Preparation And Application Of Targeted R₉-PEC-NP Drug Carriers With Cell-penetrating Effect

Drug delivery system（DDS）is a method of delivering appropriate amounts of chemotherapeutic drugs to tumor tissues via drug carriers,and the delivery efficiency can be improved by targeting modifications to drug carriers.However,there are large differences in receptor expression on the surface of different tumor cells,and certain receptors that are overexpressed are also expressed in normal cells,which limit the application of targeted drug carriers.Therefore,the p H-sensitive controlled-release drug delivery system,which was not dependent on the expression of receptors on the surface of tumor cells,was constructed with the unique micro-acidic environment of tumor tissue as a specific recognition target.It can be applied to a variety of tumor cells to achieve targeted delivery of drugs and reduce damage to normal cells.Furthermore,the cell-penetrating ability of existing drug carriers needs to be enhanced.In this paper,a safe,non-toxic,biocompatible and degradable pectin（PEC）and calcium carbonate（Ca CO₃）were selected as raw materials,and poly-arginine-9（R₉）with transmembrane transport ability was modified on their surfaces by covalent coupling.Thus,a drug carrier with cell-penetrating function and p H-responsive performance was constructed for the targeted delivery of doxorubicin（DOX）.The main content and experimental results of this topic are as follows:（1）Preparation and characterization of R₉-modified PEC nanosphere（R₉-PEC-NP）.Firstly,the pectin derivatives coupled with Poly-arginine-9（R₉-PEC）were obtained by esterification reaction using PEC and R₉ as the substrate materials.The substitution degree of R₉ was determined by elemental analyzer（EA）and functional groups were analyzed by Fourier infrared spectroscopy（FT-IR）.The results showed that R₉ and PEC were successfully linked and the substitution degree of R₉ in the synthesized product was 7.01%.Then,Ca CO₃ self-assembles in R₉-PEC solution to form a pectin nanoparticle（R₉-PEC-NP）with a p H-responsive character.The size,potential and morphology of R₉-PEC-NP were characterized by scanning electron microscope（SEM）,zeta potential and particle size analyzer.The polysaccharide organic content and crystalline phase composition in R₉-PEC-NP were analyzed by thermogravimetric analyzer（TGA）and X-ray diffractometer（XRD）.The results indicated that R₉-PEC-NP was showed regular spherical shape with an average particle size of 215 nm.It presents a negative state under the condition of p H>5,which was conducive to binding with positively charged DOX through electrostatic interactions.R₉-PEC-NP was consists of calcite,sphalerite and polysaccharide organic matter,and the organic matter content is 38.75%.（2）In vitro drug release study of R₉-modified PEC nanosphere（R₉-PEC-NP）.The loading and release properties of the nanosphere were investigated by Fluorescent Inverted microscope and ultraviolet-visible spectroscopy（UV-Vis）using DOX as a model drug.The results indicated that R₉-PEC-NP could effectively load DOX with an entrapment efficiency of（88.70±3.56）wt%and a drug loading of（8.15±0.12）wt%.The release of DOX by R₉-PEC-NP was about80%after 100 h in a simulated tumor tissue environment（p H 5.0）,which was about 2 times that of normal tissue（p H 7.4）.These suggest that such the nanospheres had a capability of effective p H-sensitivity controlled drug release in vitro drug release,offering great potential for smart drug delivery systems.（3）In vitro therapeutic evaluation of R₉-modified PEC nanosphere（R₉-PEC-NP）.DOX@R₉-PEC-NP and DOX@PEC-NP uptake by 4T1 cells under different p H conditions was analyzed by flow cytometry（FCM）and confocal laser scanning microscopy（CLSM）.The killing of 4T1 cells by DOX@R₉-PEC-NP and DOX@PEC-NP under different p H conditions was analyzed using the MTT method.The results showed that the inhibitory effect of both groups on tumor cells was dose-dependent in the range of DOX concentration from 2 to 8μg/m L,but the DOX@R₉-PEC-NP showed significantly higher cytostatic rate compared to DOX@PEC-NP.It was revealed by qualitative and quantitative observations that Cellular uptake of DOX@R₉-PEC-NP was significantly increased compared to the DOX@PEC-NP.The fluorescence intensity of DOX@R₉-PEC-NP was 2 times higher than that of group DOX@PEC-NP in 4T1 cells.The above results indicate that DOX@R₉-PEC-NP can effectively increase the uptake of drugs by cells after the introduction of the cell-penetrating peptide R₉.