Study On The Cathepsin B-sensitive Drug-loaded Polymeric Micelles

Cathepsin B is a lysosomal cysteine protease. The expression of cathepsin B in many kinds of malignant tumor tissues, such as lung cancer, gastric cancer, prostate cancer and breast cancer, is multiplied and even three to nine times higher than the adjacent normal tissues. Cathepsin B can selectively recognize and degrade some kinds of peptides, such as Val-Cit, Phe-Lys and Gly-Phe-Leu-Gly. Based on the above characteristics of cathepsin B, the peptides which are sensitive to cathepsin B are usually used in the design of antibody-drug conjugates (ADCs) and prodrugs. Moreover, PABC is indispensable to achieve the release of drugs. The aim of this article is to fabricate the cathepsin B-sensitive drug-loaded polymeric micelles and study the effect of PABC spacer in this drug delivery system.At first, a kind of amphiphilic diblock copolymer mPEG-VC-PABC-C18 was synthesized. MPEG-VC-C18 without PABC was synthesized as control. The aqueous micelles solutions were prepared by solvent volatilization. The critical micelle concentration (CMC) values of mPEG-VC-PABC-C18 and mPEG-VC-C18 in aqueous media were determined, and pyrene was used as a probe in the measurement. The particle sizes and morphology were also measured by dynamic light scattering (DLS) and transmission electron microscope (TEM). Since mPEG-VC-PABC-C18 and mPEG-VC-C18 were not soluble in deuterium oxide. So, the two model compounds, mPEG-VC-PABC-C4 and mPEG-VC-C4, soluble in deuterium oxide were synthesized, and used to monitor the degradation behavior real-timely by 1H NMR in a mimical lysosome condition with cathepsin B. Results showed that the two kinds of model compounds could both degraded by cathepsin B. The degradation percentage of mPEG-VC-PABC-C4 is 50% while the degradation percentage of mPEG-VC-C4 is 33% in 24 h. The existence of PABC could accelerate the degradation of mPEG-VC-PABC-C18 and result in the faster degradation.Secondly, the degradation of mPEG-VC-PABC-C18 and mPEG-VC-C18 micelles was also investigated. The sizes changes of the two kinds of micelles were monitored by DLS with or without cathepsin B. Results indicated that the sizes just had little increase in the first and the fourth day and kept around 200 nm. Until the ninth day, the shift to greater dimension could be clearly observed, and the average sizes of the mPEG-VC-PABC-C18 and mPEG-VC-C18 assemblies increased up to 704 nm and 414 nm. The micelles were degraded by cathepsin B and aggregated after the detachment of the PEG shell, which resulted in the increased sizes. The size change of mPEG-VC-PABC-C18 was much more remarkable than that of mPEG-VC-C18, which indicated that the existence of PABC accelerated the degradation of the micelles. SN-38 is the active metabolite of irinotecan and its antitumor activity is much higher than irinotecan. SN-38 has two kinds of forms, the lactone form and the carboxylate form. The lactone form of SN-38 can easily hydrolyze into the carboxylate form which is toxic. To overcome the shortcoming, two kinds of SN-38-loaded micelles (SN38-VC-PABC-CM, SN38-VC-CM) were fabricated and the stability of SN-38 in micelles was also studied using reverse-phase HPLC. Results showed that after 9 h of incubation in PBS, only 11.5% of SN-38 remained in the lactone ring form which indicated that unprotected SN-38 went through the rapid conversion into the inactive carboxylate form. On the other hand, after 9 h of incubation in the same condition, 94.1% and 86.9% of the lactone rings remained when SN-38 were incorporated into SN38-VC-PABC-CM and SN38-VC-CM micelles, which confirmed the improved stability of SN-38 when protected by micelles. The drug release was also investigated by HPLC. It turned out that the total drug release of SN38-VC-PABC-CM and SN38-VC-CM in the presence of cathepsin B was 55% and 30%, respectively. When no cathepsin B was added, the total drug release of SN38-VC-PABC-CM and SN38-VC-CM was 27% and 28%, respectively. The release of SN-38 was faster in the presence of cathepsin B. Morever, the introduction of PABC accelerated the degradation of mPEG-VC-PABC-C18 which resulted in the total release of SN-38 from SN38-VC-PABC-CM micelles was much more than that from SN38-VC-CM micelles.At last, the cytotoxicity of the two kinds of bare micelles and the two kinds of SN38-loade micelles was also studied. Results showed that the cell viabilities treated with the two kinds of bare micelles still kept the level higher than 90%. Compared with free SN-38, both SN38-VC-PABC-CM and SN38-VC-CM showed less cytotoxicity. And the cytotoxicity of SN38-VC-PABC-CM was much higher than that of SN38-VC-CM. The existence of PABC accelerated the degradation and drug release which further led to the increase of cytotoxicity. The result indirectly proved that when PABC was introduced in, the rate of drug release was faster.As a drug delivery system, mPEG-VC-PABC-C18 can realize the tumor-targeted SN-38 release by introduction of Val-Cit. Morever, the existence of PABC could accelerate the degradation and drug release of cathepsin B degradable micelles.