USAG-1 recombinant protein targeted delivery system based on multi-enzyme logic gating and preparation method

Abstract

The invention discloses a USAG-1 recombinant protein targeted delivery system based on multi-enzyme logic gating and a preparation method, and mainly relates to crossing of biological medicine and nanotechnology. Comprising the following steps: S1, a C-terminal fusion nuclear localization sequence PKKKRKV of a recombinant USAG-1 protein expressed by CHO cells, and the purity of the recombinant USAG-1 protein is greater than or equal to 98%; s2, preparing core-shell structure nanoparticles, wherein the core is PLGA loaded BMP-2, and the drug loading capacity is 2.5-4.2 [mu] g / mg; the outer-layer lipid membrane is composed of DSPC / DSPG according to the ratio of 7: 3, and the surface embedded USAG-1 molecular density is greater than or equal to 1 * 10 / mu m < 2 >; s3, the surface co-modified EGFR antibody and the RGDfK peptide are connected through a thiol-maleimide bond and a PEG2000 spacer arm according to the molecular ratio of 1: 1.6 + / -0.2, the coding sequence of the recombinant USAG-1 is regulated and controlled by an MMP-9 response promoter, and the promoter is located in MMP-9gt; and the expression is activated at 20nM. The system has the beneficial effects that time-space precise delivery of the bone metabolism related protein USAG-1 is realized through a multi-enzyme logic gating mechanism, and the system is suitable for treatment of bone metabolism disorder diseases such as osteosarcoma and alveolar bone defect.

Description

Technical Field

[0001] This invention relates to the field of interdisciplinary technology of biomedicine and nanotechnology, and in particular to a multi-enzyme logic-gated USAG-1 recombinant protein targeted delivery system and its preparation method. Background Technology

[0002] The intersection of biomedicine and nanotechnology is one of the important development directions in the fields of medicine and science and technology in recent years. The application of nanotechnology has brought revolutionary progress to biomedicine, especially in drug delivery, disease diagnosis, treatment and disease prevention.

[0003] The above findings reveal the following shortcomings in the current treatment of bone metabolic diseases: existing chemotherapy regimens for malignant bone tumors such as osteosarcoma suffer from significant systemic toxicity and poor targeting; while conventional delivery systems for bone repair proteins such as BMP-2 exhibit burst release effects, leading to side effects such as ectopic ossification. Summary of the Invention

[0004] The purpose of this invention is to address the problems of high systemic toxicity and poor targeting of existing chemotherapy regimens for malignant bone tumors such as osteosarcoma; and the shortcomings of conventional delivery systems for bone repair proteins such as BMP-2, which have burst release effects leading to side effects such as ectopic ossification. The invention proposes a multi-enzyme logic-gated USAG-1 recombinant protein targeted delivery system and its preparation method.

[0005] To achieve the above objectives, the present invention adopts the following technical solution, comprising the following steps:

[0006] S1. Recombinant USAG-1 protein expressed by CHO cells, with its C-terminus fused to the nuclear localization sequence PKKKRKV, and a purity ≥98%;

[0007] S2. Core-shell structured nanoparticles: The core is PLGA loaded with BMP-2, with a drug loading of 2.5-4.2 μg / mg; the outer lipid membrane is composed of DSPC / DSPG in a 7:3 ratio, and the surface is embedded with USAG-1 molecules at a density ≥1×10 / μm. 2 ;

[0008] S3 consists of surface-modified EGFR antibody and RGDfK peptide in a molecular ratio of 1:1.6±0.2, linked by a thiol-maleimide bond and a PEG2000 spacer arm.

[0009] Preferably, the coding sequence of the recombinant USAG-1 is regulated by the MMP-9 responsive promoter, which activates expression when MMP-9 > 20 nM.

[0010] Preferably, the nanoparticles have a two-stage release characteristic:

[0011] MMP-9 > 20 nM, release > 60% within 24 h BMP-2;

[0012] USAG-1, > 80% release within 48 h intracellular GSH > 10 mM.

[0013] Preferably, the PLGA inner core contains a disulfide crosslinking agent with the following structure:

[0014] HOOC-CH-S-S-CH-COO-(CH)-NHS.

[0015] Preferably, the amino acid sequence of the RGDfK peptide is:

[0016] c(RGDfK) (cyclic structure: Arg-Gly-Asp-D-Phe-Lys).

[0017] Preferably, the preparation method of the USAG-1 recombinant protein targeted delivery system based on multi-enzyme logic gate includes the following steps:

[0018] a) Preparation of BMP-2-loaded PLGA inner core by double emulsion method:

[0019] Inner aqueous phase: BMP-2 / PBS solution containing 5 mM disulfide crosslinking agent;

[0020] Oil phase: 50 mg / mL PLGA solution in ethyl acetate;

[0021] Ultrasonic emulsification power 200 W x 1 min;

[0022] b) Lipid membrane coating: mix DSPC / DSPG lipid membrane material with the outer aqueous phase and ultrasonic 200 W x 5 min;

[0023] c) USAG-1 insertion: insert the recombinant protein into the lipid bilayer by hydrophobic interaction;

[0024] d) Surface double-target modification: sequentially conjugate EGFR antibody and RGDfK-PEG2000 after EDC / NHS activation.

[0025] Preferably, the molecular weight of PLGA in step a) is 15-20 kDa, and the ratio of lactic acid / hydroxyacetic acid is 50:50.

[0026] Preferably, the coupling density is controlled in step d):

[0027] EGFR antibody: 400-600 molecules / μm 2 ;

[0028] RGDfK peptide: 700-900 molecules / μm 2 .

[0029] Preferably, the system comprises

[0030] A multi-enzyme logic-gated targeted delivery system comprises three functional modules:

[0031] Genetically engineered recombinant USAG-1 protein: containing MMP-9 responsive promoter and C-terminal nuclear localization sequence (PKKKRKV)

[0032] Lipid-polymer hybrid nanoparticles:

[0033] Core: PLGA (50:50) loaded with BMP-2 (drug loading capacity 2.5-4.2 μg / mg)

[0034] Outer layer: DSPC / DSPG (7:3) lipid bilayer embedded with USAG-1 (density ≥ 1 × 10 molecules / μm 2 )

[0035] Dual-targeting surface modification:

[0036] EGFR antibody (Cetuximab clone): thiol-maleimide bond coupling (500 molecules / μm 2 )

[0037] RGDfK peptide: PEG2000 spacer arm connection (800 molecules / μm 2 )

[0038] In summary, the beneficial effects of the present application are:

[0039] In the present application, the system realizes the spatiotemporal precise delivery of bone metabolism related protein USAG-1 through multi-enzyme logic-gated mechanism, and is suitable for the treatment of osteosarcoma, alveolar bone defect and other bone metabolism abnormal diseases. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 is a schematic diagram of the multi-enzyme logic-gated release mechanism of the present application; Figure 1 Figure 2 is a flow chart of the double emulsion method in the preparation process of the nanoparticles of the present application;

[0041] Figure 2 Figure 3 is a flow chart of the surface modification process in the preparation process of the nanoparticles of the present application;

[0042] Figure 4 is a genetic construction map of the recombinant USAG-1 of the present application. Figure 3

[0043] Figure 4 DETAILED DESCRIPTION

[0044] ​​​​The application will be further described in connection with the following specific embodiments. It should be understood that the embodiments are only used for illustrating but not for limiting the scope of the application. Furthermore, it should be understood that after reading the content of the present application, those skilled in the art can make various modifications or changes to the present application, and these equivalent forms also fall within the scope defined by the appended claims.

[0045] Embodiment 1: Reference Figure 1 As shown in the figure, the present application provides a technical solution: a USAG-1 recombinant protein targeted delivery system based on multi-enzyme logic gate, comprising the following steps:

[0046] S1, recombinant USAG-1 protein expressed by CHO cells, C-terminal fusion of nuclear localization sequence PKKKRKV, purity ≥98%;

[0047] S2, core-shell structure nanoparticles: the inner core is PLGA loaded with BMP-2, the drug loading is 2.5-4.2 μg / mg; the outer lipid membrane is composed of DSPC / DSPG at a ratio of 7:3, and the surface is embedded with USAG-1 molecules with a density of ≥1×10 / μm 2 ;

[0048] S3, surface co-modification of EGFR antibody and RGDfK peptide, molecular ratio 1:1.6±0.2, connected by thiol-maleimide bond and PEG2000 spacer arm.

[0049] The specific settings and functions will be described in detail below.

[0050] In this embodiment: the coding sequence of recombinant USAG-1 is regulated by MMP-9 responsive promoter, which activates expression when MMP-9>20nM.

[0051] The nanoparticles have a dual-stage release property:

[0052] ≥60% of BMP-2 is released within 24h when MMP-9>20nM;

[0053] ≥80% of USAG-1 is released within 48h when intracellular GSH>10mM.

[0054] The PLGA inner core contains a disulfide bond crosslinking agent, and its structural formula is:

[0055] HOOC-CH-S-S-CH-COO-(CH)-NHS.

[0056] The amino acid sequence of RGDfK peptide is:

[0057] c(RGDfK) (cyclic structure: Arg-Gly-Asp-D-Phe-Lys).

[0058] The preparation method of the USAG-1 recombinant protein targeted delivery system based on multi-enzyme logic gate control comprises the following steps:

[0059] a) Preparation of BMP-2 loaded PLGA inner core by double emulsion method:

[0060] Inner aqueous phase: BMP-2 / PBS solution containing 5mM disulfide crosslinking agent;

[0061] Oil phase: 50mg / mL PLGA solution in ethyl acetate;

[0062] Ultrasonic emulsification power 200Wx1min;

[0063] b) Lipid membrane coating: mix DSPC / DSPG lipid membrane material with outer aqueous phase, ultrasonic 200Wx5min;

[0064] c) USAG-1 embedding: insert the recombinant protein into the lipid bilayer by hydrophobic interaction;

[0065] d) Surface double-target modification: sequentially conjugate EGFR antibody and RGDfK-PEG2000 after EDC / NHS activation.

[0066] The molecular weight of PLGA in step a) is 15-20kDa, and the ratio of lactic acid / hydroxyacetic acid is 50:50.

[0067] Coupling density control in step d):

[0068] EGFR antibody: 400-600 molecules / μm 2 ;

[0069] RGDfK peptide: 700-900 molecules / μm 2 .

[0070] Preparation of recombinant USAG-1

[0071] Gene transfection:

[0072] Vector construction: pcDNA3.1(+) inserts MMP-9 response promoter, USAG-1 CDS, PKKKRKV nuclear localization sequence;

[0073] CHO cell electroporation parameters:

[0074] Voltage 250V, capacitance 950μF, pulse time 20ms;

[0075] Screening conditions: 800μg / mL G418 continuous culture for 14 days;

[0076] High expression clone screening:

[0077] Yield: 58.7 ± 3.2 mg / L (ELISA detection);

[0078] Specific activity: 1.5 x 10 5 IU / mg;

[0079] Protein purification:

[0080] Ni-NTA chromatography:

[0081] Binding buffer: 20 mM Tris-HCl, 300 mM NaCl, 20 mM imidazole, pH 8.0;

[0082] Elution gradient: 50-500 mM imidazole linear gradient;

[0083] SEC-HPLC verification:

[0084] Column: Superdex 200 10 / 300 GL;

[0085] Purity: 98.7% (monomer peak area ratio);

[0086] Comparison between traditional and present protocol:

[0087] Technical pain points Traditional scheme Invention scheme Tumor enrichment rate <15% (liposomes) 85% (double targeting) BMP-2 drug loading <2% (PLGA microspheres) 4.2 μg / mg (improved by 180%) 24h leakage rate >30％ <8% (GSH-responsive structure) Normal tissue uptake rate >25％ <5% (logic gating) Timing control capability None Double-stage release (r = 0.93)

[0088] Example 2: Reference Figure 2 、 Figure 3 Key parameters control:

[0089] Internal aqueous phase / oil phase volume ratio: 1:10;

[0090] PVA concentration: 2% (w / v);

[0091] Secondary emulsification power: 200 W x 5 min;

[0092] Example 3:

[0093] 1. In vitro experiment:

[0094] Uptake kinetics:

[0095] Saos-2 cells: 2 h uptake rate reached 82.3% peak;

[0096] Competitive inhibition experiment: uptake rate decreased to 41% after EGFR antibody blocking;

[0097] ALP activity inhibition: 78.2 ± 3.5% (10 nM USAG-1); Apoptosis induction: Annexin V+ cell ratio 68.9%;

[0098] 2. In vivo experiment:

[0099] Osteosarcoma nude mice model:

[0100] Dosing regimen: tail vein injection of 5mg / kg, twice a week; tumor volume on day 28: 152±25mm 3 (control group 892±103mm 3 ); biodistribution:

[0101] 125I labeling tracer: tumor tissue radioactivity count was 25 times that of muscle tissue; safety:

[0102] Liver and kidney function indicators: ALT (38±5U / L), AST (42±7U / L);

[0103] No abnormal inflammatory infiltration was observed histologically.

Claims

1. A multi-enzyme logic-gated USAG-1 recombinant protein targeted delivery system, characterized in that, The method comprises the following steps: S1, recombinant USAG-1 protein expressed by CHO cells, C-terminal fusion of nuclear localization sequence PKKKRKV, purity ≥98%; S2, core-shell structure nanoparticles: the inner core is PLGA loaded with BMP-2, the drug loading is 2.5-4.2 μg / mg; the outer lipid membrane is composed of DSPC / DSPG at a ratio of 7:3, and the surface is embedded with USAG-1 molecules with a density of ≥1×10 / μm 2 ; S3, surface co-modification of EGFR antibody and RGDfK peptide, molecular ratio 1:1.6±0.2, connected by thiol-maleimide bond and PEG2000 spacer.

2. The system of claim 1, wherein, The coding sequence of the recombinant USAG-1 is regulated by an MMP-9 responsive promoter, which activates expression when MMP-9>20nM.

3. The system of claim 1, wherein, The nanoparticles have a dual-stage release property: ≥60% of BMP-2 is released within 24h when MMP-9>20nM; ≥80% of USAG-1 is released within 48h when intracellular GSH>10mM.

4. The system of claim 1, wherein, The inner core of PLGA contains a disulfide crosslinking agent with the following structure: HOOC-CH-S-S-CH-COO-(CH)-NHS.

5. The system of claim 1, wherein, The amino acid sequence of RGDfK peptide is: c(RGDfK) (cyclic structure: Arg-Gly-Asp-D-Phe-Lys).

6. A method for preparing a USAG-1 recombinant protein targeted delivery system based on multi-enzyme logic gate control, using the system according to any one of claims 1 to 5, characterized in that, The method comprises the following steps: a) Preparation of BMP-2-loaded PLGA inner core by double emulsion method: Inner aqueous phase: BMP-2 / PBS solution containing 5mM disulfide crosslinking agent; Oil phase: 50mg / mL PLGA solution in ethyl acetate; Ultrasonic emulsification power 200W×1min; b) Lipid membrane coating: mix DSPC / DSPG lipid membrane material with outer aqueous phase, ultrasonic 200W×5min; c) USAG-1 embedding: insert recombinant protein into lipid bilayer by hydrophobic interaction; d) Surface double-target modification: sequentially couple EGFR antibody and RGDfK-PEG2000 after EDC / NHS activation.

7. The production method according to claim 6, characterized by, The molecular weight of PLGA in step a) is 15-20kDa, and the ratio of lactic acid / hydroxyacetic acid is 50:

50.

8. The preparation method according to claim 6, characterized in that, Coupling density control in step d): EGFR antibody: 400-600 molecules per pm 2 ; RGDfK peptide: 700-900 molecules / μm 2 .

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