Objective: To increase targeting effect and reverse the multiple drug resistance(MDR) on cancer cells by building and investigating polymeric nanoparticles that Low density lipoprotein(LDL) was used as a targeting molecule as well as a vehicle of siRNA to coupled with N-succinyl chitosan nanoparticles loaded with doxorubicin(Dox).Methods: LDL was isolated from human plasma by density gradient ultracentrifugation.The properties and tumor-targeting characters were investigated.The MDR1 chol-siRNA/LDL complex was optimized by gel retarding assay.N-succinyl chitosan was synthesized and used to prepare N-succinyl chitosan nanoparticles loaded with Dox, then coupled with chol-siRNA/LDL to give polymeric nanoparticles.In vitro drug release and anti-tumor activity of Dox and its nanoparticles were investigated.The silencing effect of chol-siRNA/LDL coupled N-succinyl chitosan nanoparticles loaded with doxorubicin (Dox-siRNA/LDL-SCS-NPs) was studied by RT-PCR.Results: The mean particle size and polydispersity index (PDI) of LDL were (23.4±5.8) nm and 0.298, respectively (Fig.l).The zeta potential of LDL was (-1.65±0.04) mV.Protein determined by the Bradford method was (0.808±0.12) mg·mL-1.Lipoprotein purity was examined by 4% polyacrylamide gel electrophoresis(PAGE).Cellular targeting of LDL was investigated by HepG2 and A549 cell lines, which overexpresses and low expresses LDL receptors, respectively.Competitive receptors inhibition test was studied in the presence of excess LDL.As shown in Fig.2a, the uptake of DiI labeled LDL in HepG2 cell line at 3h after treatment was obviously higher than that in A549 cell line, proving LDLr-mediated endocytosis.The uptake of DiI-LDL was decreased in the presence of 20 fold LDL(Fig.2b), which demonstrated DiI-LDL and LDL were uptaken by LDLr route.Fig.3 Gel retarding assay of chol-siRNA/LDL complex.Electrophoretic mobility was examined using a 12% polyacrylamide gel after 0.1 nmol of each siRNA was incubated with LDL fraction in different ratios.The lipophilic MDR1 siRNA was obtained by modifying with cholesterol.As shown in Fig.3, when the ratio of chol-siRNA and LDL was under 30∶1, siRNA was combined firmly with LDL.A few siRNA were free to come out when the ratio increased, which meant about 30 siRNA molecules could be firmly loaded into one LDL molecule.The FT-IR spectra of chitosan(CS) and N-succinyl chitosan(Suc-CS) are shown in Fig.4A.The bending vibrations of-NH2 and amide Ⅲ are showed at 1599 cm-1 and 1379 cm-1, respectively(Fig.4A.a).There are strong absorption peaks at 1657 cm-1(stretching vibration of-C=O, namely amide Ⅰ), 1557 cm-1(in-plane bending vibration of-N-H-, namely amide Ⅱ) and 1403 cm-1(symmetric vibration of carboxylic group), suggesting the formation of NH-CO and-COOH(Fig.4A.b).Furtherly, 2916 cm-1 (-CH2) demonstrates the succinylation of-NH2.The chemical structure of CS and Suc-CS is shown in Fig.4B.Suc-CS∶δ=3.43~3.77(H1, H3, H4, H5, H6), δ=2.30(-CH2-).Compared to the spectrum of CS (δ=2.95, H2), H2 signal shifts to δ=3.4, presumably the electron cloud density of H2 is changed by succinylation of-NH2.As shown in Fig.5, Dox-siRNA/LDL-SCS-NPs are uniform and round with a size of 200nm.The surface of nanoparticles are surrounded by siRNA/LDL.The FT-IR spectra and PAGE were utilized to demonstrate siRNA/LDL complex was covalently attached to Dox-SCS-NPs (pictures not shown).The in vitro Dox release data are shown in Fig.6.The amount of drug release from Dox-LDL-SCS-NPs was higher at pH5.0 than pH7.4, for nanoparticles were unstable under acidic solution.Dox release from nanoparticles was lower than crude drug with 50% released amount at 4h under pH7.4, while crude drug was released by 70%.And then the drug release from nanoparticles turned to slow period.Cytotoxicity was determined with an MTT assay on HepG2 cells (human hepatocarcinoma cell) and L-02 cells (human normal hepatocyte).As shown in Fig.7, different formulations inhibited the cells growth in a dose-dependent manner.To HepG2 cells, Dox-LDL-SCS-NPs displayed the highest level of inhibition, followed by Dox-SCS-NPs and Dox.Dox-LDL-SCS-NPs showed far lower cytotoxicity against L-02 cells than that against HepG2 cells.Meanwhile, the cytotoxicity of blank nanoparticles against L-02 cells was significantly lower than that against HepG2 cells, demonstrating the targeting effect and safety of the vehicle.Fig.8 shows the silencing effect of MDR1 siRNA in drug-resistant HepG2 cells.As compared with mock transcription, the siRNA duplex reduced the levels of MDR1 mRNA at 24h after treatment, and the siRNA did not affect GAPDH RNA.Conclusion: This study demonstrates the effectiveness of chol-siRNA/LDL complex coupled N-succinyl chitosan nanoparticles loaded with doxorubicin in tumor-targeting and MDR reversing.And the new preparation form and approach may hold promise for gene and drug co-delivery system.