Protamine derivative and application thereof
By modifying the amino group of the side chain of protamine with phenylboronic acid to form an amide bond, a protamine derivative was prepared, which solved the problem of protamine's lack of targeting and precise release, achieved stable delivery and precise release of nucleic acid drugs, and enhanced the effect of gene therapy.
Patent Information
- Application Number
- CN202510625454.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-15
- Publication Date
- 2025-10-17
AI Technical Summary
When protamine is used as a carrier for nucleic acid drugs, it lacks targeting and precise release capabilities, resulting in limited efficacy of nucleic acid drugs.
By modifying the amino group on the side chain of protamine with phenylboronic acid to form an amide bond, a protamine derivative was prepared, and the precise release of nucleic acid drugs was achieved by utilizing the ATP-responsive boron ester bond.
It improves the delivery stability and transfection effect of nucleic acid drugs, enhances the efficacy of gene therapy, and has good safety and clinical application prospects.
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Figure CN120795113A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the technical field of medicine, and particularly relates to a protamine derivative and application thereof. BACKGROUND
[0002] Gene therapy is a treatment method for correcting or compensating for abnormalities or deficiencies of target genes to achieve the purpose of treating diseases. The advantage of gene therapy is that, compared with traditional drug therapy, gene therapy can target the root cause of the disease and achieve specific and long-term effects through gene editing. The stability of nucleic acid drug delivery, how to protect nucleic acid drugs from degradation, and accurate release at the lesion site are the keys to gene therapy. Therefore, it is necessary to develop a safe, effective and accurate nucleic acid drug delivery system.
[0003] Protamine is a protein rich in arginine extracted from mature sperm of fish, which has a high positive charge on the surface and can combine with nucleic acid drugs through electrostatic interaction to help nucleic acid drugs pass through the cell membrane, and is one of the commonly used carriers for nucleic acid drugs. However, protamine itself has no targeting property, and is too tightly combined with nucleic acid drugs, which makes it impossible to accurately release the nucleic acid drugs during delivery, resulting in limited drug efficacy. Therefore, modifying protamine to enhance the release capacity of gene drugs and improve the transfection efficiency is a means to improve the application prospect of protamine.
[0004] Benzene boronic acid modification is an effective controlled release modification strategy. The boronic acid group on benzene boronic acid can reversibly combine with the diol structure on the nucleic acid drug to form a boron ester bond, which can be broken when encountering a more competitive diol structure. There is a high concentration of ATP inside the tumor, and ATP has a diol structure similar to nucleic acid. Under the condition of high concentration of ATP, the original boron ester bond breaks, releasing the nucleic acid drug. This ATP-responsive nucleic acid drug delivery system effectively enhances the transfection efficiency of nucleic acid drugs by accurate release. SUMMARY
[0005] One of the purposes of the present application is to provide a protamine derivative, which is obtained by modifying benzene boronic acid on the side chain amino group of protamine, and the benzene boronic acid is selected from 4-carboxybenzene boronic acid, 2-aminobenzene boronic acid, 3-carboxy-4-fluorobenzene boronic acid, 4-formyl chloride benzene boronic acid, or a combination of any two or more of the above.
[0006] Further, the molecular weight of the protamine is in the range of 2000-5000 g / mol.
[0007] In one embodiment of the present application, the carboxyl group of 4-carboxybenzene boronic acid is activated using NHS and EDC, and then protamine is added for reaction, and the benzene boronic acid is modified on the side chain amino group of protamine through an amide bond.
[0008] The second object of the present application is to provide the application of the above-mentioned protamine derivative as a delivery carrier in the preparation of a nucleic acid drug delivery system.
[0009] Further, the nucleic acid drug is DNA, mRNA, siRNA, microRNA, saRNA, circRNA, or a combination of any two or more of the above.
[0010] In an embodiment of the present application, 4-carboxyphenylboronic acid is used as a reactant, which is connected to the protamine side chain amino group through an amide bond to obtain the protamine derivative. The specific preparation method comprises the following steps:
[0011] Step 1: Dissolve 4-carboxyphenylboronic acid (PBA), NHS and EDC in HEPES buffer, the molar ratio of NHS to EDC is 1:1, the molar ratio of 4-carboxyphenylboronic acid to NHS is 1:1-1:10, and the reaction is carried out for 30-60 min;
[0012] Step 2: Dissolve protamine (PRM, 5000 g / mol) in the reaction system of step 1, the molar ratio of PRM to PBA is 1:10-1:40, and the reaction is carried out for 12-36 h;
[0013] Step 3: The solution obtained in step 2 is dialyzed, freeze-dried and stored to obtain PRM-PBA;
[0014] The reaction formula is as follows:
[0015]
[0016] Then, siRNA is used as a nucleic acid drug, and the ribose vicinal dihydroxyl group on the siRNA is combined with the boronic acid group on PRM-PBA to form a nanoparticle PP / siRNA with ATP responsiveness.
[0017] In an embodiment of the present application, the mass ratio of protamine derivative PRM-PBA and siRNA is 5-40:1.
[0018] The present application designs and constructs a protamine derivative, which uses protamine (PRM) as a basic carrier material, connects phenylboronic acid or its derivative (PBA) to the protamine side chain amino group through an amide bond to synthesize protamine derivative (PRM-PBA), and then combines the boronic acid group with the nucleic acid drug to form the final preparation. The functionalized protamine as a nucleic acid drug carrier improves the stability of the delivered nucleic acid drug and precise release, and enhances the efficacy of gene therapy.
[0019] Beneficial effects:
[0020] 1. The application first proposes a preparation scheme of phenylboronic acid modified protamine, and applies it to nucleic acid drug delivery. The modified protamine can improve the transfection effect on genes, and has good safety.
[0021] 2. The protamine derivative carrier prepared in the application has simple synthesis, strong universality, good reproducibility of preparation process, good stability of carrier and easy clinical transformation.
[0022] 3. The preparation prepared in the application can improve the stability of nucleic acid drug delivery, and can release the nucleic acid drug in response to ATP, improve the curative effect of nucleic acid drug, has good development prospect and high clinical use value. DETAILED DESCRIPTION
[0023] Figure 1 It is the nuclear magnetic resonance hydrogen spectrum of 4-carboxyphenylboronic acid modified protamine.
[0024] Figure 2 It is the gene encapsulation detection result of siNC encapsulated by 4-carboxyphenylboronic acid modified protamine.
[0025] Figure 3 It is the particle size and potential characterization result of siNC encapsulated by 4-carboxyphenylboronic acid modified protamine.
[0026] Figure 4 It is the RNase stability result of PP / siNC in test example 1.
[0027] Figure 5 It is the cell uptake result of PP / FAM-siNC in test example 2.
[0028] Figure 6 It is the lysosome escape result of PP / FAM-siNC in test example 3.
[0029] Figure 7 It is the ATP-responsive release result of PP / siNC in test example 4.
[0030] Figure 8 It is the silencing result of PRMT1 gene in test example 5. DETAILED DESCRIPTION
[0031] The preferred embodiments of the application will be described in detail below with reference to the embodiments. It should be understood that the following embodiments are given only for the purpose of illustration, and are not intended to limit the scope of the application. Those skilled in the art can make various modifications and replacements to the application without departing from the spirit and principles of the application.
[0032] The experimental methods used in the following examples are conventional methods unless otherwise specified.
[0033] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.
[0034] The molecular weight of protamine used in the following examples is 5000 g / mol.
[0035] The gene drugs used in the following examples were all provided by Shanghai Gene Pharmaceutical.
[0036] The gene drug sequences used in the following examples are as follows:
[0037] siNC: sense, 5'-UUCUCCGAACGUGUCACGUTT-3'
[0038] FAM-siNC: sense, 5'-UUCUCCGAACGUGUCACGUTT-3'
[0039] siPRMT1: sense, 5'-GACUACCUAACAGUGAAGATT-3'.
[0040] Example 1
[0041] 1. Synthetic protamine derivatives
[0042] 1.1 Synthesis of 4-carboxyphenylboronic acid-modified protamine
[0043] Using NHS and EDC as catalysts, 4-carboxyphenylboronic acid (PBA) was modified onto protamine (PRM, 5000g / mol) through an amide reaction to synthesize PRM-PBA. The specific process is: 0.4mmol PBA, 1mmol NHS and 1mmol EDC were dissolved in about 10mL HEPES buffer and activated for 30-60min; then 0.01mmol PRM was added to the above activation system to initiate the amide reaction, stirred for 24h, and the molar ratio of PRM to PBA was 1:40. After the reaction was completed, the reaction solution was dialyzed using a 1kDa dialysis bag for 48h, then freeze-dried, and 1 H-NMR analysis.
[0044] like Figure 1 As shown, according to 1 H-NMR results showed that the proton peak of the PBA benzene ring appeared at 7.5-8.0 ppm, proving that PBA was successfully grafted onto PRM.
[0045] 1.2 Synthesis of 2-aminophenylboronic acid-modified protamine
[0046] Using NHS and EDC as catalysts, 2-aminophenylboronic acid (PBA) was modified onto protamine via an amide reaction to synthesize PRM-PBA. The specific process involved dissolving 0.4 mmol of PBA, 1 mmol of NHS, and 1 mmol of EDC in approximately 10 mL of HEPES buffer and activating the mixture for 30-60 minutes. Then, 0.01 mmol of PRM was added to the activated mixture to initiate the amide reaction, which was stirred for 24 hours. The molar ratio of PRM to PBA was 1:40. After completion of the reaction, the solution was dialyzed using a 1 kDa dialysis bag for 48 hours and then lyophilized.
[0047] 1.3 Synthesis of 3-carboxy-4-fluorophenylboronic acid-modified protamine
[0048] Using NHS and EDC as catalysts, 3-carboxy-4-fluorophenylboronic acid (PBA) was modified onto protamine via an amide reaction to synthesize PRM-PBA. The specific process involved dissolving 0.4 mmol of PBA, 1 mmol of NHS, and 1 mmol of EDC in approximately 10 mL of HEPES buffer and activating the mixture for 30-60 minutes. Then, 0.01 mmol of PRM was added to the activated system to initiate the amide reaction, which was stirred for 24 hours. The molar ratio of PRM to PBA was 1:40. After completion of the reaction, the reaction solution was dialyzed using a 1 kDa dialysis bag for 48 hours and then lyophilized.
[0049] 2. Preparation of protamine derivatives loaded with nucleic acid drugs
[0050] 2.1 siNC encapsulation using protamine modified with 4-carboxyphenylboronic acid
[0051] W were prepared by self-assembly method. pp / W siNC PP / siNC ratios of 0, 0.5, 1, 2, 5, 10, and 20 were used. Specifically, a 50 μg / mL siNC solution was prepared, followed by a gradient dilution method to prepare PRM-PBA solutions of varying concentrations. Equal volumes of the two solutions were mixed to obtain different PP / siNC ratios. Agarose gel electrophoresis was then performed at 100 V for 15 minutes, and the exposed siNC bands were observed and photographed using a gel imager.
[0052] like Figure 2 As shown, in W pp / W siNC When it is greater than 2, PRM-PBA can completely encapsulate siNC.
[0053] 2.2 Using 4-carboxyphenylboronic acid-modified protamine to encapsulate CRISPR / Cas
[0054] W were prepared by self-assembly method.pp / W CRISPR PP / circRNA with ratio of 0, 0.5, 1, 2, 5, 10, 20. Specifically: circRNA solution was prepared at 50 μg / mL, then PRM-PBA solution was prepared at different concentrations by gradient dilution method, and then mixed with equal volume of the two solutions to obtain PP / circRNA with different ratios.
[0055] 2.3 Preparation of circRNA encapsulated by protamine modified by 4-carboxyphenylboronic acid
[0056] W pp / W circRNA PP / circRNA with ratio of 0, 0.5, 1, 2, 5, 10, 20. Specifically: circRNA solution was prepared at 50 μg / mL, then PRM-PBA solution was prepared at different concentrations by gradient dilution method, and then mixed with equal volume of the two solutions to obtain PP / circRNA with different ratios.
[0057] 2.4 Preparation of PRM / siNC (W PRM / W siNC = 10:1) and PP / siNC (W pp / W siNC = 5:1, 10:1, 20:1, 40:1, 60:1) according to the method of 2.1 using protamine (PRM, 5000 g / mol) and protamine modified by 4-carboxyphenylboronic acid (PRM-PBA), respectively, and then the particle size and zeta potential of siRNA nanocomplexes were determined by dynamic light scattering (DLS) analyzer (Brookhaven) and ZetaPlus-zeta potential analyzer (Brookhaven).
[0058] As shown in Figure 3 , compared with PRM / siNC, the particle size of PP / siNC decreased from 280 nm to 200 nm and the potential decreased from +22.3 mV to +18.2 mV after modification of phenylboronic acid.
[0059] Test Example 1
[0060] Preparation of PP / siNC (W pp / W siNC=10:1). The nanoparticles were then incubated in the presence of RNase for varying periods of time. The siNCs were displaced by SDS and analyzed by agarose gel electrophoresis. The displaced siNCs were then examined to assess the extent of RNase degradation of the siNCs in the formulation, thereby evaluating the RNase stability of the PP / siNCs. Specifically, the nanoparticles were incubated in a 37°C water bath containing 10 mg / mL RNase. Samples were taken at different time points (0, 2, 4, 8, 12, and 24 hours). 2% SDS was then added to displace the siNCs encapsulated within the nanoparticles. The exposed siNC bands were then visualized and photographed by agarose gel electrophoresis to assess the RNase stability of the formulation.
[0061] like Figure 4 As shown in Figure 3, after 24 h of incubation with RNase, there was no obvious leakage of siNC, demonstrating the good RNase stability of the preparation.
[0062] Test Example 2
[0063] The cellular uptake of nanoparticles was investigated by laser confocal microscopy. 4 The cells were evenly seeded in a confocal dish at a density of 100 μg / well and cultured for 24 h. PRM / FAM-siNC and PP / FAM-siNC (FAM-siNC 1.50 μg / mL, W PRM / W siNC =10:1, W pp / W siNC =10:1, prepared as in 2.1) of Example 1, and incubated for 4 hours. After incubation, the cells were washed twice with PBS, fixed with 4% paraformaldehyde for 15 minutes, and then washed twice again with PBS. The cells were then stained with DAPI for 10 minutes, washed twice more with PBS, and observed and photographed using a laser confocal microscope.
[0064] like Figure 5 As shown, PP / FAM-siNC showed higher cellular uptake than PRM / FAM-siNC.
[0065] Test Example 3
[0066] The lysosomal escape of the preparations was investigated by laser confocal microscopy. 4 The cells were seeded into confocal culture dishes at 37°C for 24 h, and PP / FAM-siNC (FAM-siNC 1.50 μg / mL, W pp / W siNC=10:1, the preparation process is as in 2.1) of Example 1, co-cultured with cells for 3 h, then washed twice with PBS, added with fresh blank culture medium and continued incubation for 0 or 3 h, then added with lysosomal red fluorescent probe LysoTrackerRed and incubated at 37°C for 40 min, then washed twice with PBS, and observed and photographed by laser confocal microscopy.
[0067] like Figure 6 As shown in the figure, after 3 hours of incubation, the green fluorescence of FAM-siNC was detected to colocalize with the red fluorescence of the lysosomal probe. After 6 hours of incubation, a clear separation of the FAM-siNC and LysoTracker fluorescence signals was observed, indicating that siNC had successfully escaped.
[0068] Test Example 4
[0069] Prepare PP / siNC (W) according to 2.1 in Example 1 pp / W siNC =10:1), and then incubated with different concentrations of ATP (0, 0.5, 1, 2, 5, 10 mM) for 10 min. Then, agarose gel electrophoresis was used to investigate the ATP-responsive release of siNCs by the preparations.
[0070] like Figure 7 As shown in the figure, as the ATP concentration increases, more siNC is released. When the ATP concentration is 5 mM, almost all siNC is released, which proves that PP / siNC has ATP-responsive release ability and is ATP concentration-dependent.
[0071] Test Example 5
[0072] Western Blot was used to investigate the silencing effect of the preparation on the PRMT1 gene. 4T1 cells were seeded in 12-well plates at a density of 1×10 5 After culturing for 24 h, PBS, Free-siPRMT1, PRM / siPRMT1, and PP / siPRMT1 (siPRMT1 concentration was 100 nM, W PRM / W siNC =10:1, W pp / W siNC =10:1, and the preparation process was as described in 2.1) of Example 1. The cells were incubated with 4T1 cells for 4 h, then replaced with blank culture medium and incubated for another 48 h. After washing twice with ice-cold PBS, the cells were lysed with RIPA lysis buffer, the lysate was collected, and the cells were centrifuged at 12000 rpm for 10 min. The supernatant was collected to collect various histones, and the PRMT1 protein expression was detected by Western Blot.
[0073] like Figure 8As shown, the PRMT1 protein level in the PP / siPRMT1 group was significantly inhibited, and the inhibition effect was better than that in the PRM / siPRMT1 group, proving that the preparation has good gene silencing effect.
Claims
1. A protamine derivative, characterized in that The protamine derivative is obtained by modifying the amino group of the protamine side chain with phenylboronic acid, wherein the phenylboronic acid is selected from 4-carboxylphenylboronic acid, 2-aminophenylboronic acid, 3-carboxyl-4-fluorophenylboronic acid, 4-formyl chloride phenylboronic acid, or a combination of any two or more thereof.
2. The protamine derivative according to claim 1, characterized in that The molecular weight of the protamine is in the range of 2000-5000 g / mol.
3. Use of the protamine derivative according to any one of claims 1 to 2 as a delivery carrier in the preparation of a nucleic acid drug delivery system.
4. The use according to claim 3, characterized in that The nucleic acid drug is DNA, mRNA, siRNA, microRNA, saRNA, circRNA, or a combination of any two or more of the above.
5. A nucleic acid drug delivery system, characterized in that: The invention comprises a delivery vector and a nucleic acid drug, wherein the delivery vector is the protamine derivative according to any one of claims 1 to 2.
6. The nucleic acid drug delivery system according to claim 5, characterized in that The nucleic acid drug is DNA, mRNA, siRNA, microRNA, saRNA, circRNA, or a combination of any two or more of the above.
7. The nucleic acid drug delivery system according to claim 6, characterized in that The mass ratio of the delivery vector to the nucleic acid drug is 1-100:1.