Smart Antitumor-drug Delivery System: Design And In Vitro Evaluation

Design of smart drug delivery systems(DDSs), which is based on the physiological differences between the tumor and normal tissues, plays an important role in effectively delivering antitumor drugs for cancer treatment. Owing to the improvement of therapeutic efficacy and reduction of severe side effect, the formulation of drug delivery system has attracted extensive attention recently. Moreover, these DDSs have been designed to possess some unique stimuli properties, including internal stimulus(such as pH-responsive and reduction-responsive properties) and external stimuli(such as photo-responsive properties), to further enhance therapeutic effect. Taken abovementioned factors into account, different materials(such as natural polyelectrolytes, block copolymers, and graphene oxide) have been utilized to fabricate drug delivery system with different structures. Meantime, the different interactions(such as electrostatic interaction, hydrophobic interaction and π-π stacking) were applied to load the antitumor drugs. More importantly, integration of targeting, biocompatibility and fluorescence into these DDSs with pH, reduction and photo-responsive properties have been designed for diagnosis and treatment of cancer, as following:Firstly, novel multilayer polyelectrolyte hollow microspheres((CSFITC/HA)4/GC) with pHsensitive fluorescence and hepatocyte-targeting functions were successfully fabricated via a layerby-layer(Lb L) assembly of fluorescein isothiocyanate(FITC)-modified chitosan(CSFITC) and sodium hyaluronate(HA)(as the polycation and polyanion respectively) on the polystyrene sulfonate(PSS) templates with galactosylated chitosan(GC) as the outermost layer; after etching the templates by dialysis, the aim was to use the microspheres to target hepatocytes specifically. They shoed hollow structure with a particle size of about 260 nm, and the DLS analysis demonstrated that they possessed pH and ionic strength dual-responsive characteristics. The hollow microspheres showed pH-sensitive fluorescence, hepatocyte-targeting and exhibited favorable cytocompatibility. Based on this work, to reduce experimental steps and shorten the preparation time, the monodisperse pH-responsive surface charge-reversible and hepatoma-targeting polyelectrolyte complex nanoparticles(GC/HA NPs) near 80 nm were prepared via the ionotropic gelification by simple and mild co-precipitation of galactosylated chitosan(GC) and hyaluronate(HA). Their surface charges could transfer from negative in neutral or basic media to positive in acidic media, indicating their potential application for delivering antitumor drugs in response to the extracellular pH of the tumor environment. The unique ‘‘electrostatic sponge’’ structure enhanced their drug-loading capacity and encapsulation efficiency towards the anticancer agent doxorubicin(DOX), as well as the pH-triggered controlled release. MTT assays and fluorescence microscopy analysis revealed that the DOX-loaded GC/HA NPs possessed the hepatoma-targeting capacity with favorable biocompatibility. Meantime, a versatile folate receptor-mediated targeting DDS with pH/reduction dual-responsive and pH-modulated fluorescent properties(DCL FA-HA-Rh 6G NPs) was fabricated via facile ionic(pH sensitive) and covalent(reduction responsive) doublecrosslinking(DCL) of the folic acid(FA) and Rhodamine 6G modified hyaluronic acid(FA-HA-Rh 6G). It not only had high drug loading capacity(DLC) and drug encapsulation efficiency(DEE)(0.7161 ± 0.0341 mg/mg and 71.60% ± 3.4%), but also possessed perfect triggered release in stimulated tumor microenvironment. The MTT assay and confocal laser scanning microscope(CLSM) analysis revealed that the proposed double-crosslinked HA-based DDS had favorable cytocompatibility and folate receptor-mediated targeting functionality for HeLa cells.Secondly, a biocompatible PEGylated chitosan was synthesized by introduction of hydrophilic PEG moieties to the skeleton of chitosan. This copolymer was an amphiphilic precursor for molecular self-assembly in PBS(pH 6.8). Based on chitosan-based amphiphilic homopolymer, the general strategy was developed via precisely modulating PEG2000-CHO feed ratio and viscosityaverage molecular weight of chitosan for fabricating a series of controllable morphology and diameter of DOX/CS-PEG micelles. More importantly, the cumulative release ratio of DOX from the DOX/ CS100K-PEG2 micelles under PBS(pH 7.4) was only 2.68 wt%, while that of PBS(pH 5.0) was 72.76 wt%. The DOX/CS100K-PEG2 micelles possessed highly stability at physiological pH, and burst release properties in a slight acidic environment(pH 5.0) to realize “off-on” switchable release properties of DOX in the microenvironment of tumor cell. As revealed by MTT assays and CLSM analysis, hydrophobic anticancer DOX could be efficiently and rapidly transported into tumor cell nuclei and showed significant cell inhibition.Thirdly, a facile and effective approach was established for fabricating core-shell-corona micelles by self-assembly of PEG43-b-P(AA30-co-tBA18)-b-PCL53 triblock copolymer, synthesized via a combination of ring-opening polymerization(ROP), atom transfer radical polymerization(ATRP), click chemistry, and hydrolyzation. After cross-linking with a disulfide bond to regulate the drug release kinetics, the spherical core-shell-corona micelles were obtained with average diameter of about 52 nm. The reduction-responsive cross-linked micelles showed a slow sustained release in normal physiological conditions and a rapid release upon exposure to the simulated tumor intracellular condition. In addition, the cytotoxic analysis and HepG2 cell growth inhibition assays demonstrated their remarkable biocompatibility and similar excellent anticancer activity as free DOX, which has also been revealed by the CLSM analysis. Moreover, a photo-responsive nitrobenzene-based amphiphilic PEG43-b-P(AA76-co-NBAc35-co-tBA9) micelles were fabricated via a combination of atom transfer radical polymerization(ATRP), hydrolyzation and esterification. The morphological change of the micelles under UV-irradiation endowed them with a tunable release and enhance DOX release efficacy in a triggered or on-demand manner. The PEG43-bP(AA76-co-NBAc35-co-tBA9)micelles presented a fast UV-sensitivity upon 365 nm UV irradiation and the photo-cleavage reaction finished within 20 min.Finally, graphene oxide with high surface areas, benzene ring of conjugate system and favorable biocompatibility is applied to load effectively DOX via π-π stacking. A novel graphene oxide(GO)-based nanocarrier has been designed for the targeting and pH-responsive controlled release of anticancer drugs via the classic amidation of the carboxyl groups of carboxylated graphene oxide(CG) with the amine end-groups of functional poly(ethylene glycol)(PEG) terminated with an amino group and a folic acid group(FA–PEG–NH2). The carboxylated graphene oxide conjugated folate-terminated poly(ethylene glycol)(CG–PEG–FA) nanocarriers showed favorable biocompatibility and perfect folate receptor-targeting functions. Based on this research, a facile strategy was established to develop a drug delivery system(DDS) based on the graphene oxide nanoparticles(GON) with suitable size and shape to deliver drug effectively, by grafting the biocompatible PEGylated alginate(ALG-PEG) brushes onto the GON via the disulfide bridge bond. Importantly, the in vitro release showed that the platform could not only prevent the leakage of the loaded DOX under physiological conditions but also detach the cytamine(Cy) modified PEGylated alginate(Cy-ALG-PEG) moieties upon response to glutathione(GSH). Moreover, the unique reduction-triggered functional graphene oxide nanoparticles(GON) with well defined size and uniform distribution were designed as an innovative drug delivery platform for cancer treatment for the first time, via the redox radical polymerization of methacrylic acid from the polyethylene glycol(PEG) modified GON(GON-PEG), following by cross-linking with cystamine. Notably, introducing the cross-linked PMAA brushes efficiently minimized the premature release of doxorubicin(DOX) in the stimulated normal tissues, and accelerated DOX release in the stimulated tumor tissues through response to reduce agent. The drug delivery system showed a 6-fold faster releasing rate at pH 5.0 in the presence of 10 mM glutathione(GSH) than at pH 7.4 with 10 μM GSH.