Preparation And Characterization Of CMCS/PEI Based Dual PH-sensitive DOX And Gene Co-delivery System

Nanotechnology promotes great potential in co-delivery of drug and gene to tumor cells for cancer therapy, which solved the problems of poor stability, solubility and selectivity in the process of payloads delivery. While, great efforts still needed to be made to promote the development of drug and gene co-delivery systems. The pH-sensitive drug and gene co-delivery systems with targeted selectivity and controllable payloads release behavior exhibit great potential, which simply used the acidic tumor tissue pH or endosomes/lyosomes pH to realize targeted delivery and controlled payloads release. The pH-sensitive drug and gene co-delivery systems could help to reduce the undesirable side-effects and enhance the therapeutic effects of cancer. The tumor tissue pH-sensitive drug and gene co-delivery systems could dissociate their outmost layer triggered by acidic pH of tumor tissue and exposed the functionalized core, then the exposed core could enhance the cellular uptake and exert antitumor effect. The endosomes/lyosomes pH-sensitive drug and gene co-delivery systems could rapidly release of parloads in a desirable controlled manner after internalized by tumor cells and transported into the endosomes. With the deeply research of drug and gene co-delivery systems, more accurately controllable payloads release appeared as the most urgent problems. Take advantage of the acidic pH of tumor tissue (6.0±7.0) and endosomes/lyosomes (4.5-6.5) in a reasonable way, to design a dual pH-sensitive drug and gene co-delivery system programmable responds to acidic tumor tissue and endosomes/lyosomes could help to realize more controllable payloads release.In this article, the multi-function integrated assemble technology was used the construct dual pH-sensitive drug and gene co-delivery system, which was expected to help realize more controllable payloads release. The design and synthesis of functionalized materials was the basis of dual pH-sensitive co-delivery system construction. PEI as the most effective polycationic gene vectors was selected as core material to co-loading of drug and gene. The hydrazone bonds, which could be ruptured when exposed in acidic endosomal/lyosomal pH conditions, was used to conjugate DOX and PEI. Then the intracellular pH-sensitive DOX-PEI (DP) was synthesized. Take advantage of acidic tumor tissue pH-triggered charge-reversal O-carboxymethyl-chitosan (CMCS, which is negative charged when the pH above 7.0 and positive charged when the pH below 6.5) with biocompatibility, the pH-sensitive material CMCS-PEG-NGR (CPN) with NGR as the targeting ligand was synthesized. Based on DP and CPN, the dual pH-sensitive drug and gene co-delivery system was prepared. DP with positive charges condensed the anionic pDNA molecules to form DP/pDNA complexes (DPD), the drug and gene were co-loaded by DPD core in a simple way. DPD with positive surface charges absorbed CPN to form dual pH-sensitive CPN/DPD (CDPD). CDPD was expected to be stable in blood circulation after administration, which could be accumulated at tumor tissue as NGR-mediated transcytosis. Then, CPN reversed their charges from negative to positive triggered by acidic tumor tissue pH and dissociated from surface of CDPD at tumor tissue (the first pH-sensitivity), DPD with intracellular pH-sensitivity was exposed. The positive surface charges of DPD were conducive to cellular uptake, the internalized DPD was transported into endosomes. Then the DOX was rapidly released triggered by acidic enosomal environments (the second pH-sensitivity). As the "proton sponge" effect of PEI, the PEI/pDNA and DOX were released into the cytoplasm and exert effective antitumor effects. The pDNA expression of enhanced green fluorescent protein was selected as model gene to investigate the dual pH-sensitive drug and gene co-delivery CDPD sequentially dissociation of CPN and programmable release of DOX and gene. The possibility of CDPD as dual pH-sensitive drug and gene co-delivery system was investigated, which was ready for the further research in drug and gene co-delivery.To further investigate the significance of dual pH-sensitive drug and gene co-delivery system CDPD, and enhancement the intracellular payloads concentrations. TAT peptide as a kind of cell penetrating peptides could help the cargos acrossing the cell membrane, thus the cellular internalization assistant material PEI-PEG-TAT (PPT) was synthesized. The plasmid tumor necrosis factor related apoptosis inducing ligands (pTRAIL) was selected as therapeutic gene. The cellular internalization assistant material PPT was used to condense pTRAIL along with DP to construct dual pH-sensitive drug and gene co-deivery core PPT/DP/pTRAIL (PDT) with enhancement of cellular uptake. The enhancement of cellular uptake of PDT as PPT was investigated in our work and the in vitro antitumor effect of PDT in HepG2 and A549 cells were also evaluated. The Western Blot analysis was used to evaluate the expression of TRAIL protein. The above research paved way to further application of dual pH-sensitive co-delivery system CDPD.Above all, the synthesis of functionalized materials, the possibility of dual pH-sensitive drug and gene co-delivery system to co-deliver gene and DOX and the antitumor effect of DOX and pTRAIL of PDT were evaluated in this article, which were ready for the further application of dual pH-sensitive drug and gene co-delivery system. The main methods and results were below:1. Determination of DOX contentThe DOX concentration was determined by UV-Vis method and its methodology in pH 5.0 and pH 7.4 PBS buffer was examined. The results showed that it was an easy, convenient and correct method to determine the DOX concentration in vitro.2 Preparation and evaluation of dual pH-sensitive drug and gene co-delivery systemThe multi-function integrated assemble technology was used to construction of dual pH-sensitive drug and gene co-loaded system in this article. DP and CPN were synthesized and the structure was also characterized by 1H NMR. Based on the functionalized DP and CPN, the intracellular pH-sensitive DPD and dual pH-sensitive CDPD were assembled. The optimal mass ratio of DP/pDNA was 25:4 to prepare DPD. The optimal mass ratio of CPN/pDNA was 25:8 to prepare CDPD. CDPD and DPD exhibited spherical shapes, uniform particle size distribution (137.4±2.7 nm and 80.0±4.2 nm, respectively), positive zeta potential (8.25 mV and 23.59 mV, respectively). The in vitro stability results suggested that the DPD was more stable than CDPD in distilled water (mean sizes of DPD and CDPD increased of 33.35 nm and 83.02 nm within 7 days), and the CDPD was more stable than DPD in DMEM with 10% FBS (average particle sizes of DPD and CDPD increased of 226.48 nm and 36.6 nm within 24 h at 37℃ in DMEM with 10% FBS). Targeted cellular uptake of CDPD was confirmed (cellular uptakes of CDPD were 92.46±2.28% and 67.82±0.07% in CD13 positive A549 cells and CD 13 negative HepG2 cells, respectively. p<0.05). The in vitro transfection results of CDPD under different pH conditions suggested that the transfection efficiency of CDPD in HepG2 cells was enhanced with the pH of culture media reduced from 7.4 to 6.0, which indicated that the CDPD dissociated their outmost layer CPN at acidic tumor tissue. The exposed DPD could rapidly release of DOX and protect pDNA from degradation in simulated endosomal/lyosomal conditions (0.1 M PBS of pH 5.0). The prepared dual pH-sensitive CDPD could dissociate the outmost layer CPN and successful co-deliver DOX and pDNA into the same tumor cells.3 Preparation and evaluation of DOX and pTRAIL co-delivery system with dual pH-sensitivity and dual enhancementTo further confirm the significance of dual pH-sensitive CDPD, and enhancement the intracellular payloads concentrations. The cellular internalization assistant material PPT was synthesized, and the structure was characterized by 1H NMR. The pTRAIL was selected as the therapeutic gene. The prepared cellular internalization assistant and intracellular pH-sensitive nanocarrier PPT/DP/pTRAIL (PDT) was used to confirm the in vitro antitumor effect of pTRAIL and DOX. The agarose gel electrophoresis assay results confirmed that the PPT, DP and C6-SANH-PEI with the similar ability in condensing the pTRAIL molecules and condensed the pTRAIL at mass ratio of 25:16. Thus the PPT, DP and C6-SANH-PEI could not affect our subsequent research as the different ability in condensing the pTRAIL molecules. The mass ratio of PDT were optimized by the particle sizes, cellular uptake and in vitro transfection study, and the optimal mass ratios of (PPT30%+70%DP)/pTRAIL was 25:4. The prepared PDT exhibited spherical shapes, uniform particle size distribution (80.29±3.66 nm). Compared with the DOX and only pTRAIL loaded nanoparticle, the cellular internalization assistant and intracellular pH-sensitive nanocarrier PDT could enhance the in vitro antitumor effect in HepG2 and A549 cells. Meanwhile, the Western Blot assay also suggested that PDT could transfect HepG2 cells successfully and enhanced the expression of TRAIL protein. Thus, the prepared DOX and pTRAIL co-delivery system with dual pH-sensitivity and dual enhancement (CPDT) not only could respond to tumor intracellular/extracellular but also could enhancement the payloads accumulation at tumor tissue/intracellular. The CPDT released payloads in a more controlled manner and enhanced intracellular payloads concentrations, exerted efficient anti-tumor effects.Above all, the prepared dual pH-sensitive DOX and gene co-delivery system in this article could dissociate their outmost layer CPN and programmable release of DOX and gene in endosomal/lyosomal environments, which released the DOX and gene in a more controlled manner and effectively killed the tumor cells. To confirm the advantage of dual pH-sensitve drug and gene co-delivery systems and enhancement the intracellular payloads concentrations, the prepared DOX and pTRAIL co-delivery system with dual pH-sensitivity and dual enhancement(CPDT) not only could respond to tumor intracellular/extracellular but also could enhancement the payloads accumulation at tumor tissue/intracellular. The CPDT released payloads in a more controlled manner and enhanced intracellular payloads concentrations, which provided a new method for cancer therapy.