Design Of PNA-loaded Nano-drug Delivery Systems Based On Modified Chitosan And Their Cellular Uptake Properties

Peptide nucleic acid（PNA）is an artificial oligonucleotide in which the sugar-phosphate back bone is replaced by N-（2-aminoethyl）glycine units.This replacement gives PNA several desirable properties including high affinity for DNA and RNA,stability towards degradation of both nucleases and peptidases and convenient structural modification.As a third generation antisense and antigene agent,PNA displays strong potential application for therapeutic,particularly for anticancer drug.However,similar to natural nucleic acids,PNA shows inefficient cellular uptake,and this undesirable property limits the use of PNA in medicinal and other applications.In order to promote the cellular uptake of PNA,a large variety of modified PNAs have been designed and synthesized.Among them,introduction of halogen atoms into the back bone of PNA is one of the typical modification.PNA containing halogen atoms has attracted extensive interest because of their enhanced hybridization.In recent years,various delivery methods,including modification of the backbone of PNA,conjugation to CPP,and using nanoparticle carrier system,have been carried out to enhance the cellular uptake of PNA.However,a major disadvantage that limits the pharmaceutical application of PNA is its poor cell permeability,despite the development of a variety of delivery techniques.Therefore,it is urgent to develop an effective and easy delivery methods of PNA.Self-assembled drug-loaded nanoparticles can be composed of chitosan（CS）amphiphilic derivatives due to their excellent drug loading property.Moreover,the lymphocyte-macrophage system is circumvented by the hydrophilic group of the nanoparticles,and the cellular uptake of the nanoparticles is enhanced by the hydrophobic grouping.Chitosan amphiphilic derivatives have recently been used as delivery carriers for gene therapy.However,there were few reports on their use with PNA.In this work,a facile approach to construct biocompatibility nano particles for PNA delivery based on the self-assembly of amphiphilic chitosan derivatives with hydrophilic quaternary ammonium cationic and hydrophobic long alkyl chains.We show,for the first time,the successful use of chitosan amphiphilic derivatives based vector for the delivery of PNAs in vitro.The main research contents are as follows:（1）In order to enhance the hybridization of PNA,5-halouracils（5-XU）have been introduced into the monomers of PNAs using the Fmoc protection strategy for the first time.Moreover,a series of 5-XU-PNA oligomers were synthesized using solid-phase protocols.The compounds were purified by silica gel column chromatogram and reverse-phase HPLC,and characterized by MS、¹H NMR、¹³C NMR and HPLC.（2）The sequence specificity of 5-XU in PNA duplex hybridizations were investigated by UV-melting experiments.The results indicated that PNAs containing one 5-XU unit showed 1.8–4.0℃（for DNA）or 2.5–3.7℃（for RNA）higher T_m values than normal PNA.Introduction of 5-XU-PNA monomers into PNA oligomers could further improve the hybridization of PNA.Furthermore,in 5-XU-PNA:DNA/RNA duplexes,the 5-XU preferred to bind to A,.and the thermal stability of the XU:A base pairs varied as FU:A>ClU:A>BrU:A>T/U:A.（3）¹H NMR spectroscopy was employed to study the influence mechanism of halogen substitution in the base of PNA on the hybridization properties of 5-XU-PNAs.The results showed that the chemical shifts of the N₃ in the XU:A were downfield 0.62–0.76 ppm relative to the T:A.The electron withdrawing properties of all three halogens resulted in the low electron density of the pyrimidine ring,and increased the acidity of N₃ protons.The increased acidity of the N₃ protons of 5-XU would be expected to strengthen the hydrogen bond between N₃–H and adenine.（4）A series of amphiphilic N,N,N-trimethyl-O-alkyl chitosans（TMACs）were designed and synthesized.The long alkyl chains as hydrophobic moieties were grafted onto the CS via its hydroxyl group with the Williamson-Hall method.The quaternary ammonium cation as hydrophilic moieties on CS was supposed to improve the solubility of CS.The compounds were purified by dialysis,and characterized by FT-IR and ¹H NMR.（5）The PNA-loaded TMACs nanoparticles（NPs）was prepared using ultrasonic-dialysis method.The NPs are spherical shape with a size range of around 100nm and a zeta potential above+20 mV.The length of alkyl chain and the weight ratio were found to significantly influence on the entrapment efficiency（EE）of PNA-loaded TMAC NPs.The drug-loaded NPs have the optimal EE at the alkyl chain length of 16and the weight ratio of 20.PNA-F was sustained release from the drug-loaded NPs,and the drug release pattern was in accorded with the first order kinetics.In addition,as the alkyl chain length increased,the PNA-loaded TMAC NPs showed better sustained release character in vitro.（6）The safety of the PNA-loaded TMAC NPs was evaluated using hemolysis and MTT assays.The cytotoxicities of TMACs were strongly influenced by the length of alkyl.The safety could be ranked as follows:TMCC>TMDC>TMBC>TMOC.The hemocompatibility and nontoxicity were confirmed when the concentration of TMCC lower than 5 mg/mL.These results indicated that TMCC might be a potential vector for drug delivery.（7）With HeLa cells as a model cell type,the cellular uptake of 5-XU-PNA and PNA-TMCC NPs was evaluated using flow cytometry（FCM）and confocal laser scanning microscopy（CLSM）.The results indicated that the cellular uptake of5-XU-PNA was inefficient,while the cellular uptake of 5-XU-PNA was improved by51-63 folds for the free PNA when delivered by the TMCC NP.The efficient cellular uptake of PNA-loaded TMCC NP was attributed to the suitable size and high positive charge of the NP.In this study,the PNA-loaded NPs with TMACs were constructed to overcome the poor cellular delivery of PNA.Firstly,a series of 5-XU-PNA monomers and oligomers were designed and synthesized.PNAs carrying 5-XU formed a duplex with complementary DNA or RNA with higher affinity than unmodified PNA.Secondly,a series of TMACs with different lengths of alkyl chains were designed and synthesized.The hemocompatibility and nontoxicity were confirmed when TMCC was utilized as PNA carrier,and the excellent PNA-loading capacity of TMCC was confirmed.Lastly,the unefficient cellular uptake of PNA could be overcomed by loading PNA into TMCC NP using FCM and CLSM.These results indicated that TMCC NP might be a novel vector for PNA delivery.