Glycyrrhizic Acid Modified Carboxymethyl Chitosan Nanoparticles To Improve The Targeting And Efficacy Of Antineoplastic Agents On Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) has turned into a major global health problem. For the management of patients with HCC, systemic chemotherapy plays a palliative role while yields unsatisfactory response rates, which is partly due to the poor selectivity and low uptake efficiency of chemotherapeutic drugs in tumor. Targeted drug delivery syetems (TDDS) could selectively deliver drug to the liver lesions through the carriers and reduce toxicity to normal tissues, reduce the dose and frequency of administration, and improve anti-tumor effect. Polymeric nanoparticles as drug carriers are employed to improve the solubility and stability of the drugs, alter biodistribution, extend the time of drug action, and control drug release, which offers the potential in a successful HCC therapy. Through the introduction of functional ligands on their surfaces, the targeting of nanoparticles can be further improved.In the present investigation, the O-carboxymethyl chitosan nanoparticles modified by GL (CMCNP-GL) with various GL substitution degrees were synthesized to evaluate their potential for HCC targeting. Their physicochemical characteristics were investigated. The comparison of their in vitro cellular uptake was performed on SMMC-7721(human liver cancer cells) and L02(human normal liver cells). Paclitaxel (PTX) as a model antitumor drug was incorporated into the CMCNP-GL. The in vitro drug release, in vivo biodistribution as well as in vitro and in vivo antitumor capability of PTX loaded CMCNP-GL (PTX/CMCNP-GL) with different GL substitution degrees were evaluated as compared to the unmodified PTX/O-carboxymethyl chitosan NPs (CMCNP) and PTX injection. In addition, toxicological examination of CMCNP-GL as liver targeting drug carrier after intravenous administration was also carried out.1Preparation and characterization of CMCNP-GLO-CMC-MMA copolymers were systhesized through the graft copolymerizion of hydrophobic methyl methacrylate (MMA) and hydrophilic O-carboxymethyl chitosan (O-CMC) under the initiation of ammonium persulfate, which spontaneously self-assembled into core-shell CMCNP in aqueous solution. The adjacent hydroxyls of GL were turned into aldehyde groups via periodate oxidation. The oxidized GL was reacted with residual amino groups on CMCNP to obtain CMCNP-GL. CMCNP-GL with various feed mass ratio of GL to O-CMC (1:1,2:1, and4:1) were denoted as CMCNP-GL1, CMCNP-GL2, and CMCNP-GL4, respectively. FTIR,1H NMR, and DSC confirmed the chemical composition of CMCNP-GL. Morphology of the nanoparticles was observed using TEM. The degree of substitution (DS), defined as the number of GL molecules per100sugar residues of CMC was determined by1H NMR and UV method. Contact angle, particle size, Zeta potential, and the stability of CMCNP-GL under different conditions were also characterized. According to the1H NMR results, the DS of GL in CMCNP-GL1, CMCNP-GL2, and CMCNP-GL4was2.4%,6.9%, and9.6%, respectively. CMCNP-GL appeared monodisperse and spherical in shape, demonstrated a narrow size distribution (P.I.<0.15) with a size range of100-250nm, and their Zeta potentials were around-30mV. The particle size of CMCNP-GL2increased compared with CMCNP-GL1, and decreased in CMCNP-GL4as compared to CMCNP-GL2. However, no significant differences in their Zeta potentials. The surface hydrophilicity gradually increased with the DS of GL increased. CMCNP-GL kept good lyophilization and storage stability, and their physicochemical properties were stable at different pH, ionic strength and physiological conditions.2Preparation and characterization of PTX-loaded CMCNP-GLPTX was incorporated into the inner core of CMCNP-GL through sonication. Average particle size of PTX/CMCNP-GLwith various DS of GL was110-205nm, and Zeta potential was around-30mV. Entrapment of PTX scarcely changed the size and surface charge of the corresponding blank CMCNP-GL. The encapsulation efficiency (EE) and drug loading (DL) of CMCNP-GL were increased with the increase of GL substitution degree. EE of CMCNP-GL for PTX was all above69.0%which reached maximal value of81.5%; DL of CMCNP-GL for PTX was all above13.0%with a maximum of15.1%. For all PTX/CMCNP-GL, release of PTX revealed a biphasic pattern:an initial burst and a following slower and continued release. GL modification could enhance the slow-release effect of CMCNP, and PTX release rate slowed down with the increase of GL substitution degree. There was no difference among the release profiles of PTX/CMCNP-GL1, PTX/CMCNP-GL2and PTX/CMCNP-GL4. 3In vitro and in vivo HCC-targeting of CMCNP-GLRhodamine B labeled nanopartiles RhB-CMCNP and RhB-CMCNP-GL were prepared for assessment. Cell adsorption, cellular uptake, uptake mechanism and subcellular localization of CMCNP-GL were studied in SMMC-7721and L02cells. The cell adsorption and uptake rate of CMCNP-GL were significant higher than CMCNP in the two cells, the adsorption and uptake rate of CMCNP-GL with various GL substitution degree in SMMC-7721cells were significant higher than them in L02cells, while there was no significant difference among CMCNP-GL1, CMCNP-GL2, and CMCNP-GL4. The uptake of CMCNP-GL by SMMC-7721cells was markedly inhibited in response to the concentration of free GL. On the contrary, the inhibitory effect on CMCNP-GL uptake was much weaker in L02cells. Under the same situation, the uptake of CMCNP was not affected after the addition of free GL in both cell types. CMCNP-GL2could carry drug into the tumor cells chiefly through energy-dependent caveolin-and clathrin-mediated endocytosis which were relevant of cytoskeleton reorganization. Realistically, GL modification did not affect the uptake mechanism of CMCNP. CMCNP-GL2were mainly accumulated in the perinulear region after internalization.Biodistribution of PTX/CMCNP-GL and the blank nanoparticles were investigated in H-22tumor bearing mice to evaluate their targeting efficiency. The results revealed that in vivo distribution of PTX could be altered significantly through CMCNP-GL2entrapment, the clearance from the blood circulation were slowed down. Comparatively, PTX/CMCNP-GL2distributed quickly and extensively in the liver and tumor, which could reduce cardiac and renal toxicity of PTX. The HCC-targeting efficiency of PTX/CMCNP-GL2was significantly higher than that of unmodified PTX/CMCNP and PTX injection. In addition, the biodistribution results of RhB-CMCNP and RhB-CMCNP-GL2without drug entrapment accorded well with their corresponding PTX loaded nanoparticles.4Anti-tumor effect and safety assessment of CMCNP-GLThe in vitro and in vivo anti-tumor effects of PTX/CMCNP-GL were evaluated in SMMC-7721cells and H-22tumor bearing mice, respectively. The influence of different DS of GL was also compared. Results showed that PTX/CMCNP-GL significantly inhibited the in vitro proliferation of liver cancer cells SMMC-7721. Within72h, the IC50of PTX/CMCNP-GL, PTX/CMCNP, and PTX injection was 2.7-3.2,8.1, and13.5mg·mL-1, respectively. There was no significant difference among PTX/CMCNP-GL with various DS of GL. PTX could induce apoptosis of SMMC-7721cells with the apoptosis ratio10-20%during24h, while CMCNP carrier and GL modification had no significant influence in the mechanism and effect of PTX. Intravenous administration of PTX/CMCNP-GL could effectively inhibit H-22tumor growth and extend the tumor doubling time. PTX/CMCNP-GL exhibited strongest tumor regression with the average tumor inhibition ratio of87.5%, promoted2.5and1.6folds compared to PTX injection and PTX/CMCNP. CMCNP-GL with different substitution degrees possessed similar targeting property and therapeutic efficacy.Furthermore, safety assessment of CMCNP-GL was explored at cellular, tissue, and animal levels in terms of cytotoxicity, blood compatibility, intravenous acute and sub-acute toxicity. Results of FDA/PI double staining assay, LDH assay, neutral red assay, and protein assay in L02and SMMC-7721cells revealed lack of cytotoxicity of blank CMCNP-GL (5mg-mL"1) with different DS of GL following short-time exposure (6or24h), and MTT assay could further evidence that CMCNP-GL did not interfere with cell proliferation following longer-time treatment (72h). Hemolysis ratio of CMCNP-GL2was lower than5%, prolonged plasma recalcification time of CMCNP-GL2was detected, and the dynamic blood clotting ratio was lower than silicated glass, suggesting its desired anticoagulant capacities. The maximum tolerance amount of intravenous CMCNP-GL2in healthy mice was1800mg·kg-1. In15-day sub-acute toxicity study (1800mg·kg-1), body weight of CMCNP-GL2treated mice gradually increased with no appreciable difference to control animals, weights of mouse hearts, livers, spleens, lungs, kidneys, and brains were found to be normal, and in the histological examination no inflammation, necrosis, edema or other pathological signs were detected. With regard to the hematological parameters, no statistically significant difference was observed against control. It did not show any significant difference in ALT/AST/ALP levels when compared with saline-treated group, indicating no damage on hepatic cells occupied. The toxicity results demonstrated that blank CMCNP-GL2did not elicit any sort of hepatic or systemic toxicity. These studies collectively apprehended the safety and suitability of CMCNP-GL for use as drug delivery vehicle for HCC.