| Summary: | 碩士 === 國立清華大學 === 生醫工程與環境科學系 === 99 === Objective: Lactoferrin (Lf) conjugated polyethylene glycolated liposome (PL) was constructed and was entrapped with 99mTc as a radiotracer for study as a novel brain drug delivery system across the blood-brain barrier.
Methods: Lf was thiolated and conjugated via maleimide functional group on the surface of polyethylene glycolated liposome to form the modified liposome, referred to Lf-PL. The biorecognitive activity of Lf-PL was investigated by Lf ELISA assay following DLS analysis. The 99mTc radionuclide by its complex with N,N-bis (2-mercaptoethyl)-N’,N’-diethyl-ethylenediamine (BMEDA) was entrapped in the core of the liposomes, PL and Lf-PL with ammonium sulfate gradient. The resulted 99mTc loaded liposomes, referred to PL[99mTc] and Lf-PL[99mTc] were taken for in vitro stability study and in vivo pharmacokinetic study. The effect of Lf-PL[99mTc] on improving brain drug delivery was assessed by in vitro bEND.3 cell uptake study and in vivo brain uptake study using BALB/c mice via intravenous injection in comparison with PL[99mTc].
Results and Discussion: According to DLS analysis, the size of PL was 91.23±17.88 nm in diameter and increased to 96±17.84 nm after Lf conjugation. The average number of Lf conjugated on each liposome was ca. 60. The Lf ELISA assay results confirmed the biorecognitive activity of Lf-PL. The 99mTc-BMEDA loaded efficiency for Lf-PL[99mTc] (26.4±4.45%) was lower than that for PL[99mTc] (75.31±5.52%), indicating that the coupled Lf ligands might somewhat obstacle the 99mTc-BMEDA trapping in the liposome. The radiochemical purities of PL[99mTc] and Lf-PL[99mTc] all maintained high above 87% during 48 h incubation in normal saline or in rat plasma at 37 °C. The particle sizes of the 99mTc-BMEDA loaded liposomes were similar to the unloaded liposomes. From pharmacokinetic study, the area under the concentration-time curve (AUC0→24h) was calculated with 653.57±40.84 h ?~ %ID/g for Lf-PL[99mTc] in comparison with 746.37±119.26 h ?~ %ID/g for PL[99mTc], being without significant difference (P-value = 0.33). From the results, Lf-PL[99mTc] likely maintained a long circulation property as PL[99mTc]. The in vitro uptake of Lf-PL[99mTc] by bEND.3 cells (26.53±0.4%) presented at ca. 15-fold and 3-fold higher than that of 99mTc-BMEDA (1.89±0.8%) and that of PL[99mTc] (8.99±1.19%), respectively at 1 h postinjection. The in vivo brain uptake of Lf-PL[99mTc] (1.02±0.06% %ID/g) presented at ca. 1.47-fold higher than that of PL[99mTc] (0.69±0.06% %ID/g) at 1 h postinjection, respectively (P-value < 0.01).
Conclusion: The aforementioned in vitro and in vivo studies suggest that Lf-PL may be a potential brain drug delivery system.
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