| Malignancy is the global public health issue threatening human health seriously.Among these cancers,the incidence of leukemia ranks sixth in the ten most common malignant tumors in the world.Leukemia is a heterogeneous malignant blood cancer,characterized by abnormal proliferation or progenitor cells and diffused infiltration into other tissues and organs.Cytarabine(cytosine arabinoside 1-?-D-Arabinofuranosylcytosine,Ara-C),a pyrimidine nucleoside analog,is one of the mainstays used in the therapy of acute myeloid leukemia and non-Hodgkin's lymphoma.Via intracellular phosphorylation,cytarabine is converted into cytarabine 5 '-triphosphate derivative(Ara-CTP),followed by inhibition of DNA polymerase and DNA chain elongation,exerting its anti-neoplastic effect.Nevertheless,there were five main drawbacks in its clinical applications,such as(1)easy to be deaminated by deaminase and converted to inactive 1-?-D-arabinofuranosyluracil(Ara-U)in vivo;(ii)the poor membrane permeability ascribing to the hydrophilicity;(iii)the extremely short plasma half-life(10-20 mim)and drug multiresistance;(iiii)ineffectiveness for solid tumors;(v)clinical side effects such as hand foot syndrome(HFS)and stomatitis.Thus,Ara-C has to be administrated by intravenous continuously in order to obtain the maximum therapeutic efficiency in clinic,which causes the poor tolerability to the patients frequently.Therefore,it is significant and valuable to research safe and efficient cytarabine nano-drug delivery systems.Small molecule assembled prodrugs are designed to form nanodelivery system by conjugating low molecular weight inert materials with drug according to the defects of the drug itself.The prodrugs could self-assemble into nanoassemblies,realizing passive targeting to tumors tissues via enhanced permeability and retention(EPR)effect.Furthermore,as a part of the carrier and without inert excipients,the drug can increase drug loading and reduce side effects.Significantly,small molecule prodrug can improve the infiltration ability of solid tumors by conjugating lipid materials to nucleoside hydrophilic drugs,which expand the clinical application of nucleoside drugs in anti-solid tumors.Based on the above principle,we have designed and synthesized three mono-chain fatty acid cytarabine prodrugs with different carbon chain lengths,aiming at the drawbacks of cytarabine and carrying out a series of in vitro anticancer efficiency evaluation.We covalent linked hexanoic acid(C6),lauric acid(C12)and oleic acid(C18)-to 4-NH2 of cytarabine by amide bonds,obtaining three multi-functional prodrugs HA-Ara,LA-Ara and OA-Ara.These prodrugs could self-assemble into different morphology,such as nanospheres,nanobelts and nanofibers depending on the length of the fatty acids,and showing excellent anti-leukemia activity.Herein,in order to further explore the prodrug formed by two-chain hydrophobic fatty acids linking with cytarabine,its assembly behavior and anti-leukemia effect,we covalently conjugated the biocompatible double-chain fatty acid(DTA)to hydrophilic cytarabine(Ara-C)to form the prodrug DTA-Ara,constructing the self-assembled nano drug delivery system of small-molecule prodrug.Compared with Ara-C and single-chain fatty acid prodrugs,DTA-Ara has the merits of increased lipid solubility and membrane permeability,enhanced plasma stability and long-term preservation.On the basis of the above studies,we compared the anti-leukemia efficiency of four different hydrophobic carbon chain lengths cytarabine prodrugs.The results showed that lauric acid-cytarabine(LA-Ara)had the strongest cytotoxicity against leukemia cells.Furthmore,the pharmacokinetic study showed that the oral bioavailability of LA-Ara was significantly improved and realized the feasibility of Ara-C for oral administration.Therefore,based the the principle that the increase of liposolubility can improve the potential of nucleoside hydrophilic drugs to infiltrate into solid tumors,we selected LA-Ara oral nanofibers as a model drug for anti solid tumors,and carried out preclinical anti-solid tumors study.We aim to provide comprehensive preclinncal data for cytarabine against solid tumors,and laying thefoundation for clinical research.The main contents include two parts:(1)We covalented carboxyl group of fatty acids to 4-NH2 of cytarabine to construct small molecule self-ssembled prodrug systems.(2)The preclinical evaluation of lauric acid-cytarabine prodrug assembled nanofibers toward solid tumors.The specific research contents are as follows:1.Synthesis and biological evaluation of the 2-decyltetradecanoic acid-cytarabine prodrug self-assembled nanoparticlesCytarabine is a hydrophilic chemotherapy drug,which interferes with metabolic process of cells by inhibiting DNA synthesis,exerting its anti-neoplastic effect.Nonetheless,its severely drawbacks such as low lipophilicity and rapid plasma degradation limit clinical applications.Here,we synthesized a new Ara-C prodrug DTA-Ara by conjugating 2-decyltetradecanoic acid(DTA),a biocompatible double-chained fatty acid with Ara-C by amido group.Ethyl chloroformate was chosen as the catalytic agent.1H-NMR,ESI-MS and FT-IR techniques were used to determine the successful synthesis of the prodrug.The saturation solubility was decreased for 10,707 times compared with free Ara-C,indicating significantly improved lipophilicity and biomembrane permeability.DTA-Ara could self-assemble into spherical nanoparticles(NPs).TEM,DLS,zeta-potential were utilized to assess physicochemical characteristics of the assembly.The DTA-Ara NPs had the average sizes of approximately 130 nm and a zeta potential around-31.6 mV.Importantly,the DTA-Ara NPs were stable in deionized water or phosphate buffer solution(PBS,pH 7.4)solution for more than one week and the hemolysis rate was less than 10%,which indicated that it could be administrated intravenously.Moreover,the in vitro cytotoxicity study further manifested that the resulting prodrug showed the marked antitumor activity against human leukemia cell line K562 and HL60 cells compared to the naked drug.Therefore,the self-assembled DTA-Ara NPs provide a new platform for leukemia therapy with high antitumor capability.2.The preclinical evaluation of oral cytarabine-lauric acid prodrug assembled nanofibers toward solid tumorsOur earlier efforts of designing an active prodrug named lauric acid-cytarabine(LA-Ara),showing enhanced biological activities toward leukemia(none solid tumor).In addition,the pharmacokinetic studies demonstrate that the oral bioavailability of LA-Ara NFs dramatically increased.Herein,we evaluate the cytotoxicity in vitro against two breast cancer cells 4T1 and MCF-7 cells.LA-Ara exhibited significantly stronger cytotoxicity in vitro.We also assess preclinical antitumor efficacy of LA-Ara NFs on 4T1 breast cancer-bearing BALB/c mice model in vivo.The results showed that LA-Ara NFs presented notably more effective therapy than that of free Ara-C for the solid tumors.The safety evaluation indicated that LA-Ara NFs could be safely administered at repetitive doses without untoward toxicity such as hepatorenal dysfunction and blood routine abnormality.Notably,the limitations of Ara-C involving HFS and stomatitis could be overcomed by LA-Ara NFs.Therefore,the LA-Ara NFs might offer a new and efficient platform in the treatment of solid tumors in clinical setting,promoting the clinical promotion of Ara-C in oncology.In summary,two self-ssembled prodrugs of cytarabine nano-drug delivery systems were systematically studied for the biological evaluation and the preclinical study.The results showed that DTA-Ara NPs realize self-delivery process and avoid the use of inert non-pharmaceutical materials,significantly increasing lipophilicity and permeability of cytarabine,prolonging the plasma half-life.LA-Ara NFs can be administered orally to enhance the therapeutic efficacy of solid tumors.It is of great significance for broadening the clinical applications of Ara-C in oncology to study the self-assembling small molecule nano-drug delivery systems. |
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