Linoleic Acid And Poly Apple Acid Grafted Chitosan New Nano-carrier Materials Enhance The Anticancer Drug Efficacy

In the case of cancer therapy, chemotherapy is often impotent in effective treatment due to unfavorable specificity towards tumors and great side effects. Therefore, development of novel drug delivery systems (DDS) which are characterized by improved anti-tumor effect, reduced side effects, decreased cost during R&D of new drugs, and favorable safety has around great attention. Among them nanoparticles delivery systems show distinct beneficial attributes, including controlled and sustained drug release and targeted drug delivery. It is a prerequisite that the polymeric nanoparticles subjected to clinical investigations and marketing should possess desired biocompatibility and safety. Chitosan shows wide applications due to its favorable biocompatibility and biodegradability, and is an approved excipient by FDA. The current investigation aims at developing a novel kind of amphiphilic chitosan derivative nanoparticles, which are further modified with PEG, folic acid (FA) and biotin (BT), thus achieving an effective, safe, and actively targeted delivery carrier for anti-tumor drugs.Linoleic acid (LA) and poly(malic acid) double-grafted chitosan (LMC) was prepared through acrylation reaction. Functionally modified LMC derivatives including PEG modified LMC (PEG-LMC), FA modified PEG-LMC (FA-PEG-LMC), and BT modified PEG-LMC (BT-PEG-LMC) were synthesized through Schiff base reaction and EDC·HCl mediated covalent bonding. The amphiphilic LMC and LMC derivatives self-assembled into nanoparticles in water. PTX as a model insoluble anticancer drug was loaded in LMC and LMC derivatives nanoparticles, which were characterized by drug loading efficiency, loading capacity, and in vitro release. In vitro cellular uptake of LMC and LMC derivatives nanoparticles were evaluated in SMMC-7721 cells and HEK-293 cells, and the tumor inhibition effect, biodistribution, and targeted effect were monitored in H22 bearing mice.1 Preparation and characterization of LMC and LMC derivativesPMLABz was synthesized through ring-open polymerization of lactone with D,L-aspartic acid as monomer and lactic acid as initiator. LA and PMLABz were activated using the acrylation method in MeSO₃H, and were conjugated with hydroxyl and amino groups on chitosan through ester and amide bonding, respectively, thus achieving the LMC. Effect of feed ratio and feed sequence of LA/poly(malic acid), reaction temperature, and reaction time on the graft reaction was assessed. The optimal molar ratio of LA/saccharide ring was set to be 0.2-1.0, molar ratio of malic acid/saccharide ring to be 1.0, reaction time to be 6 h at room temperature, and MeSO₃H was used for hydrolysis of benzyl groups.mPEG aldehyde with an activation degree of above 80% was synthesized using the DMSO-acetic anhydride method, and the effect of acetic anhydride/mPEG molar ratio, reaction temperature and reaction time on the activation degree was evaluated. Optimally, mPEG aldehyde was allowed to conjugate with LMC in a mixed solution of DMSO and pH 8.0 borate suffer, which was deoxidized with NaBH₄ and purified through centrifugation.Folic acid and biotin were separately conjugated to PEG-LMC via amide bond mediated by EDC·HCl, achieving the FA-PEG-LMC and BT-PEG-LMC.FTIR, ¹H NMR, XRD, DSC, and elemental analysis confirmed synthesis of LMC, PEG-LMC, FA-PEG-LMC, and BT-PEG-LMC. Elemental analysis demonstrated that the degree of substitution (DS) of LA was 36.1-60.5, DS of PMLA was 0.7-1.1, DS of PEG was 12.4, DS of FA was 6.0, and DS of BT was 6.1.2 Preparation, characterization of blank and PTX-loaded LMC and LMC derivatives nanoparticlesLMC and LMC derivatives self-assembled nanoparticles with different LA and PMLA substitution degrees were prepared by sonication. Morphology of the nanoparticles was observed using TEM and SEM, and particle size and Zeta potential were monitored using DLLS. CAC was determined using fluorescence spectroscopy with pyrene as a hydrophobic probe. PTX as a model anticancer drug was loaded in the nanoparticles, and the effect of nanoparticle composition and modification of PEG, FA, and BT on encapsulation efficiency, loading capacity and in vitro release behavior was investigated.Average particle size of LMC and its derivatives nanoparticles at pH 7.4 was 190-350 nm, and Zeta potential was -2～-20 mV. An increase in DS of LA or decrease in PMLA chain length led to decreased particle size of LMC, and particle size in alkaline conditions was larger than in acidic conditions. Zeta potential of the LMC nanoparticles was sensitive towards pH values in that they were positively charged at pH lower than 6.0, nearly uncharged within the pH range of 6.0-7.0, and negatively charged at pH higher than 7.0. The modification of PEG led to an increase in particle size and a decrease in the absolute value of Zeta potential. The modification of FA or BT had no significant effect on the particle size and Zeta potential of PEG-LMC NP. The CAC of LMC was deceased as the DS of LA enhanced and the modification of PEG, FA, and BT didn't increase CAC. Loading efficiency of LMC and its derivatives nanoparticles for PTX was above 70%, and the loading capacity increased with LA substitution degree which reached maximal value of 9.9%. The modification of PEG slightly decreased the loading capacity of LMC. A sustained release of PTX was achieved within 24 h, and an increase in LA substitution degree and PMLA chain length could slow down the release rate of PTX. The modification of PEG could enhance the slow-release effect of LMC.3 Anti-tumor effect, tumor-targeting and safety assessment of LMC and LMC derivatives nanoparticlesAnti-tumor effect of PTX loaded LMC and LMC derivatives nanoparticles were investigated in H22 bearing mice. Both unmodified and modified PTX loaded LMC nanoparticles showed significant anti-tumor effect, while PEG modification and ligand modification resulted in improved anti-tumor effect. The tumor inhibition rate (TIR) was: FA-PEG-LMC>BT-PEG-LMC>PEG-LMC>LMC>PTX solution, with TIR of 82.5% and 80.6% for FA-PEG-LMC and BT-PEG-LMC, respectively.Cellular uptake study of LMC and LMC derivatives nanoparticles were performed in SMMC-7721 cells and HEK-293 cells to evaluate the in vitro tumor targeting effect. Rhodamine B labeled LMC (RB-LMC), PEG-LMC (RB-PEG-LMC), FA-LMC (RB-FA-LMC), FA-PEG-LMC (RB-FA-PEG-LMC), BT-LMC (RB-BT-LMC), and BT-PEG-LMC (RB-BT-PEG-LMC) nanoparticles were used for assessment. The uptake of LMC and its derivatives nanoparticles in SMMC-7721 cells was much higher than that in HEK-293 cells. Cellular uptake of SMMC-7721 increased with nanoparticle concentration within 1000μg·mL^-1, and cellular uptake of HEK-293 increased with nanoparticle concentration within 500μg·mL^-1. Cellular uptake of SMMC-7721 and HEK-293 increased with incubation time in within 4 h. The uptake amount of FA-LMC and BT-LMC nanoparticles was 3-5 fold and 2-3 fold of that of LMC nanoparticls and the uptake amount of FA and BT modified LMC nanoparticles in HEK-293 was significantly lower than that of SMMC-7721. It meant that FA and BT modified LMC nanoparticles could actively target to tumor cells. The modification of PEG had no obviously effect on the uptake of LMC nanoparticles, but the uptake amount of FA-PEG-LMC and BT-PEG-LMC was lower than that of FA-LMC and BT-LMC. Free folic acid and biotin inhibited uptake of FA and BT modified LMC nanoparticles, respectively. The inhibition on FA-PEG-LMC and BT-PEG-LMC was stronger than that on FA-LMC and BT-LMC, which meant that FA-LMC and BT-LMC had higher affinity with FA and BT receptors on cell surface.Biodistribution of PTX loaded LMC, PEG-LMC, FA-PEG-LMC, and BT-PEG-LMC nanoparticles were investigated in normal and H22 bearing mice. HPLC was used for PTX quantification, and the method was precise and reliable. PEG modification prolonged retention of LMC nanoparticles in the blood circulation and decreased phagocytosis by macrophages in the liver. However, phagocytotis by the spleen was not prevented. Both unmodified and modified LMC nanoparticles showed tumor-targeting effect, while FA and BT modification yielded an additional active targeting towards tumor with FA-PEG-LMC showing the maximal tumor-target effect of R_e of 3.8. Besides, distribution of LMC and LMC derivatives nanoparticles in the heart and spleen was remarkably lower than PTX injection, indicating that toxicity of PTX towards heart and spleen was successfully reduced following encapsulation in LMC and LMC derivatives nanoparticles.Finally, safety assessment was carried out on LMC nanoparticles in terms of hemolysis and acute toxicity. Hemolysis ratio of LMC was below 5%, and the maximum tolerance amount of LMC in mice was 1250 mg·kg^-1, which suggested it a safety drug carrier for intravenous injection.