Construction And In Vitro Anti-tumor Evaluation Of Nanoassemblies From Amphiphilic Small Molecular Prodrugs Of Cytarabine

Leukemia is a disease characterized by abnormal proliferation of hematopoietic stem cells and the morbidity of leukemia is ranked sixth among the various tumors in our country.1-(β-D-Arabinofuranosyl)cytosine(cytarabine,Ara-C)a pyrimidine nucleoside analogue,has been the backbone of majority therapeutic regimens in acute lymphoblastic leukemia(ALL)treatment.However,the low lipophilicity and membrane permeability as well as rapid deamination to,the biologically inactive 1-β-D-arabinofuranosyluracil(Ara-U)by cytidine deaminase lead to an extremely short plasma half-life and low oral bioavailability.Consequently,Ara-C has to be administrated in continuous infusion to maintain therapeutic plasma level which causes the high adverse effects.The clinical application of Ara-C is severely restricted by the above defects and it is significant to develop a novel drug delivery system of Ara-C with high drug loading and safety.Amphiphilic small molecular weight prodrugs are a kind of conjugates in which the carrier materials commonly bear low molecular weight,and drugs themselves play a vital role in adjusting the hydrophilic-lipophilic balance of the compounds.On account of the intrinsic amphiphilic property,the prodrug could self-assemble into nanoassemblies in aqueous phase,realizing self-delivery of the drug to tumor tissues through EPR effect.In addition to avoiding the introduction of inert excipients,this type of pure nanoassemblies possesses a remarkable high drug-loading and the chemical bond between drug and carrier materials makes the sustained release more obvious.Herein,in order to overcome the defects of cytarabine,two kinds of hydrophobic fatty chain-oleic acid(OA)and hexanoic acid(HA)were covalent to 4-NH2 of Ara-C,designing two types of amphiphilic small molecular weight prodrug of cytarabine,OA-Ara and HA-Ara.The advantages of the prodrugs are displayed as following:(ⅰ)markedly increasing the lipophilicity of Ara-C so that the permeability of cell and biological membrane was improved strikingly;(ⅱ)preventing NH2 group from deactivation by deamination to inactive metabolin;(ⅲ)the proper amphiphilic structure endows the prodrug self-assemble into nanoformulations with high drug-loading and self-delivery function.In this paper,OA-Ara oral administration and HA-Ara active targeting nano-drug delivery system were developed and the research contents are as follows.1.Preparation and biological evaluation of OA-Ara oral formulationHere,we have conjugated the biocompatible material,OA to 4-NH2 of Ara-C obtaining oleic acid-cytarabine(OA-Ara)prodrug with a high yield(61.3%)and the 1H-NMR spectrum confirmed the successful synthesis.The results of oil/water partition coefficient(Ko/w)and parallel artificial membrane permeability assay indicated that the lipophilicity and permeability of the prodrug were increased significantly.Meanwhile,OA-Ara molecules were stable in various pH solutions and artificial digestives,suggesting that it could be orally administrated.In addition,OA-Ara molecules could assemble into spiral assembly in aqueous phase through both "top-down" and "bottom-up" approaches under probe ultrasonication with BSA as stabilizer.Transmission electron microscopy(TEM),circular dichroism(CD)and fourier transform infrared(FTIR)were applied to speculate the formation mechanism of the spiral assemblies.From the results of MTT assay,the prodrug exhibited stronger cytotoxicity on leukemia cells K562 and HL60 due to its higher lipophilicity and membrane permeability.In conclusion,OA-Ara spiral assemblies could enhance the therapeutic index of leukemia and provided a safer and more effective treatment for Ara-C’s clinical application.2.Studies on FR-targeting HA-Ara nano-drug delivery systemHexanoic acid(HA)containing 6 carbons were covalent to the NH2 group of Ara-C and the novel prodrug,hexanoic acid-cytarabine(HA-Ara)were synthesized.1H-NMR and MS were utilized to prove the successful synthesis.Based on the proper amphiphilicity of the prodrug,HA-Ara could self-assemble into nanoasseblies around 90 nm in water with a drug loading as high as 71%via nanoprecipitation method.The morphology,size distribution and critical aggregation concentration(CAC)of HA-Ara nanoassemblies(NAs)were characterized.Besides,FA-BSA conjugate was synthesized successfully which could be adsorbed on the surface of HA-Ara NAs,obtaining FR-targeting nanoassemblies(NAs/FA-BSA)and for comparison,non-targeting NAs with BSA adsorption was prepared,too.Hemolysis assay was carried out to verify the biocompatibility of the NAs and they could be administrated intravenously,In vitro release studies results illustrated that HA-Ara NAs exhibited an obvious sustained-release effect.MTT assays against K562[FR(+)]and A549[FR(-)]cells were performed and the results showed that HA-Ara NAs markedly increased the cytotoxicity of the native drug.For K562 cells,NAs/FA-BSA had the strongest proliferation inhibition capacity on the cells.In cellular uptake assays,FITC was used to label the NAs and fluorescence microscopy as well as flow cytometric analysis were applied.The results validated that NAs could promote the cellular uptake by endocytosis and NAs/FA-BSA showed highest accumulation in K562 cells.The in vitro tests indicated that FR-mediated endocytosis could increase the cytotoxicity and internalization of active-targeting HA-Ara NAs.All in all,amphiphilic small molecular prodrug associated with targeting delivery would further enhance therapeutic effect of leukemia.In summary,we designed and synthesized two kinds of novel amphiphilic small molecular prodrug for cytarabine’s delivery.The results showed that both the prodrugs could not only improve the lipophilicity and permeability of the parent drug,but also self-assemble into nanoassemblies in aqueous phase with a prominent high drug loading.Contrast with the native drug,prodrugs molecules and their nanoformulations exerted a significantly higher anti-tumor capacity.OA-Ara spiral nanoassemblies were suitable for oral administration while FR-targeted HA-Ara nano-drug delivery system could deliver the drug into tumor cells directly,which supplied a further improvement of leukemia therapeutic index.Consequently,the studies on the properties of the two prodrugs in this paper build the theoretical foundation for the construction of amphiphilic small molecule prodrugs and have important practical significance.