RGD Mediated Docetaxel-Loaded Ph Sensitive Liposomes: Synthesis, Preparation, Characterization And Antitumor Effiency In Vivo And In Vitro

Docetaxel (DTX), an analog of paclitaxel, is broadly used in treating a various human malignancies in clinical application, such as human ovarian, breast cancer and non-small-cell lung carcinoma. DTX take a unique mechanism of antitumor:it promotes the assemble of tubulin into nonfunctional tubules, thus blocking cell division in the G2/M-phases of the cell cycle. However, DTX have the characteristics of low aqueous solubility, limited bioavailability and poor targeting specificity which are main obstacles for effective cancer therapy. Clinically applicated formulation (Duopafei(?) and Taxotere(?) is formulated in a mount of Tween-80 and ethanol. A high risk of generating serious side effects, such as neurotoxicity, allergic reactions and nephrotoxicity, is seriously affect its antitumor effects. Therefore, the development of one targeted drug delivery system for DTX is urgently required.Liposomes are deemed as one of the most promising vehicles for clinical cancer therapy due to its merits, including biocompatibility, targeted and controlled release potential, longer blood circulation time. We attempt to add pH responsive molecules linonic acia (LA) into liposome formulation and liposomes will simultaneously disrupt the lipid bilayer membrane and controlled release the payload drugs upon external mildly acidic conditions. Recent expectation regarding the role of RGD (Arg-Gly-Asp), as active targeting ligand,has been growing. For the first time, we synthesis the molecule RGD-PEG-LA. Small molecular probes RGD were decorated on the surface of liposomes from grafting on the top of PEG. In the present study, PSL was decorated with RGD-PEG and evaluated for pharmacy and biology properties. The main research content as follows.1 Preparation and characterization of DTX-PSLThis study established a method of High Performance Liquid Chromatography (HPLC) for the determination of the amount of DTX. DTX as the model drug, was loaded into the pH sensitive liposome (DTX-PSL) which were made up of phosphatidyl ethanolamine (PE), cholesterol (CHOL) and linoleic acid (LA) as a pH sensitive material and preparated by traditional thin film dispersion method. The final optimized formulation and the best preparation technology was determined by the single factor investigation and orthogonal design method concerning the entrapment efficiency (EE%) as the main index. The physical and chemical properties of the DTX-PSL were evaluated, including encapsulation efficiency, drug loading efficiency (DL%), particle size and particle size distribution, Zeta potential and Transmission Electron Microscopy (TEM). DTX-PSL was lyophilized by freeze drying method and lactose as the freeze in order to improve the stability of formulation during the period of storage.The optimized DTX-PSL formulation is sphere and the surface is free of adhesions. The average particle size is 127.2±3.58 nm, PDI 0.218±0.15 by dynamic light scattering method. The potential is-35.19±1.27 mV with the appropriate electronegativity. EE%and DL%is 4.8±0.1% and 81.9±2.2%, respectively.2 The synthesis of molecule RGD-PEG-LA and the study of RGD/DTX-PSL formulationIn this section, linear RGD peptide were successfully synthesized by solid-phase peptide synthesis method (SPPS) with the help of 9-Fluorenylmethoxycarbonyl (Fmoc)-protected chemical molecule. Electrospray ionization mass spectrometry analysis (ESI-MS) and Matrix assisted laser desorption time of flight mass spectrometry (MALDI-TOF-MS) were also used to confirm the structure of RGD, RGD-PEG-NH2 and RGD-PEG2000-LA for sure, respectively. The physical and chemical properties of the RGD/DTX-PSL were also evaluated as above. In vitro release of Duopafei(?)、DTX-PSL and RGD/DTX-PSL were investigated in 0.5% Tween-80 (w/v) phosphate buffer solution (PBS, pH 5.0 and pH 7.4) by a dialysis technique.The molecular masses calculated (346.3) and detected (346.1) by ESI-MS which were unanimous with the expected level. The signal peaks of RGD (693.4[2M+H], IV route at single dose, was carried out on Male Sprague-Dawley(SD) rats (200±20 g), and DAS 2.0 software package was used to calculate parameters and chose the statistical moment method. Liquid-liquid extraction method was applied to extract drug from the plasma for High Performance Liquid Chromatography (HPLC) analysis.The t1/2z of DTX in DTX-PSL (2.455) and RGD/DTX-PSL (3.478) formulation which respectively is 2-fold and 3-fold compared with Duopafei(?) group (1.187) give rise to a longer blood circulation. The prolonged t1/2z、.increased MRT0-∞ and declined CLz suggest that both developed formulations could postpone the removed from the rats blood circulation and the bioavailability is greatly improved compared with Duopafei(?) group.In general, we have successfully synthesized molecule RGD-PEG-LA and RGD targeting factor is decorated on the surface of pH sensitive liposomes with the help of PEG mediating. The developed liposomes were prepared in simple preparation and the solubility of Docetaxel is greatly improved. The targeted-pH-sensitive liposome equipping with physical chemical and active targeted, could improve the antitumor effiency and decrease side effects by increasing the amount of docetaxel into tumor tissue rather than normal tissue. In addition to, RGD mediated pH sensitive liposomes provide a new way of developing a safe, stable, effective drug delivery system.