Hepatitis B virus-like particle nano-carrier and drug delivery system
A hepatitis B virus, nano-carrier technology, applied in nano-drugs, viruses/phages, viruses, etc., to achieve the effects of good stability, high encapsulation stability, and not easy to leak
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Embodiment 1
[0046] Embodiment 1 Preparation of the hepatitis B virus-like particle nanocarrier of the present invention
[0047] Include the following steps:
[0048] (1) According to the amino acid sequence of the hepatitis B virus-like particle nanocarrier of the present invention, the nucleotide sequence encoding the hepatitis B virus-like particle nanocarrier is obtained by gene synthesis, and the expression vector plasmid pEt43 is constructed by Xho I / Nde I restriction site .1(a)—insert the RGD sequence between amino acids 78 and 82 of the HBc-170 protein (delete amino acids 79-81), and insert a connecting peptide between amino acid 78 and the RGD sequence GTSGTSGSSGSGGT, insert connecting peptide GGSGSSGSTG between RGD and the 82nd amino acid, obtain the amino acid sequence of RGD-HBc-170 protein (as shown in SEQ ID No.1); Add poly Histidine polypeptide (HHHHHH); according to the above description, synthesize the nucleotide sequence encoding the hepatitis B virus-like particle nano...
Embodiment 2
[0056] Embodiment 2 builds the drug delivery system of the present invention
[0057] 2.1 Drug loading
[0058] Take 100mL of the target protein (0.5mg / mL) and incubate with 50mL of pre-prepared dissociation solution at 25°C for 2h, the total volume of the solution is 150mL. At this point, 37.5 mg of doxorubicin was added to the above mixed solution, and the resulting mixed solution was incubated at 4° C. for 30 min with shaking. Afterwards, it was moved to a dialysis bag with a molecular weight of 3500 Da, first placed in 10% polymer solution (1 L) and dialyzed overnight at 4°C, and then replaced with 10% polymer solution after 12h and dialyzed at 4°C, After 2 hours, change to 0% polymer solution and continue dialysis at 4° C., during which the polymer solution is replaced 5 times until the dialysate is clear and transparent, and the dialysis can be stopped when the dialysate no longer turns red, that is, the encapsulation complex (delivery agent of the present invention) is...
Embodiment 3
[0073] Embodiment 3: Verify the purity of complex (drug delivery system of the present invention) by means of HPLC
[0074] (1) The detection wavelength of the detector of the high performance liquid chromatograph is 280nm (hepatitis B virus-like particle nanocarrier absorption peak of the present invention), 480nm (DOX absorption peak);
[0075] (2) the chromatographic column is an Ultrahydrogel 2000 chromatographic column;
[0076] (3) The column length of the chromatographic column is 300mm
[0077] (4) The inner diameter of the chromatographic column is 7.8mm
[0078] (5) The column temperature of the chromatographic column is 25°C
[0079] (6) The mobile phase of the chromatographic column is: 20mmol / L PBS, 150mmol / L NaCl;
[0080] (7) The chromatographic column flow rate: 0.6mL / min;
[0081] (8) By detecting the ultraviolet absorption value of the complex at 280nm and 480nm, it can be seen that there is a single absorption peak at about 14min, without other miscellan...
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