| Biopolymer NPs formed by self-assembly of natural protein and polysaccharide, featured by their nontoxicity, easy preparation, good chemical, high drug encapsulation efficiency, controlled drug release manner, has gained much more focus. Nevertheless, almost all of these biopolymer NPs can only accumulate spontaneously in solid tumors through the leaky tumor neovasculature by the enhanced permeability and retention(EPR) effect, known as the passive targeting. To further improve delivery efficiency and reduce the side effects, we decorated the protein/polysaccharide NPs with specific ligands as new active targeting nanoparticles for drug delivery.In this research, a simple and green approach was developed to produce a novel nanogel via self-assembly of low density lipoproteins(LDL) and sodium carboxymethyl cellulose(CMC). Doxorubicin(DOX) was used as the drug model to study the loading, p H-triggered release, cellular uptake and antitumor activity of DOX-LDL/CMC nanogels. In order to enhance the efficacy of DOX-LDL/CMC nanogels, sodium carboxymethyl cellulose(CMC) was modified with folic acid(FA) through the esterification. Then, two types of novel FA-targeted nanogels were prepared for drug delivery. DOX-LDL/CMC-FA NPs was formed by heating method and Ca2+-DOX-FA-CMC/LDL NPs was prepared by ionic cross-linking method. The characterization, in vitro drug release and the active targeting efficiency were studied. The results demonstrated that the prepared DOX-FA-CMC/LDL and Ca2+-DOX-FA-CMC/LDL were able to achieve p H dependent release of DOX and capable of active targeting. They could promote tumor targeting delivery of DOX, increase tumor cell uptake and enhance the drug efficacy. Hence, such excellent targeted nanoparticle has a good application prospect.The main results are as follows:1. A simple and green approach was developed to produce a novel nanogel via self-assembly of low density lipoproteins(LDL) and sodium carboxymethyl cellulose(CMC). Experiments were performed to find out the most suitable conditions(weight ratio, p H, heating time and temperature) for forming stable nanogels. The well-defined LDL/CMC nanogels was about 90 nm spherical nanoparticles with PDI~ 0.3, a zeta potential~-35 m V. LDL and CMC could assemble into functional biopolymer nanogels based on electrostatic attraction and intermolecular hydrophobic association. The nanogels had a polysaccharide surface that made the nanogels stable in various p H conditions(3.0-10.0). Meanwhile, the nanogels stored in isotonic solution at 4℃ did not change their sizes much after 90 days. In addition, the nanogel sizes were almost the same as the sizes of freshly prepared ones after lyophilization and rehydration.2. Doxorubicin(DOX) was used as the drug model to study the loading, p H-triggered release, cellular uptake and antitumor activity of DOX-LDL/CMC nanogels. DOX was effectively encapsulated into the LDL/CMC nanogels via electrostatic and hydrophobic interactions between LDL/CMC and DOX. Notably, DOX-LDL/CMC nanogels prepared using doxorubicin concentration 100 μg/mg had the highest EE 98.219% and lower size 123.7 nm, a zeta potential~-38.20 m V. In the meantime, the in vitro studies demonstrated that DOX release from LDL/CMC nanogels was p H-dependent: DOX was slowly released from nanogels under physiological condition(p H 7.4), whereas its release rates were much higher at mildly acidic environments(p H 6.2). Furthermore, the DOX-LDL/CMC nanogels were shown to effectively kill cancer cells in vitro. The IC50 of the DOX-LDL/CMC nanogels in He La and Hep G2 cells was approximately 2.45 and 1.72 times higher than that of free DOX. The slightly reduced antitumor efficacy was primarily due to the less cellular uptake of the DOX-LDL/CMC nanogels, which was confirmed by CLSM and FCM.3. Sodium carboxymethyl cellulose(CMC) was modified with folic acid(FA) through EDC coupling. The structure of grafting FA was confirmed by UV-vis, FT-IR, 1H NMR, XPS, DSC and XRD. When the mass ratio of FA and CMC was 3:1, the coupling ratio(CR) was 4.1%. Under the best conditions, the optimal FA-CMC/LDL nanoparticle was about 124 nm spherical nanoparticles with PDI~ 0.3, a zeta potential~-35 m V; and the best Ca2+-FA-CMC/LDL nanoparticle was about 152 nm spherical nanoparticles with PDI~ 0.26, a zeta potential~-30 m V. DLS and TEM showed that Ca2+-FA-CMC/LDL nanoparticle had a better spherical morphology and dispersion than FA-CMC/LDL nanoparticle. Besides, these nanoparticles possessed an excellent physical stability after 90 days of storage at 4℃.4. The optimal FA-CMC/LDL and Ca2+-FA-CMC/LDL nanogels were chosen for loading DOX to study the loading, p H-triggered release and the active targeting efficiency. When the doxorubicin concentration was 80 μg/m L, DOX-FA-CMC/LDL nanoparticle had an exceptionally high EE of 96.576% with size~ 136.07 nm, a zeta potential~-28.50 m V and PDI~ 0.431. While Ca2+-DOX-FA-CMC/LDL nanoparticle was about 196.93 nm spherical nanoparticles with EE of 97.245%, PDI~ 0.278, a zeta potential~-24.30 m V. Importantly, the release of DOX from DOX-FA-CMC/LDL and DOX-Ca2+-FA-CMC/LDL were p H-dependent, and DOX-Ca2+-FA-CMC/LDL exhibited better controlled release ability than DOX-FA-CMC/LDL. In addition, MTT assay demonstrated that both DOX-FA-CMC/LDL and DOX-Ca2+-FA-CMC/LDL were significantly more cytotoxic than DOX-LDL/CMC against KB cells. But the free DOX was more cytotoxic than all DOX-loaded nanopartcles(DOX-LDL/CMC, DOX-FA-CMC/LDL and DOX-Ca2+-FA-CMC/LDL) against A549 cells. Furthermore, CLSM and FCM measurements confirmed that folic acid conjugation markedly increased the cellular uptake of nanoparticles by FR-positive KB cells. The cellular uptake of targeted NPs was approximately 1.3 times more than untargeted NPs. Finally, FA competition test confirmed that the cellular uptake of the DOX-FA-CMC/LDL and DOX-Ca2+-FA-CMC/LDL were mainly based on a folate-receptor-mediated endocytosis mechanism. It might increase tumor cell uptake and enhance the drug efficacy. |
PDF Full Text Request