| Wogonin(Wog)is a natural flavonoid isolated from the root extract of the Chinese herb Scutellaria baicalensis.Wogonin has received much attention for its antioxidant,antiinflammatory,neuroprotective and apoptosis-inducing effects on cancer cells.However,similar to most flavonoids,its poor water solubility,rapid clearance rate in vivo,low bioavailability and lack of targeting when used as an anti-tumour drug have greatly limited the clinical potential of baicalin as a broad-spectrum anti-cancer drug.Ferritin is a spherical cage-like protein formed by the self-assembly of 24 identical or similar subunits,which are found in plants,animals and microorganisms.In addition to the properties of protein-based nanoparticles,human heavy chain ferritin(HFtn)specifically recognises the transferrin receptor 1(Tf R1),which is overexpressed in most tumour cells,and enters tumour cells via cytocytosis mediated by this receptor.This unique characteristic highlights the potential of HFtn as a targeted delivery vehicle.PAS polypeptides,composed of natural L-amino acid proline(Pro),alanine(Ala),serine(Ser),are hydrophilic,uncharged natural disordered biopolymers,which can increase the hydrodynamic volume of conjugated drugs and prolong drug half-life.In this study,two PAS short peptides(PAS10,PAS30)with different amino acid lengths were modified at the N-terminus of human heavy chain ferritin by genetic engineering,and used as a nanocarrier to encapsulate wogonin to construct wogonin-ferritin nanoparticles(PAS10/30-HFtn-Wog)to improve the water solubility and bioavailability of wogonin.The stability,targeting,anti-tumor effect and pharmacokinetics of the nanodrug were studied and evaluated.The main results are as follows:(1)The p ET-20b(+)-PAS10-HFtn and p ET-20b(+)-PAS30-HFt recombinant plasmids were successfully constructed,and the target proteins were obtained with high purity by heat treatment,affinity chromatography and FPLC.The characterization results showed that the modification of PAS peptides did not alter the original structure and properties of ferritin,and the size exclusion chromatography(SEC)and Dynamic Light Scattering(DLS)results demonstrated that the modification of PAS10 and PAS30 peptides increased the hydrodynamics radius of the protein cage from 14.21± 2.13 nm to 19.87± 2.81 nm and 23.43 ± 3.24 nm,providing a prerequisite for prolonging the half-life of the nanoparticles in vivo.(2)HFtn-Wog,PAS10-HFtn-Wog and PAS30-HFtn-Wog were successfully prepared by encapsulating wogonin into ferritin-based nanocages by expanding/ shrinking protein channels by adjusting temperature.HPLC analysis showed that each PAS30-HFtn protein cage could load approximately 60 Wog molecules,almost twice that of HFtn,and the protein recovery of the modified nanocarriers was also higher.The loading of wogonin did not affect the secondary structure(α-helix),polymerisation state(24 polymer)and morphological characteristics(cage structure)of the protein carriers.In vitro release and storage stability results showed that wogonin-ferritin nanodrugs were relatively stable at physiological p H conditions(p H = 7.4),and the release of Wog was significantly increased under slightly acidic tumour conditions(p H = 5.0),which indicated that the release of wogonin-ferritin nanomachine was p H-dependent.Furthermore,the stability of PAS-modified wogonin-ferritin nanoparticles was much higher than that of HFtn-Wog,indicating that PAS peptide modification significantly improved the stability of the protein,which was beneficial to prolong the retention time of wogonin in vivo.(3)In vitro biological activity and in vivo pharmacokinetic studies of the prepared wogoninferritin-based nanoparticles.Cytotoxicity test results showed that the protein carriers HFtn,PAS10-HFtn and PAS30-HFtn were almost non-toxic to cells,demonstrating their good biocompatibility.HFtn-Wog,PAS10-HFtn-Wog and PAS30-HFtn-Wog nanodrugs showed concentration-dependent killing effects,with PAS10-HFtn-Wog showing the greatest cytotoxicity at higher concentrations.In addition,Hep G2 cells were more sensitive to nanodrugs compared to MCF-7 cells.The toxicity of Wog-ferritin based nanoparticles on Hep G2 and Mc F-7 cells was further demonstrated by Calcein AM/PI double staining for live and dead cells.The wound healing analysis showed that nanoparticles could effectively enhance the inhibitory ability of Wog on Hep G2 and Mc F-7 cells.The mobility of Hep G2 and Mc F-7 cells after PAS10-HFtn-Wog treatment was 15.6% and 17.2%,respectively.Confocal laser and flow cytometry analyses showed that the modification of PAS peptides enhanced the uptake of the nanocarriers,which may stem from the unique biological activity of PAS peptides.Pharmacokinetic studies showed that PAS peptide modified nanocarriers significantly prolonged the retention time of Wog in vivo compared to free wogonin and wild-type HFtn,thus enhancing its toxicity to cancer cells and compensating for its rapid clearance in plasma as a protein-based nanocarrier.These results demonstrate that PAS peptide-modified ferritin-based nanoparticles are a promising drug carrier system and that PAS-HFtn-Wog has potential for application in cancer therapy. |
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