Based On The Rational Design Of The Polymer Nano-drug System Used To Improve The Protein Peptide Drugs And SiRNA In Vitro And In Vivo Efficacy

Nanoparticle-based drug delivery systems can improve the drug stability, release the drug in a controlled manner, promote the drug absorption, enhanced the therapeutic effect, and lower the side effects. Therefore, it is demanded to systemically investigate their in vitro and in vivo mechanisms in the purpose of providing guidelines to the rational design of nanoparticulate vehicles. Biomacromolecules such as protein, polypeptide, and nucleic acids are vulnerable in physiological environment. Nanoparticles have been demonstrated to efficiently deliver biomacromolecule drugs in vivo, especially via oral administration. The physicochemical properties of nanoparticles significantly affect the cellular uptake, oral absorption, and biodistribution. In this regard, the development of nanoparticle delivery systems for improved drug efficacy should be based on the understanding of the effects of physicochemical properties of nanoparticles on their in vitro and in vivo fate.The present investigation adopted chitosan derivative based polymeric nanoparticles as models to assess the effects of particle size and surface charge on the absorption and biodistribution of biomacromolecule drugs. Multifunctional chitosan derivative based polymeric nanoparticles, termed as mannose modified trimethyl chitosan-cystein (MTC) NPs, were developed and encapsulated with tumor necrosis factor-a (TNF-a) siRNA, which possessed active targeting, enhance the oral absorption of drugs, facilitate cellular uptake, and overcome the biological barreirs for siRNA delivery, thus mediating highly eficient in vitro and in vivo RNAi via oral administration. The in vivo gene silencing effects and therapeutic efficacies mediated by orally delivered MTC nanoparticles were evaluated in lipopolyssachride (LPS)/D-galactosamine (D-Ga1N) induced acute hepatic injury animal models and dextran sulfate sodium (DSS) induced ulcerative colitis mouse models.1Effects of particle size and Zeta potential on cellualr uptake and biodistribution of polymeric nanoparticlesThe effects of particle size and Zeta potential on cellular uptake and biodistribution of polymeric nanoparticles were investigated. Rhodamine B (RhB) labeled carboxymethyl chitosan grafted nanoparticles (RhB-CMCNP) were developed of which the particle size (150,500, and1500nm) and Zeta potential (-15,-25, and-40mV) were precisely controlled and encapsulated with model protein drug as FITC labeled protamine sulfate (FITC-PS), thus obtaining double fluorescence labeled drug loaded polymeric nanoparticles (RhB-CMCNP-PS). The fluoresence of RhB-CMCNP-PS was stable in plasma and aqueous solutions containing I-, which guaranteed the validation for the following quantitative and qualitative assessment. RhB-CMCNP-PS with more negative charges and higher particle sizes was tended to be phagocytosized by murine peritoneal macrophages while RhB-CMCNP-PS with less negative charges and smaller particle sizes was more readily taken up by non-phagocytic cells. The cellular uptake of RhB-CMCNP-PS was cell line dependent. The internalization process of RhB-CMCNP-PS in non-phagocytic cells was energy-dependent and actin-mediated. After intravenous administration of RhB-CMCNP-PS to H-22tumor-bearing Kunming mice, nanoparticles with less negative charges and smaller particle sizes were more efficiently accumulated in the tumor.2Size-dependent absorption mechanisms of polymeric nanoparticles for oral delivery of protein drugsThe effects of particle size on the oral absorption of protein drugs delivered by polymeric nanoparticles were investigated in terms of drug release, cellular uptake, intestinal transport, and biodistribution. Rhodamine B labeled carboxylated chitosan grafted nanoparticles (RhB-CCNP) with different particle sizes (300,600, and1000nm) and similar Zeta potentials (-35mV) were developed. FITC labeled bovine serum albumin (FITC-BSA) was encapsulated into RhB-CCNP to form drug loaded polymeric nanoparticles (RhB-CCNP-BSA). RhB-CCNP-BSA with uniform particle size and similar surface charge possessed desired structural stability and fluorescence stability in plasma and solutions containing various chemical quenchers. RhB-CCNP-BSA with smaller sizes (300nm) demonstrated elevated intestinal absorption, as mechanistically evidenced by higher mucoadhesion in rat ileum, release amount of the payload into the mucus layer, Caco-2cell internalization, uptake and transport across Caco-2cell monolayers and rat ileum, and systemic biodistribution after oral gavage. Peyer’s patches could play a role in the mucoadhesion of nanoparticles, resulting in their close association with the intestinal absorption of nanoparticles. Orally administered RhB-CCNP-BSA(-35,300) could accumulate in the tumor via efficiently intestinal transport followed by translocation to blood and major tissues in H-22tumor bearing mice.3Mannose modified trimethyl chitosan-cystein nanoparticles for oral delivery of siRNA to suppress systemic inflammationChitosan (Mw200kDa and500kDa) was sequentially modified with trimethyl, mannose, and thiol to achieve MTC conjugate, termed as MTC200and MTC500, respectively, of which the mannose modification degrees were15%and11%, respectively. MTC was ionically crosslinked with TPP to yield MTC NPs and encapsulate TNF-a siRNA. MTC NPs possessed particle sizes around150nm and Zeta potential of30mV. MTC NPs could maintain structural stability in physiological environment, thus protecting their payload from degradation by nucleases. MTC NPs enhanced the intestinal permeation and absorption of orally delivered siRNA, thereby transloacting siRNA to the systemic circulation and major tissues via blood circulaton, lymphatic circulation, and macrophage migration. The uptake and cytoplasmic release of siRNA were promoted by MTC NPs, wherein the internalization mechanims involved caveolae-mediated endocytosis and macropinocytosis rather than clathrin-mediated endocytosis, thus avoiding the endolysosomal degradation. MTC NPs mediated highly efficient in vitro RNAi in Raw264.7cells and murine peritoneal exudate macrophage cells (PECs). Serum TNF-a, ALT, AST, and TNF-a mRNA expression in macrophage-enriched organs of LPS/D-GalN induced acute hepatic injury mice were significantly decreased after oral gavage of MTC NPs. Histological assessment of liver tissue slices suggested that the hepatic injury of mice was alleviated and the survival rate of mice was remarakably elevated. Compared to intraperitoneal injection, MTC NPs delivered via oral administration mediated preferable in vivo RNAi in C57BL/6mice which might be due to the higher systemic biodistribution levels of siRNA. Serum TNF-a and TNF-a mRNA expression in macrophage-enriched organs of LPS/D-GalN induced acute hepatic injury rat were significantly decreased after oral gavage of MTC NPs. Histological assessment of liver tissue slices suggested that the hepatic injury of rat was alleviated. In addition, orally administered MTC200NPs induced no toxicity in C57BL/6mice, suggesting their high bioavailability. 4Mannose modified trimethyl chitosan-cystein nanoparticles for oral delivery of siRNA to suppress local ulcerative colitisThrough adjusting the weight ratio of MTC200to TPP, MTC200NPs of150nm and MTC200LNPs of1000nm were prepared through ionic gelation and encapsulated with TNF-a siRNA. The Zeta potential of MTC200NPs and MTC200NPs were30mV. MTC200NPs and MTC200LNPs could maintained the structural stability in physiological environment, and no obvious disparity in siRNA release, uptake in Raw264.7cells, and in vitro gene silencing between these two NPs was observed. However, the intestinal transport and absorption levels of MTC200NPs was significantly higher than MTC200LNPs, as evidenced by the higher systemic biodistribution levels in healthy C57BL/6mice receiving oral administration of MTC200NPs than MTC200LNPs. On the other hand, after oral administration, the distribution percent of siRNA loaded into MTC200LNPs in inflammatory colon site was significantly than MTC200NPs, which might be due to the diminished siRNA transport of MTC200LNPs that promoted the specific accumulation in inflammatory colon tissue and activated macrophage uptake. Orally administered MTC200NPs and MTC200LNPs significantly decreased the colonic TNF-α, colonic MPO, and TNF-α, IL-1β, IL-6, and IFN-γ mRNA expression level in colon of ulcerative colitis mice. The body weight loss had been alleviated. Histological assessment of colon tissue of ulcerative colitis mice suggested the preferable therapeutic effects in local inflammation, where MTC200LNPs outperformed MTC200NPs. Furthermore, orally administered MTC200LNPs induced no toxicity in C57BL/6mice, suggesting their high bioavailability.5siRNA binding-release of nanoparticles correlates to RNAi efficiency in vitro and in vivoSeven kinds of NPs containing TNF-a siRNA based on MTC were prepared, of which the siRNA binding-release balance was tuned through adjusting the kinds and contents of the crosslinkers (sodium tripolyphosphate (TPP), hyaluronic acid (HA, Mw100kDa), and Eudragit S100(ES)) involved. The seven kinds of MTC NPs possessed particle sizes and Zeta potentials ranging from120-225nm and18-37mV, respectively, with narrow size distributution (polydispersity index<0.3). The encapsulation efficiency of MTC NPs was higher than98%. MTC NPs could maintain their structural stability in physiological environment to protect siRNA from nuclease degradation. The different siRNA binding-release balances of MTC NPs were confirmed by gel retardation, siRNA integrity in physiological fluids, minimal heparin sodium concentration for nanoparticle dissociation, and in vitro siRNA release profiles. The7kinds of MTC NPs exhibited no obvious differences in the Raw264.7cell uptake levels and the cytoplasmic dissociation was directly observed. NPs releasing siRNA either too slowly or too rapidly both evoked no RNAi effects in vitro. Efficient in vitro short-term and long-term RNAi required NPs with relatively high release rate and sustained release manner, respectively, which further correlated to in vivo RNAi mediated by orally-given NPs in acute and chronic murine inflammatory models, respectively. Collectively, the present investigation might provide broad insights into the optimization of siRNA carriers in respect to their binding-release balance considering improved RNAi efficacies for different disease types.