Preparation And Anti-tumor Activity Of Doxorubicin-loaded Histidine-hyaluronic Acid Nanoparticles

Hyaluronic acid (HA) is a naturally polysaccharide. Owing to itsbiocompatibility, biodegradability, HA has been extensively investigated forbiomedical applications such as drug delivery, tissue engineering. In this paper,histidine–hyaluronic acid conjugates were synthesized at first. Doxorubicin loadednanoparticles were prepared using a probe-type sonifer, the cytotoxicity and cellularuptake of drug-loaded nanoparticles were tested by MTT assay. In addition, thepharmacokinetics, tissue distribution, and antitumor activity of drug-loadednanoparticles were investigated by H22tumor-bearing mice.Histidine–hyaluronic acid (His–HA) conjugates were synthesized using histidine(His) as hydrophobic segment by cross-linking method. The structural characteristicsof the His–HA conjugates were investigated using1H NMR. The critical aggregationconcentration (CAC) of His-HA nanoparticles was tested by fuorescencespectroscopy, and the change of micro-structure at different pHs was analyzed. Theresults showed that His was successfully modified to HA chain by1H NMR, and theCAC value decreased with increase in the DS of the His.His-HA nanoparticles were prepared using a probe-type sonifer. The particlesize and Zeta potential of His-HA nanoparticles were evaluated by Malvern dynamiclaser light scattering at different pHs. The morphology of the His-HA nanoparticleswas observed using transmission electron microscopy (TEM). The results showed thatthe average particle size of His-HA nanoparticles decreased from780±56nm to230±21nm with an increase in the DS of the His, and increased with the decrease of pHvalue. The Zeta potential of His-HA nanopartecles was negative, its absolute valuedecreased with an increase of DS, and increased at acidic pHs. TEM images indicated that the morphology of His-HA nanopaticles was spherical in shape.To prepare drug-loaded His-HA nanoparticles using doxorubicin (DOX) as amodel drug, and test the loading efficiency (LE) and encapsulation efficiency (EE). Inaddition, we explored in vitro drug release at different DS and pHs. The resultsindicated that the LE and EE increased with the increase of DS. In vitro DOX releaseexperiments indicated that DOX/His-HA nanoparticles showed DOX sustainedrelease from nanoparticles，and the effect of DOX sustained release increased with anincrease in the DS. DOX/His-HA nanoparticles released large amount of drug atacidic environments, the accumulative release of DOX/HH-3was32.92%(8h) and51.87%(48h) at pH6.0.The cytotoxicity of DOX/His-HA nanoparticles on MCF-7was investigated usingMTT assay. Cellular uptake of FITC-labeled His-HA nanoparticles and DOX/His-HAnanoparticles were examined by fuorescent microscopy. Furthermore, the quantitativeanalysis of DOX uptake was explored at different temperature, pH value and inpresence of HA. The cytotoxicity results showed that DOX/His–HA nanoparticlesexhibited similar dose and time-dependent cytotoxicity against MCF-7cells with freeDOX. The cellular uptake showed that FITC-His-HA nanoparticles were readilyinternalized into MCF-7cells. On the other hand, the auto-fuorescence of DOX wasobserved in the cytoplasm as well as the nucleus when the cells were incubated withthe DOX/His-HA nanoparticles. The quantitative study showed that amount of DOXuptake increased with the decrease of pH value at4℃, and was lower than thetreatment at37℃. This indicated that the uptake of DOX depended on HAreceptor-mediated endocytosis and extracellular DOX release from DOX/His-HAnanoparticles.To study the biocompability of the His-HA nanoparticles, the cytotoxicity andhemolysis tests were conducted. The cytotoxicity was tested by MTT assay using L02as cell model, the results showed that His-HA nanoparticles demonstrated nocytotoxicity even in1mg/ml. The hemolysis test of His-HA nanoparticles was detected by direct contact method. The results showed that the hemolysis rate of1mg/ml of His-HA nanoparticles solution was fewer than5%. These results indicatedthat His-HA nanoparticles had no cytotoxicity.To study the pharmacokinetics and tissue distribution of DOX/His-HAnanoparticles using H22tumor-bearing mice as an animal model. The experimentalresults showed that t1/2βand AUC increased significantly, and MRT was significantlyprolonged, while CL and Vc significantly deceased compared with free DOX. Thetissue distribution experiments showed that the amounts of DOX in tumorsignificantly increased via tail vein injection compared with free DOX, this indicatedthat His-HA nanoparticles were tumor-targeting.To examine the antitumor effect of DOX/His-HA nanoparticles using H22tumoranimal as models. Compared with the control group, the body weight in mice forHis-HA nanoparticles treatment had not statistically different (P>0.05), while itsignificantly reduced for DOX treatment (P <0.05). The tumor was significantlyinhibited after DOX and DOX/His-HA nanoparticles administration, the inhibitionrate of DOX/His-HA nanoparticles was67.07%. Compared with the control group,thymus index and spleen index for DOX/His-HA nanoparticles has no significantchange (P>0.05), while they significantly decreased for DOX treatment (P <0.05).The results indicated that the DOX/His-HA nanoparticles can effectively inhibit tumorgrowth, and reduced drug toxicity.