| Anticancer drug can be used in chemotherapy process for patients withcancer. The chemotherapy does not achieve the desired results for lack of specifictoxicity, tumor selectivity and multidrug resistance(MDR). In order to achieve themaximum efficacy, the delivery system of anticancer drug have been used intumor targeted therapy with the promising prospect.In decades, scientists have been achieved many breakthroughs in drugdelivery systems, such as liposomes, microspheres, nanoparticles and micellesetc.. These techniques can improve the efficacy of anti-cancer drug, entrapmentefficacy with chemical or physical methods, solubility and prolong the half-live.Meanwhile, we can modify the antibodies or ligands on the surface ofcarriers to achieve the active target. These carriers with the characters of higherspecificity, selectivity, affinity and efficacy. So, active targeting drug deliverysystem is very popular both at home and abroad.Although many drug delivery systems showed a very good tumor targetingthrough antigen-antibody, receptor-ligand compounds, the capacity of endocytosis is weak after the carrier bonding to target cells. The cell-toxicitydrugs can not reach the enough concentration to inhibit the tumor cell especiallythe drug resistant tumor cell.The carrier outside the cancer cells will release the drug and redistribute thedrug to normal tissues. Now bioactive molecular transport to intracellular is a keyproblem in drug delivery.So we chose poly (β-malic acid) as the substrate for its biocompatibility andbiodegradation. Cell-penetrating peptides(CPP) which can carry macromoleculesinto cells with high membrane affinity, fast penetrating speed and rapidlydegraded.PEG with different molecular weight and anticancer drug can link to poly(β-malic acid), respectively. PEG with different length can carry ligand, CPP,biotin, monoclonal antibodies or targeting molecules, respectively. And this kindof copolymer can active-targeted accumulate and be intracellular in acidicenvironment of the tumor.In normal systemic circulation (PH>7.0), CPP linked to the short-chain ofPEG hides in protection of long-chain PEG; in acidic environment of the tumortissues, acid sensitivity site linked to the long-chain PEG cleavage to lose theprotective layer, then CPP exposes on the surface of carrier, and internalization oftumor cells will be achieved at last.A large number of literature report that targeting molecules can bond tocarrier through amide bond or ester bond, this kind of carriers have a goodstablitivity in physical pH and weak acidic environment of tumor. In order tomake the idea become reality, we chose the hydrazone bond as acid sensitivitysite because it can cleavage easier in weak acidic environment. Targetingmolecular linked to α-OH-ω-CHO-PEG can bond to PMLA through hydrazonebond to achieve the cleavage of long-chain PEG from PMLA in the weak acidic environment of tumor.In this article, PMLA as the main chain is modified by hydrazine hydrate toform hydrazide keys and bonded to CHO-PEG-FA with hydrazone bond. Wediscussed the cleavage character of hydrazone bond in acidic environment. Theresults revealed that we can improve the progress of high-efficiency intracellularof targeted anti-cancer drug carrier.1Synthesis of poly (β-malic acid)L-aspartic acid was chosen to synthesize β-alkaneoxycarbonyl-β-propiolactone, which was chosen as the monomer to carry out the ring-openingpolymerization with initiator to synthesize poly (β-benzyl-malate). And poly (β-malic acid) was synthesized by hydrogenolysis of poly (β-benzyl-malate),palladium-carbon as catalyst, in hydrogen at room temperature. IR spectra andNMR spectra characterize the structure of the product.2Synthesis of CHO-PEG-FAA certain amount of folic acid and α-OH-ω-CHO-PEG were reacting inanhydrous DMSO in the presence of EDC HCl and DMAP. Then the productwas dialyzed with distilled water for2days and saved by freeze-drying.3Synthesis of hydrazide-decorated poly (β-malic acid)(β-PMLA-NH2)β-PMLA was reacted with NHS in the DMF for a moment. AddingEDC HCl the reaction was progressing in ice bath. Then5%hydrazinehydrate solution was added dropwise within one hour after the reaction at roomtemperature. The solution was dialyzed with distilled water for two days and theproduct was saved by freeze-drying. 4Preparation of β-PMLA-PEG-FAA certain amount of CHO-PEG-FA andβ-PMLA-NH2were added into DMFwith the catalyzation of EDC HCl. The product was dialyzed with distilledwater for2days and saved by freeze-drying. The nano-copolymers wasconfirmed by1H-NMR analysis. The folic acid conjugation efficiency wasdetermined by using ultraviolet spectrophotometry.5In vitro the release of FA from the nano-copolymerDynamic dialysis method was simulated in vitro hydrazone bond cleavage tostudy the stability of folic acid in different pH buffer. The release concentrationof folic acid was determined by UV spectrophotometer at wavelength of280nm.The lower the pH value was, the faster folic acid released. After6h of incubationat pH5.5, pH6.5and pH7.4the released free FA was about88.1%,85.3%and41.6%.6Cytotoxicity evaluation of β-PMLA-PEG-FA nano-copolymerThe vitro cytotoxicity of the β-PMLA-PEG-FA nano-copolymer wasassessed by using human hepatocarcinoma SMMC-7721cells as in vitro cellmodel. The results showed that the nano-copolymer does not have significantcytotoxicity. |
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