Studies On Self-Aggregated Nanoparticles From Deoxycholic Acid Modified Carboxymethyl Chitosan As Drug Delivery System For Doxorubicin

In this paper, we prepared pH-sensitive self-aggregated nanoparticles (SNPs) based on amphiphilic deoxycholic acid modified carboxymethyl chitosan (DCMC) for delivery of the anticancer drug doxorubicin (DOX). With negatively charged hydrophilic carboxymethyl groups covering the shells, the DCMC SNPs could remain stable at physiologic pH 7.4 and show improved adhesion to tumor cells with the shells becoming less hydrophilic as a result of the partial protonation of carboxymethyl groups in the mildly acidic extracellular environment of tumor tissues. In the acidic intracellular environment, the carboxymethyl groups and amine groups of DCMC are expected to be protonated, causing deformation and aggregation of the SNPs with the shell becoming hydrophobic and the core getting loose, which may further trigger the faster release of enclosed drugs, endosomal disruption and eventual release of drugs into the cytosol. Such a system would be an effective delivery mode for cancer cells.Using chitosan with different viscosity molecular weights, water soluble carboxymethyl chitosan was fiirstly synthesized, then the DCMCs with different degrees of substitution (DS) of DOCA were synthesized, by the reaction between the animo groups of CMCS and the activated DOCA-NHS. The structure of the conjugates were confirmed using Fourier transform infrared (FT-IR) spectrophotometer and 1H NMR spectrometer as well as the the colloidal titration method. The synthetic condition for carboxymethylation was optimized. Three kinds of DCMC conjugtes with different viscosity average molecular weights and different degrees of substitution (DS) of DOCA were prepared by controlling the feed ratio of DOCA-NHS to sugar residues of chitosan.The synthetic DCMC conjugtes can form spherical and well-dispersed self-aggregated nanoparticles in aqueous solution under probe-type sonication and the size of the SNPs was in the range of 87.7-174.5 nm. The critical aggregation concentration (CAC) of the resulting SNPs decreased with the enhancing DS of DOCA and the decreasing molecular weight of chitosan. Moreover, the DCMC SNPs showed an acidic pH-induced aggregation and deformation behavior. After 3 months of storage at 4℃, no significant changes in the particle size of nanoparticles occurred and the stability of the DCMC SNPs increased with the enhancing DS of DOCA and the decreasing molecular weight of chitosan.Doxorubicin (DOX) was incorporated into the DCMC SNPs by dialysis method. The influences of formulation factors on drug loading were investigated in detail. With the increasing of feed ratio of DOX to DCMC, concentration of DCMC and DOX, the DOX-loading content (LC) and encapsulation efficiency (EE) increased. Meanwhile, the particle size increased and the absolute value of the zeta potential decreased with an increase in the DOX feed ratio. It was also noted that the higher the DS of DOCA and the molecular weight of CS, the higher the drug loading content and entrapment efficiency, while the particle size decreased and increased, respectively. The TEM images indicate that the nanoparticles retain a spherical and well-dispersed structure. After a burst effect, the in vitro release of DOX from [D]NP was consistent with a Higuchi, Ritger-Peppas or Weibull diffusion mechanism. After 3 months of storage at at 4 ℃ and shakening at 37 ℃ for 7 days, no significant changes in particle size of nanoparticles occurred.The cytotoxicity of blank and DOX-loaded DCMC nanoparticles ([D]NP) against MCF-7 (human breast carcinoma cells) and MCF-7/Adr (multi-drug resistant variant) was evaluated by MTT assay. The [D]NP with different molecular weights of CS exhibited much lower cytotoxicity against MCF-7 than free DOX, with that of the [D]NP-600 kDa being much stronger than the [D]NP-50 kDa. In contrast, the [D]NP with different moleculars of CS were much more effective than the free DOX against MCF-7/Adr, presenting significant reversal activity against DOX resistant cancer cells with time-dependent resistance reversion index (RRI). Images acquired by the confocal laser scanning microscope (CLSM) showed that the free DOX accumulated quickly in the nucleus of MCF-7 cells, but showed a much lower and punctate fluorescence in the cytoplasm of MCF-7/Adr cells after incubation and no significant increase in fluorescence intensity was observed with a prolonged incubation time. In contrast, the [D]NP accumulated throughout the cytoplasm of MCF-7 and MCF-7/Adr cells after 0.5 h incubation, exhibiting release-limited and time-dependent nucleus distribution. The enhanced cellular uptake and greater retention of [D]NP in drug-resistant cells, as evidenced by flow cytometry, contributed to a superior efficacy of [D]NP over free DOX.An assay method was established for the determination of DOX in rat plasma and tissues of H22 tumor-bearing mice. The results of intravenous pharmacokinetic behaviors in rats showed that the [D]NP with different molecular weights of CS exhibited an prolonged elimination half-life (t1/2), an increased AUC and MRT (P< 0.05), compared with the free DOX. With the molecular weight of CS increasing, the AUC(0-∞) increased (P< 0.01) and the CL decreased (P< 0.05). Therefore, [D]NP could prolong the circulation time in rat plasma by a slower elimination, and the [D]NP with higer molecular weight of CS presented a better long circulation effect. [D]NP displayed a much longer systemic circulation time in H22 tumor-bearing mice than free DOX. Moreover, in comparison with free DOX, [D]NP exhibited significantly increased accumulation and affinity in liver, spleen, tumor and plasma than free DOX, as evidenced by the three targetability indexes, with decreased accumulation in the heart, lung and kidney of mice. In addition, [D]NP with higer molecular weight of CS produced more evident targetability.The results of in vivo antitumor activity in H22 tumor-bearing mice showed [D]NP-600 kDa and [D]NP-300 kDa showed higher tumor inhibition rates among the [D]NP with different molecular weights of CS. [D]NP-600 kDa exhibited a dose-dependent tumor inhibitory effect with much lower side effects, which produced similar tumor inhibitory rates to that of free DOX at the same dose and was more effective than the free DOX with the enhanced dose. These results suggest the potential of DCMC SNPs as carriers for the hydrophobic drug DOX for effective cancer therapy.