The Study Of Receptor-mediated Tumor Targeting Nanoparticles Based On Hyaluronic Acid As Drug Delivery System

Hydrophobically modified hyaluronic acid-doxycholic acid (HD) conjugates were prepared with doxycholic acid (DOCA) as the hydrophobic groups. The self-assembled behavior of the amphiphiles in water solution was investigated. Adriamycin (ADR) was chosen as a model drug to fabricate the drug-loaded hyaluronic acid (HA) nanoparticles and the in vitro release of ADR form the nanoparticles was analyzed. Human epithelioid cervical cancer cell line (HeLa) was used to image and measure the cell uptake of nanoparticles. Cancer cell viability of the ADR-loaded HD nanoparticles was measured by MTT assay.The chemical conjugates of HA and DOCA were synthesized by covalent attachment of DOCA to HA with amide formation though EDC-mediated reaction. Various HD conjugates with different degree of substitution (DS) were prepared by changing the free mole ratios of DOCA to HA and the DS, defined as the number of DOCA per one HA molecule, was determined by a titration method. The structure of HD conjugates was identified by 1H NMR spectra. The self-assembled HD nanoparticles solution was obtained by sonication and the self-assembling properties were analyzed by the fluorescence probe test. The critical aggregation concentration (CAC) of HD nanoparticles decreased with the increasing of DS and the CAC determined for HD conjugates HD6, HD7 and HD9 were 0.056mg/ml, 0.037mg/ml and 0.025mg/ml respectively. The hydrophobicity of the core of nanoparticles was increased as the increasing of DS. To investigate the associating number of hydrophobic moieties in one self-aggregate and to identify the microstructure of nanoparticles, a fluorescence quenching method was used. The mean aggregation number of DOCA groups (NDOCA) and the e number of polymer chains (Nchain) per single hydrophobic domain increased with increasing DS of DOCA. The results indicated that one HD nanoparticle could have multiple hydrophobic inter-cores. The formation of HD nanoparticles was characterized by dynamic laser light scattering and transmission electron microscopy (TEM). The particle size was decreased with DS increased and the mean diameter of HD nanoparticles with a DS of 5.9, 7.4 and 9.6 were 1144nm, 447nm and 208nm, respectively. The TEM observation demonstrated the near spherical shape of self-aggregates with a integrated form.ADR was used as a model drug to incorporate into hydrophobic intercore of HD nanoparticles though hydrophobic interaction to prepare drug-loaded nanoparticles. various swatches were synthesized by change the feed weight ratio of ADR to HD conjugates. The drug-loaded nanoparticles with different DS were synthesized with same feed weight ratio of ADR to HD conjugates. The drug release profile of all the samples was investigated. The results indicated that the drug loading content increased with increasing the feed weight ratio of ADR to HD conjugates and the maximum loading content of HD6 nanoparticles was determined to 11.58 wt.%. The loading efficiency reduced with the increasing feed weight ratio of ADR. The drug loading content and efficiency were increased with the increasing DS. The drug was sustained released form all the three HD nanoparticles with different DS and the cumulated release time could achieve 5 days. The release behavior of ADR exhibited a triphasic pattern characterized by a fast initial release, followed by a constant release and the a slower and continuous release. The release profile was significantly influenced by the DS and the drug loading content. The hydrophobic drug in nanoparticles partially crystallized at a higher drug content and a higher DS which enhanced the interaction between ADR and the nanoparticles. The initial burst content decreased and the sustained release improved owing to the crystallization.The in vitro cytotoxicity of blank HD nanoparticles and cytostatic activity of ADR-loaded nanoparticles with different DS were analyzed by the MTT mothod. The cellular uptake of fluorescent HD nanoparticles was assayed and visualized by using fluorescence microplate reader and fluorescence microscopy. The blank nanoparticles exhibited no cytotoxicity for tumor cells and the cell viability was higher than 95% within 3 days. All the three different nanoparticle types of ADR appeared a certain extent anticancer activity, which increased with increasing of DS. The kill rate of HeLa with three of the drug-loaded nanoparticles were 33.72%, 41.27%, 49.76% respectively within incubating 3 days at the 10μg/ml drug concentration. ADR-loaded HD9 nanoparticles showed higher cytotoxicity versus free ADR at the drug concentration range of 1-5μg/ml and achieved equivalently anticancer activity the 10μg/ml drug concentration. Cellular uptake of HD nanoparticles depended on nanoparticle concentration and incubation time. The uptake rate of HD nanoparticles to HeLa cells showed a linear increasing within 4h. The lower nanoparticle concentration the higher cellular uptake efficiency, the maximum rate was above 50%. The DS was another key factor influencing the cellular uptake of nanoparticles. The degree of the interaction between the nanoparticles and the cells increased with increasing DS under identical condition. Above 50% HD9 nanoaparticles with the highest DS were internalized into cells within incubation time. Visual evidence of specific coupling between nanoparticle and cell and the nanoparticles's uptake by HeLa cells was obtained with fluorescence microscopy. The internalized process exhibited high dependence of incubation time corresponded to the quantitative test with fluorescence microplate reader.