Dual Transporter Co-mediated Polymeric Liposomes For Efficient Oral Delivery Of Paclitaxel

Paclitaxel（PTX）,a paclitaxel-based antitumor drug,has a good chemotherapeutic effect on a variety of malignant tumors,especially invasive and metastatic breast cancer.Currently,the clinical administration of PTX is mainly intravenous injection.Although the effect of injection is rapid,the administration is inconvenient and easy to cause pain,leading to poor patients’compliance.Compared with injection administration,oral administration has higher safety,more convenient administration,and better patient compliance.Therefore,it is urgent to develop a PTX oral preparation.However,the poor water solubility and intestinal permeability of PTX limit its intestinal absorption.Therefore,the study of oral formulations of PTX remains a major challenge for our pharmaceutical researchers.The small intestine,which has a large specific surface area,is the main site of oral absorption of drugs,so promoting the uptake of PTX by small intestinal epithelial cells is an important way to increase its oral absorption.Studies have shown that the apical membrane of small intestinal epithelial cells contains a variety of transporter proteins,such as glucose transporters（GLUTs）,monocarboxylate transporters（MCTs）,and amino acid transporters,etc.Modifying nanoparticles with transporter ligands is an effective strategy to promote intestinal absorption and improve the bioavailability of oral drugs.However,relevant studies have shown that the use of single transporter mediated drug transport is prone to saturation,which is therefore not conducive to long-term drug delivery.In the previous study,the research group found that increasing the density of the modified transporter ligands on nanoparticles after reaching a certain density would not enhance the effect of transport.This further indicates that the use of single transporter to mediate oral drug absorption is prone to saturation,and this has prompted us to further investigate the use of dual transporter to co-mediate oral drug absorption.Based on the results of the group’s previous research,in this study,both MCTs and GLUT transporters were used to promote the small intestinal absorption of PTX to efficiently improve the oral bioavailability of PTX.Among them,MCTs mediated the transmembrane uptake and transport of monocarboxylic acids such as acetic acid,propionic acid,and butyric acid,and GLUT mediated the transmembrane uptake and transport of small molecules such as glucose,fructose,and galactose.In order to increase the adequate binding of the nano-drug delivery system to the small intestinal transporter,modified chitosan derivatives were used in the modification of the nano-formulation to enhance its adhesion to the intestine and further promote the absorption effect of the small intestine.In this work,succinic anhydride（SAA）and D-fructose（D-Fru）were grafted onto chitosan to produce p H-responsive chitosan derivatives（NSCF）,and then the liposomes were modified with NSCF to make an oral nanoformulation of PTX（NSCF-PTX-PLip）.The prepared nano-system can increase the uptake of PTX liposomes by intestinal cells via strong affinity effects with MCTs and GLUT and enhance the oral bioavailability and antitumor effects of PTX.In this work,N-succinyl chitosan（NSC）was synthesized by grafting succinic anhydride onto the amino group of chitosan through the acylation reaction,and D-fructose was grafted onto the synthesized NSC through the Maillard reaction to obtain D-fructose-grafted N-succinyl chitosan（NSCF）.The generation of new functional groups was characterized by infrared and ultraviolet spectroscopy,and the effects of succinylation and the Maillard reaction on the crystalline structure of CS were investigated by X-ray diffraction.The NSCF-modified PTX liposomes（NSCF-PTX-PLip）were prepared by thin film dispersion method,and the optimal formulation was screened by single-factor and orthogonal experiments.The results of formulation characterization showed that NSCF-PTX-PLip had smaller particle size,smaller PDI,improved drug loading,and better stability.The results of in vitro release showed that the modified liposomes of NSCF showed significant p H sensitivity and slow release in simulated gastrointestinal fluid.The results of pharmacokinetic experiments in rats showed that after oral administration,the peak time of the 30.78%NSCF-PTX-PLip group was delayed to 3h,the C_max was increased 1.59-fold and the elimination was slowed down compared with the PTX-Lip group,and the blood concentration was still above 200 ng/m L after24 h of administration.In addition,the absolute oral bioavailability of the 30.78%NSCF-PTX-PLip group was 6.07-fold and 0.59-fold higher than that of the PTX-Lip group and 30.78%NSC-PTX-PLip group,respectively.These results suggest that the modification of NSCF increased the blood concentration of PTX liposomes,prolonged the retention time in vivo,and improved its oral bioavailability.Cytocompatibility assays showed that NSC and NSCF are safe and can be used as drug delivery materials.Both qualitative and quantitative cellular uptake results showed that modification of NSC and NSCF increased the uptake of PTX liposomes by Caco-2 and 4T1 cells compared to unmodified liposomes.Among them,the effect of NSCF-modified liposomes was more pronounced,suggesting a stronger cellular uptake mediated by the MCTs and GLUT dual transporters together.The results of competitive inhibition assay in Caco-2 and 4T1 cells also confirmed that both dual transporters were involved in the cellular uptake of liposomes.In addition,30.78%NSCF-PTX-PLip group showed significantly enhanced inhibition of 4T1 cells with an IC₅₀ value of 1.18μg/m L,which was significantly lower than that of the PTX-Lip group（4.58μg/m L）and PTX Solution group（14.44μg/m L）.In vivo imaging of mice and ex vivo imaging of tissues and organs showed that the modification of NSC and NSCF prolonged the intestinal retention time of PTX-Lip,enhanced its intestinal absorption,and increased the accumulation of drugs in the tumor site.Moreover,the effect of NSCF was stronger,confirming the advantage of dual transporter transport.The tumor bearing Balb/C mouse model was constructed by4T1 cell line,and the antitumor ability of different PTX preparations in vivo was studied.The results showed that the modification of NSCF enhanced the antitumor effect of PTX-Lip without significant toxicity.The tumor growth inhibition rates were83.28%,70.76%and 41.52%in the 30.78%NSCF-PTX-PLip group,30.78%NSC-PTX-PLip group and PTX-Lip group,respectively.The above experimental results confirm that dual transporter-mediated transport can lead to better anti-tumor effects of oral PTX.