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Carrier-free protein intracellular delivery prodrug as well as preparation method and application thereof

A technology for delivering prodrugs and proteins, applied in the field of protein modification and transport, to achieve the effect of reducing toxic and side effects

Pending Publication Date: 2021-11-05
SUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It solves the problem that the protein drugs used in the prior art all function by targeting cell surface receptors or extracellular specific structures

Method used

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  • Carrier-free protein intracellular delivery prodrug as well as preparation method and application thereof
  • Carrier-free protein intracellular delivery prodrug as well as preparation method and application thereof
  • Carrier-free protein intracellular delivery prodrug as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Synthetic example

[0045] (1) 4-(Hydroxymethyl)phenylboronic acid pinacol ester (0.5 g, 2.1 mmol) was dissolved in 20 mL of anhydrous THF. Triethylamine (0.6 mL, 4.5 mmol) was added, followed by 4-nitrophenyl chloroformate (0.47 g, 2.3 mmol), followed by stirring at room temperature for 1 hour. The reaction mixture was diluted with ethyl acetate and washed with 1.0 M HCl followed by saturated NaHCO 3 Wash with aqueous solution. The organic layer was treated with MgSO 4 Drying, filtration and concentration, followed by purification on a silica gel column eluting with 5% ethyl acetate in hexanes gave a white solid as monomer N:

[0046]

[0047] pass 1 H NMR (400 MHz, CDCl3) characterization, δ = 8.25 (d, J = 9.2 Hz, 2H), 7.85(d, J = 8.0 Hz, 2H), 7.43 (d, J = 8.0 Hz, 2H), 7.36 ( d, J = 9.2 Hz, 2H), 5.31(s, 2H), 1.35 (s, 12H);

[0048] Monomer P is the structure shown in the following formula:

[0049]

[0050] Monomer N and monomer P are used in the following examples.

Embodiment 1

[0052] Dissolve monomer N in dimethyl sulfoxide solution to a final concentration of (32 mg / mL) to obtain monomer N solution; dissolve bovine serum protein (BSA) in NaHCO at a concentration of 0.1 M 3 aqueous solution (6 mg / mL) to obtain bovine serum protein solution; then mix the two solutions according to the molar ratio of monomer N and amino group of bovine serum protein as 2:1, stir at room temperature for 10 hours, and transfer the reaction solution into a dialysis bag (MWCO = 1 kDa), dialyzed with ultrapure water for 3 days, and freeze-dried to obtain N-modified protein monomer (N-BSA).

[0053] Dissolve monomer P and N-BSA in water respectively to obtain monomer P solution (1 mg / mL) and N-BSA solution (6 mg / mL). Mix the monomer P solution and the N-BSA solution in a molar ratio of 2:3 according to the molar ratio of the monomer P and the primary amino group of the protein BSA, stir at room temperature for 30 minutes, and then transfer the reaction solution to an ultraf...

Embodiment 2

[0056] BSA was labeled with fluorescein isothiocyanate (FITC) in 0.1 M NaHCO 3 According to protein: FITC = 1:4 (mass ratio) in the buffer solution, react overnight under dark conditions, and then use ultrapure water dialysis (MWCO = 3.5kDa) for two days to remove unreacted FITC molecules and obtain FITC-labeled protein , and then use the method in Example 1 to modify the protein to obtain PN-BSA-FITC. Then human cervical cancer cells (HeLa) were divided into 5×10 cells per well 4 Inoculated into 24-well plates and cultured in DMEM medium containing 10% FBS for 24 hours. After the cells were completely attached to the wall, different modifications were made according to the concentration of 4 μg / mL and the final concentration of 100 μM H 2 o 2 Protein samples processed 12 hours in advance were added to the wells at 37 °C and 5% CO 2 Incubate under conditions for 12 hours. After washing twice with PBS, incubate with trypan blue solution for 3 minutes, continue to wash thr...

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Abstract

The invention provides a carrier-free protein intracellular delivery prodrug as well as a preparation method and application thereof. The carrier-free protein intracellular delivery prodrug is a carrier-free protein intracellular delivery system which is transferred through L-type amino acid transporter protein 1 (LAT1). The delivery system can directly deliver proteins with different charges / molecular weights into cells to prevent inclusion of endosomes / lysosomes and maintain protein activity, has excellent tumor targeting property, and reduces toxic and side effects. The delivery system is the first case of transmembrane transporter mediated carrier-free protein prodrug intracellular transport, and highly sensitive and highly selective regulation and control of protein activity in tumor cells are realized. The simple and efficient technology provides a new strategy for potential clinical application of anti-tumor protein drugs.

Description

technical field [0001] The invention relates to protein modification and transport technology, more specifically to a protein / polypeptide delivery system, specifically a carrier-free protein intracellular delivery prodrug and its preparation method and application. Background technique [0002] Proteins are an important class of biomaterials with good biocompatibility, and many proteins (such as enzymes) also have special biological functions, so they are expected to be used in a variety of applications such as: cancer therapy, immunotherapy, and biological imaging, etc. . The vast majority of biological reactions are carried out in cells, and protein preparations acting in cells have broad application prospects. However, the protein has a large molecular weight and cannot pass through the biomembrane barrier. The current protein preparations mostly target extracellular receptors or domains, which largely limits the application of protein preparations. It is of great signi...

Claims

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Application Information

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IPC IPC(8): A61K38/38A61K38/16A61K38/44A61K47/54A61P35/00
CPCA61K38/385A61K38/168A61K38/44A61K47/545A61K47/542A61P35/00
Inventor 殷黎晨赵子印刘寻
Owner SUZHOU UNIV
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