Preparation Of Drug-loaded Polymeric Micelles Based On PLA-PEG-COOH And Its In- Vitro Anticancer Activities

As active pharmaceutical ingredients（APIs）,triptolide（TP）,cyclopamine（CYP）and chrysin（CHR）all have a variety of pharmacological effects,including potent anti-tumor effects,but poor water solubility of these APIs,usually resulting in low bioavailability for traditional dosage forms,seriously restrict their development and application.Overcoming the common shortcomings of these APIs by preparing functional dosage forms with higher bioavailability and lower doses has become a research hotspot in recent years.Polymer micelles（PM）are self-assembled in water,consisting of ordered molecular aggregates of polymer molecules with shell-core structure,and the shell of which is hydrophilic chains exposed to the outer layer in water,and the core of which is formed by hydrophobic chains.The shell not only effectively avoids the direct contact of drugs with water but also circumvents the recognition by the reticuloendothelial system in the body and the core is a hydrophobic environment which can increase the apparent solubility of poorly soluble drugs in water.Therefore,using an amphiphilic polymer as a carrier material,the poorly soluble APIs can be encapsulated into micelles,which not only can improve the water solubility of the drug and improve the release characteristics of the drug,but also serves as a target for drug delivery.The following researches have been carried out:（1）As an initiator,hydroxycarboxyethylpolyethylene glycol（HO-PEG-COOH）was derived from PEG with the carboxyethyl group in the one end of polyethylene glycol（PEG）and the original hydroxyl group in the other.HO-PEG-COOH and L-lactide or D,L-lactide were used to synthesize diblock copolymers by a ring-opening polymerization,and the diblock copolymer was poly（L-lactic acid）-（carboxyethylpolyethylene glycol）（PLLA-PEG-COOH）or poly（racemic lactic acid）-（carboxyethylpolyethylene glycol）（PDLA-PEG-COOH）.The chemical structures and properties of HO-PEG-COOH,PLLA-PEG-COOH and PDLA-PEG-COOH were characterized by FT-IR,H¹-NMR,C¹³-NMR,and GPC,and the critical micelle concentration（CMC）was determined using pyrene as a fluorescent prob.The results showed that HO-PEG-COOH,PLLA-PEG-COOH and PDLA-PEG-COOH were synthesized successfully.The CMC of PLLA-PEG-COOH（Mw:2.44×10⁴）and PDLA-PEG-COOH（Mw:3.68×10⁴）was 2.57×10^-4mg/mL and 9.73×10^-3mg/mL,respectively.（2）To evaluate the effect of different preparation methods on polymer micelles,four methods,dialysis,film hydration,solvent evaporation and ultrasonic emulsification,were used to prepare polymer micelles using PLLA-PEG-COOH and PDLA-PEG-COOH diblock copolymers as carrier materials and the particle size（PS）and polydispersity index（PDI）of PM served as evaluation indicators for these methods.It was found that the ultrasonic emulsification method was the most optimal method to prepare PM,the particle size and PDI of which was relatively small.Therefore,the TP and CYP-loaded in PM（（TP+CYP）-PM）and the CHR-loaded PM（CHR-PM）were prepared using the optimal method.The PS of the drug loaded in PM was around 100nm,and the PDI was less than 0.4.The morphology of PM was nearly spherical and the particles of PM was not aggregated.The encapsulation efficiency（EE）of（TP+CYP）-PM for TP and CYP was 65.44%and 78.16%,respectively and the EE of CHR-PM for CHR was 6.22%.The in-vitro cumulative release of CHR from CHR-PM reached 31.13%in pH 7.4 PBS at 37℃within 48h.（3）MTT proliferation inhibition assay,Hoechst 33258 fluorescence staining assay and flow cytometry assay showed that（TP+CPA）-PM inhibited A2780,A549,HepG2and SKOV3 cells,and its inhibitory intensity was similar to that of free drug.The activity of the drug was not reduced after encapsulated into polymer micelles.In summary,it is feasible to prepare PM using PLA-PEG-COOH to encapsulate such poorly soluble APIs as TP and CPA to exert anti-tumor effects.The drug-loaded PM based on PLA-PEG-COOH provides a starting-point for preparing active targeted PM,and the preparation of which needs to be optimized.