of RGD-HSA/Lysine-CpG-ODN according to the present invention;
FIG. 3 is a high pressure liquid phase detection map of RGD-HSA/Lysine-CpG-ODN.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
EXAMPLE preparation of human serum Albumin/lysine-oligonucleotide nanoparticles
1. Preparation of positively charged oligonucleotide (Lys-CpG-ODN):
(1) n () -trifluorooctyl-L-lysine Benzyl Ester 2(N () -trifluoroacetyl-L-lysine Benzyl Ester 2) was synthesized using toluenesulfonic acid monohydrate (p-toluenesulfonic acid monohydrate 480mg,2.52mmol) and N () -trifluorolysine (N () -trifluoroacetyl-L-lysine 1,300mg,1.24 mmol).
(2) Oligonucleotide synthesis was performed on ABI 392 using a biological system DNA/RNA synthesizer to obtain resin bound oligonucleotides which were then sulfurized and purified prior to use to obtain sulfurized-resin bound oligonucleotides;
(3) deprotection: transferring the vulcanized-resin-bonded oligonucleotide obtained in the step (2) into a 5ml reaction tube of a Quest 210 manual synthesizer, taking polyvinylpyrrolidone stable palladium nanoparticles (0.7ml) and 1, 4-cyclohexadiene (0.05ml) as hydrogen donors, and realizing the dissociation of a benzyl protecting group on the resin-bonded oligonucleotide through phase transfer hydrogenation, wherein the method comprises the following specific steps: adding cyclohexadiene to the pvc-stable suspension of palladium and microfiltering it (0.22mm, ensuring no precipitated palladium was applied to the resin-bound oligonucleotide), mixing the filtrate with the resin-bound oligonucleotide and effecting cleavage of the benzyl protecting group on the thio-resin-bound oligonucleotide by phase transfer hydrogenation; after the reaction was completed, the resin was washed three times with methanol and three times with DMF to obtain a debenzylated protecting group-resin conjugated oligonucleotide.
(4) Prior to the coupling reaction, the carboxyl group of the oligonucleotide prepared in step (3) was activated with a solution of 1-hydroxy-1H-benzotriazole (HOBT, 0.4mg, 3.0. mu. mol) and N, N' -diisopropyl-carbodiimide (DIC, 1.1mg, 8.7. mu. mol) in 1ml of DMF. The mixture was stirred for 3h, and then the solution was filtered off again to give carboxyl-activated oligonucleotide.
(5) Coupling: a solution of the carboxyl-activated oligonucleotide prepared in step (4), 1-hydroxy-1H-benzotriazole (0.4mg, 3.0. mu. mol), N' -diisopropylcarbodiimide (0.4mg, 8.7. mu. mol) and N () -trifluorooctyl-L-lysine benzyl ester 2(2.0mg, 6.0. mu. mol) was added to 1ml of DMF, stirred for 18 hours and coupled. The resin was washed three times with DMF and three times with water.
For the preparation of conjugates containing two and three lysines, the deprotection and coupling procedures were repeated. For cleavage and deprotection of the oligonucleotide, 2ml of concentrated ammonia were added and the mixture was heated to 55 ℃ for 18 h. The solution was filtered and the solvent was evaporated in vacuo to give Lys-CpG-ODN. Finally, the conjugate is purified and detected by HPLC.
2. Preparation of HSA nanoparticle empty capsules: adding 200mg glutathione powder into 100ml water solution containing 500mg HSA (human serum albumin), stirring or shaking for 2h, mixing, adjusting pH to 3.5 to obtain HSA/r-GSH mixed solution; dripping the ethanol solution into the uniformly mixed HSA/r-GSH mixed solution, vibrating (200rpm) and magnetically stirring (500rpm) for curing for 3h, and then separating and purifying (100,000 cutoff) by membrane filtration; adding 5% mannitol into the obtained filtrate, and freeze-drying to obtain HSA nanoparticle empty capsule.
3. Adding 10mg of prepared Lysine-CpG-ODN into an aqueous solution for dissolving, adjusting the zeta potential to 5.2, then dropwise adding the Lysine-CpG-ODN solution into 10mg of centrifuged HSA freeze-dried powder, uniformly mixing, centrifuging, adding an acidification solution (20mM acetate buffer solution, pH5-5.5), and dispersing for 2s by using ultrasound to realize loading of Lysine-CpG-ODN in HSA nano-particle empty capsules, thereby obtaining HSA/Lysine-CpG-ODN.
4. After the drug loading is finished, modifying the surface of the HSA nano-particles with RGD: adding 20mM excessive sulfo-SMCC solution into the prepared HSA/Lysine-CpG-ODN at the pH of 7.8, uniformly mixing, and freeze-drying to obtain freeze-dried powder A; respectively dissolving the freeze-dried powder A and RGD cyclopeptide with sulfydryl in a PBS solution to obtain a freeze-dried powder A-PBS solution and an RGD cyclopeptide-PBS solution with sulfydryl; mixing the freeze-dried powder A-PBS solution and the RGD cyclopeptide-PBS solution with sulfydryl, and performing freeze-drying treatment to obtain freeze-dried powder B, wherein the freeze-dried powder B is RGD-HSA/Lysine-CpG-ODN, and the structural formula of the freeze-dried powder B is shown in figure 1.
In fig. 1: 1 is reduced glutathione, 2 is HSA nano empty capsule, red part refers to Lysine, irregular curve part connected with red part is CPG-ODN (Lysine-CpG-ODN in the circle), 3 is targeting ligand RGD
5. The human serum albumin nano vacant shell with the targeting ligand RGD can also be prepared according to the following method: adding reduced glutathione into human serum albumin + RGD solution with sulfydryl (also can be PEG-cRGD, RGD with sulfydryl is easier to modify), so that the human serum albumin forms a nano empty capsule in a disulfide bond opening mode, meanwhile, RGD (PEG-cRGD) with sulfydryl is loaded on the surface of the nano empty capsule through a disulfide bond, then adding ethanol for solidification, exposing nanoparticles in the air for 3h, finally purifying, freeze-drying, and finally forming the RGD-HSA nano empty capsule. And (3) connecting lysine serving as an end-capping reagent to the CPG to form lysine-CPG so as to be embedded by the RGD-HSA nano empty capsule finally. The method comprises the steps of utilizing the difference of isoelectric points of lysine-CPG and RGD-HSA nano empty capsule, and embedding lysine-CPG into the RGD-HSA nano empty capsule by adjusting the isoelectric points to form the RGD-HSA-CPG nano particles shown in figure 1.
Example two
1. Screening the reaction conditions of the human serum albumin solution and the r-GSH:
the particle size of the HSA/Lysine-CpG-ODN nano composite medicine can finally influence the clinical application and the in vivo drug release behavior, and the particle size and the dispersion degree of the nano particles can be influenced by the preparation process and the solution concentration.
(1) Investigating the influence of the stirring rotating speed and the rotating speed of the shaking table: weighing 25mg of r-GSH powder, adding the powder into 5ml of human albumin solution, pretreating the solution according to the following different requirements, and finding that the human albumin solution is placed on a shaking table to be pretreated for 4 hours at the speed of 80rpm to generate light blue opalescence, and the r-GSH powder and the human albumin solution are mixed and then shaken for 2 hours at the speed of 200rpm on the shaking table to generate light blue opalescence. Detailed experimental data are detailed in table 1 below.
TABLE 1
(2) The effect of human serum albumin concentration and pH was examined: in the preparation process, the concentration of the human serum albumin solution influences the size of nanoparticles formed by the human serum albumin solution, and experiments prove that the human serum albumin solution is optimal when the concentration of the human serum albumin solution is 4 mg/ml; after the human serum albumin solution and the R-GSH are uniformly mixed, NaOH is added to adjust the pH value of the solution, then ethanol is dropwise added, and the ethanol is removed by reduced pressure distillation at the temperature of 50 ℃. The particle size of the nanoparticles was at a minimum at pH 3.5 as the optimum pH. The specific experimental data are detailed in table 2 below.
TABLE 2
2. The prepared nano-particles (RGD-HSA/Lysine-CpG-ODN) need to be purified by a tangential dialysis method and then freeze-dried for long-time storage, and the selection of a freeze-drying protective agent influences the particle size, the appearance and the rehydration performance of the nano-particles. Finally, selecting levo-glycoside as the best freeze-drying protective agent. Specific experimental data are detailed in table 3 below.
TABLE 3
| Freeze-drying protective agent | Particle size before lyophilization (nm) | Lyophilized particle size (nm) | Appearance of the product | Phenomenon of rehydration |
| Albumin | 86.92±5.9 | 92.33±5.93 | Flocculent powder | Rapid rehydration |
| Mannitol | 86.92±5.9 | 94.13±6.91 | Flocculent powder | Rapid rehydration |
| Xylitol, its preparation method and use | 86.92±5.9 | 99.13±5.97 | Granular powder | Slow rate of rehydration |
| Polyethylene glycol | 86.92±5.9 | 102.33±4.45 | Slight caking | Slow rate of rehydration |
| Glycine | 86.92±5.9 | 96.39±7.42 | Flocculent powder | Rapid rehydration |
| L-glycoside | 86.92±5.9 | 90.83±4.91 | Flocculent powder | Rapid rehydration |
3. Since the CPG-ODN can not be directly embedded by using the human serum albumin nanoparticle empty capsule, the complex is embedded after the CPG-ODN is modified by Lysine. The ratio of positively charged Lysine to negatively charged CPG-ODN will ultimately affect its polarity. The particle size and encapsulation efficiency of Lysine to CPG-ODN performed best at a mass ratio of 0.5: 6. The specific experimental data are detailed in table 4 below.
TABLE 4
| Lysine：CPG-ODN(0.5mg) | HSA-NP(mg) | Particle size (nm) | Encapsulation efficiency |
| 0.5:2 | 9.29 | 91.2 | 99.2％ |
| 0.5:4 | 9.25 | 92.8 | 99.2％ |
| 0.5:6 | 9.18 | 91.5 | 98.9％ |
| 0.5:8 | 9.22 | 94.8 | 97.1％ |
4. 0.05. mu.l, 0.5. mu.l, 1. mu.l, 5. mu.l, 10. mu.l, 15. mu.l, 25. mu.l of an aqueous solution of lysine-CPG-ODN (1mg/ml) were added to the HSA nanoparticle powder, the pH of the solution was adjusted to 4 after mechanical mixing, the solution was dispersed under ultrasound, the particle size and physical appearance of the composite nano-drug were observed, and the encapsulation efficiency was examined. Experimental results show that the encapsulation efficiency obtained by 1mg of Lysine-CPG-ODN and 9.5mg of HSA-NPs is optimal. See table 5 below for detailed experimental data.
TABLE 5
5. After sterilization, the nano composite particles are stored at 4 ℃ and 40 ℃, and the particle size change and the particle size distribution of the nano medicament are respectively observed. Over time, the particle size and particle size distribution of the nanoparticles increased, with the appearance performing best within 4 months. See table 6 below for detailed experimental data.
TABLE 6
In clinical administration, the human serum albumin nanocomposite drug is diluted and then used, so that the stability of the composite drug in a 5% glucose solution and a 0.9% sodium chloride solution is tested. See table 7 below for detailed experimental data.
TABLE 7
6. The RGD-HSA/Lysine-CpG-ODN of the invention is targeted to research
Blowing 4T1 breast cancer cell with sterile pipette, and adjusting cell concentration to 5 × 106The purchased mice are fed for one week in SPF grade, no abnormal phenomenon appears, and are used for establishing a 4T1 breast cancer tumor model, female babl/c mice are randomly picked, 75% alcohol disinfection is carried out on injection sites, and 1ml syringes are used for blowing and beating uniform cells with the cell concentration of 5 × 106Injecting 4T1 breast cancer cells into hind limb armpit back by subcutaneous injection, observing skin bulge of injection part after injection, indicating successful injection, feeding the injected mice in SPF clean area, detecting tumor size by vernier caliper every 2 days, calculating the tumor volume size (a × b)2)/2
6.1 imaging
When the tumor volume of the breast cancer tumor-bearing mouse is 100mm3And when the fluorescent probe is positioned on the left and the right, near-infrared fluorescence living body imaging can be performed. 200ul of the injection solution was injected into the tail vein by using a 1ml syringe, and near infrared fluorescence in vivo imaging was performed 4 hours after the injection by using IVIS Lumina III (see the attached figure 2 of the specification for details).
6.2 treatment: when the tumor volume of the breast cancer tumor-bearing mouse is 100mm3The treatment effect is verified on the left and right. 200ul of the injection was injected in the tail vein using a 1ml syringe,the injection is injected once every 2 days for three doses.Volume (× length by × height) was recorded 14 days after 1 course of treatment (3 times). The experimental results are detailed in Table 8 below:
TABLE 8
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (10)
1. A lysine-oligonucleotide conjugate prepared by capping lysine at a 2 'position from the 3' terminus of an oligonucleotide, wherein the lysine-oligonucleotide conjugate has a positive charge.
2. The lysine-oligonucleotide conjugate according to claim 1, wherein the lysine-oligonucleotide conjugate is prepared by a method comprising the steps of: (1) synthesizing N () -trifluorooctyl-L-lysine benzyl ester 2 by taking toluenesulfonic acid monohydrate and N () -trifluorolysine as raw materials; (2) synthesizing oligonucleotides on a DNA/RNA synthesizer, and vulcanizing the oligonucleotides to obtain resin-bonded oligonucleotides; (3) transferring the resin-bound oligonucleotide prepared in the step (2) into a 5ml reaction tube of a Quest 210 manual synthesizer, adding polyvinylpyrrolidone stable palladium nanoparticles and 1, 4-cyclohexadiene as hydrogen donors, and performing phase transfer hydrogenation to realize the dissociation of a benzyl protecting group on the resin-bound oligonucleotide to obtain a debenzyl protecting group-resin-bound oligonucleotide; (4) activating the carboxyl group of the oligonucleotide prepared in the step (3) in DMF by using 1-hydroxy-1H-benzotriazole and N, N' -diisopropyl-carbodiimide; (5) putting the product obtained in the step (4), N () -trifluorooctyl-L-lysine benzyl ester 2, 1-hydroxy-1H-benzotriazole, N' -diisopropyl-carbodiimide and DMF into a reaction vessel, stirring and reacting for 15-20H, and carrying out coupling reaction; after the reaction was complete, the resin-containing product was washed with DMF and water, respectively.
3. The lysine-oligonucleotide conjugate according to claim 2, wherein the mass ratio of the trifluorolysine to the oligonucleotide is 1: (4-16); preferably, after adding 1, 4-cyclohexadiene to the PVP-stabilised palladium suspension and filtering the solution, the filtrate obtained is then used as hydrogen donor for the cleavage of the benzyl protecting group on the resin-bound oligonucleotide; preferably, the lysine-oligonucleotide conjugates containing two and three lysines can be prepared by repeating the deprotection and coupling procedures.
4. A human serum albumin/lysine-oligonucleotide nanoparticle, wherein the main raw materials of the human serum albumin/lysine-oligonucleotide nanoparticle comprise a human serum albumin nano empty capsule and the lysine-oligonucleotide conjugate according to any one of claims 1 to 3, and the lysine-oligonucleotide conjugate can be embedded in the human serum albumin nano empty capsule through electrostatic attraction.
5. The human serum albumin/lysine-oligonucleotide nanoparticle according to claim 4, wherein the human serum albumin/lysine-oligonucleotide nanoparticle is a sphere having a particle size of 70-100 nm; preferably, the preparation method of the human serum albumin nano empty capsule comprises the following steps: (1) adding glutathione into human serum albumin water solution, and adjusting the pH value to 3-4; (2) dropwise adding an ethanol solution into the solution obtained in the step (1), and curing for 3 hours under the conditions of shaking and magnetic stirring; (3) membrane filtration, separation and purification of the mixed solution obtained in the step (2); (4) adding arginine into the filtrate obtained in the step (3), and then freeze-drying to obtain human serum albumin nano empty capsules; preferably, the concentration of the human serum albumin solution is 4mg/mL, and the pH is 3.5.
6. The human serum albumin/lysine-oligonucleotide nanoparticle according to claim 4, wherein the human serum albumin nanocapsule surface is further modified with a targeting group RGD.
7. The method for preparing human serum albumin/lysine-oligonucleotide nanoparticles according to any one of claims 4 to 6, comprising the steps of: (1) adding the lysine-oligonucleotide conjugate into an aqueous solution for dissolving and adjusting the zeta potential to be 5.2; (2) dropwise adding the lysine-oligonucleotide conjugate solution prepared in the step (1) into the human serum albumin nano empty capsule, uniformly mixing, centrifuging, and storing supernatant for later use; (3) and (3) adding an acidified solution into the supernatant obtained in the step (2), and performing ultrasonic dispersion for 2s to encapsulate the lysine-oligonucleotide conjugate in the human serum albumin nano empty capsule to complete drug loading, so as to obtain the human serum albumin/lysine-oligonucleotide nanoparticles.
8. The method for preparing human serum albumin/oligonucleotide nanoparticles according to claim 7, further comprising the steps of: adding 20mM sulfo-SMCC solution into the human serum albumin/lysine-oligonucleotide, mixing and then carrying out freeze-drying treatment to obtain freeze-dried powder A; dissolving the freeze-dried powder A in a PBS solution to obtain a freeze-dried powder A-PBS solution, and dissolving RGD cyclopeptide containing sulfydryl in the PBS solution to obtain RGD cyclopeptide-PBS solution with sulfydryl; mixing the freeze-dried powder A-PBS solution with RGD cyclopeptide-PBS solution with sulfydryl, and carrying out freeze-drying treatment to obtain freeze-dried powder B, wherein the freeze-dried powder B is human serum albumin/lysine-oligonucleotide with an RGD targeting group modified on the surface; the freeze-drying protective agent is selected from arginine, albumin, mannitol, glycine and levo-glucoside.
9. The method of claim 7, wherein the lysine-oligonucleotide to HSA nanocapsule mass ratio is (0.05-25): 9.5; preferably, the mass ratio of the lysine-oligonucleotide conjugate to the human serum albumin nano empty capsule is 1: 9.5.
10. use of the human serum albumin/lysine-oligonucleotide nanoparticles according to claims 1-6 for the preparation of an antitumor drug.
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| CA2261562A1 (en) * | 1996-07-26 | 1998-02-05 | Novartis Ag | Fusion polypeptides comprising an ige-binding domain and a hsa component, and their diagnostic and therapeutic uses |
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| CA2261562A1 (en) * | 1996-07-26 | 1998-02-05 | Novartis Ag | Fusion polypeptides comprising an ige-binding domain and a hsa component, and their diagnostic and therapeutic uses |
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