Chunbai He

Effects of particle size and surface charge on cellular uptake and biodistribution of polymeric nanoparticles.

To elucidate the effects of particle size and surface charge on cellular uptake and biodistribution of polymeric nanoparticles (NPs), rhodamine B (RhB) labeled carboxymethyl chitosan grafted NPs (RhB-CMCNP) and chitosan hydrochloride grafted NPs (RhB-CHNP) were developed as the model negatively and positively charged polymeric NPs, respectively. These NPs owned well defined particle sizes (150-500 nm) and Zeta potentials (-40 mV - +35 mV). FITC labeled protamine sulfate (FITC-PS) loaded RhB-CMCNP and camptothecin (CPT) loaded RhB-CHNP with high encapsulation efficiency were prepared. The fluorescence stability in plasma and towards I(-) was investigated, and the result indicated it was sufficient for qualitative and quantitative analysis. NPs with high surface charge and large particle size were phagocytized more efficiently by murine macrophage. Slight particle size and surface charge differences and different cell lines had significant implications in the cellular uptake of NPs, and various mechanisms were involved in the uptake process. In vivo biodistribution suggested that NPs with slight negative charges and particle size of 150 nm were tended to accumulate in tumor more efficiently. These results could serve as a guideline in the rational design of drug nanocarriers with maximized therapeutic efficacy and predictable in vivo properties, in which the control of particle size and surface charge was of significance.

Drug permeability and mucoadhesion properties of thiolated trimethyl chitosan nanoparticles in oral insulin delivery

Trimethyl chitosan-cysteine conjugate (TMC-Cys) was synthesized in an attempt to combine the mucoadhesion and the permeation enhancing effects of TMC and thiolated polymers related to different mechanisms for oral absorption. TMC-Cys with various molecular weights (30, 200, and 500 kDa) and quaternization degrees (15 and 30%) was allowed to form polyelectrolyte nanoparticles with insulin through self-assembly, which demonstrated particle size of 100-200 nm, zeta potential of +12 to +18 mV, and high encapsulation efficiency. TMC-Cys/insulin nanoparticles (TMC-Cys NP) showed a 2.1-4.7-fold increase in mucoadhesion compared to TMC/insulin nanoparticles (TMC NP), which might be partly attributed to disulfide formation between TMC-Cys and mucin as evidenced by DSC measurement. Compared to insulin solution and TMC NP, TMC-Cys NP induced increased insulin transport through rat intestine by 3.3-11.7 and 1.7-2.6 folds, promoted Caco-2 cell internalization by 7.5-12.7 and 1.7-3.0 folds, and augmented uptake in Peyers patches by 14.7-20.9 and 1.7-5.0 folds, respectively. Such results were further confirmed by in vivo experiment with the optimal TMC-Cys NP. Biocompatibility assessment revealed lack of toxicity of TMC-Cys NP. Therefore, self-assembled nanoparticles between TMC-Cys and protein drugs could be an effective and safe oral delivery system.

Effects of hydrophobic and hydrophilic modifications on gene delivery of amphiphilic chitosan based nanocarriers

The structure-activity relationships between hydrophobic and hydrophilic modification on chitosan and resultant physicochemical properties along with performances in dealing with critical gene delivery barriers were investigated through amphiphilic linoleic acid(LA) and poly (β-malic acid) (PMLA) double grafted chitosan (LMC)/plasmid DNA (pDNA) nanocomplexes. LMC polymers with various LA and PMLA substitution degrees were synthesized and their hydrophilicity/hydrophobicity was characterized. Compared to chitosan, LMC nanoparticles retained the pDNA binding ability at pH 5.5 when they formed nanocomplexes with pDNA encoding enhanced green fluorescence protein (pEGFP) and the resultant complexes showed diameters below 300 nm. Hydrophobic LA and hydrophilic PMLA substitution contributed to suppressed non-specific adsorption, reduced interactions inside LMC/pDNA nanocomplexes, and enhanced pDNA dissociation. However, enzymatic degradation resistance, cell adsorption, and cellular uptake through clathrin-mediated pathway were promoted by hydrophobic LA grafting while being inhibited by hydrophilic PMLA substitution. In vitro transfection assay suggested the optimal LMC/pEGFP nanocomplexes mediated an 8.0-fold improved transfection compared to chitosan/pEGFP nanocomplexes. The 4.2-fold and 2.2-fold higher intramuscular gene expression in mice compared to chitosan/pEGFP and polyethyleneimine (PEI)/pEGFP nanocomplexes further demonstrated the superiority of LMC/pDNA nanocomplexes. Therefore, amphiphilic chitosan derivates with appropriate combination of hydrophobic and hydrophilic modification would be promising gene delivery nanocarriers.

Size-dependent absorption mechanism of polymeric nanoparticles for oral delivery of protein drugs

Polymeric nanoparticles have been widely applied to oral delivery of protein drugs, however, few studies focused on the systematical elucidation of the size-dependent oral absorption mechanism with well-defined polymeric nanoparticles. Rhodamine B labeled carboxylated chitosan grafted nanoparticles (RhB-CCNP) with different particle sizes (300, 600, and 1000 nm) and similar Zeta potentials (-35 mV) 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 in simulated physiological environment to substantially guarantee the validation of elucidation on size-dependent absorption mechanisms of polymeric nanoparticles using in vitro, in situ, and ex vivo models. RhB-CCNP-BSA with smaller sizes (300 nm) demonstrated elevated intestinal absorption, as mechanistically evidenced by higher mucoadhesion in rat ileum, release amount of the payload into the mucus layer, Caco-2 cell internalization, transport across Caco-2 cell monolayers and rat ileum, and systemic biodistribution after oral gavage. Peyers patches could play a role in the mucoadhesion of nanoparticles, resulting in their close association with the intestinal absorption of nanoparticles. These results provided guidelines for the rational design of oral nanocarriers for protein drugs in terms of particle size.

Multifunctional polymeric nanoparticles for oral delivery of TNF-α siRNA to macrophages

Oral delivery of therapeutic siRNA is an appealing strategy for the treatment of many diseases, however poses numerous challenges to escort siRNA from the site of administration to the cytoplasm of the target cells. Mannose-modified trimethyl chitosan-cysteine (MTC) conjugate nanoparticles (NPs) were developed via ionic gelation and performed as highly effective polymeric vehicles for oral delivery of TNF-α siRNA. The chitosan backbone as well as trimethyl, thiol, and mannose groups of MTC NPs could be activated at proper time and location to overcome the extracellular and intracellular barriers to oral siRNA delivery, thereby promoting gene silencing efficiency. MTC NPs effectively improved siRNA integrity in physiological environment, enhanced siRNA permeation across the intestinal epithelium, facilitated siRNA uptake by macrophages through clathrin-independent endocytosis, and promoted cytoplasmic siRNA release. At equivalent TNF-α siRNA dose, MTC NPs notably outperformed Lipofectamine2000 in terms of in vitro knockdown of TNF-α production in macrophages. Orally delivered MTC NPs containing low amount of TNF-α siRNA (3.75 nm/kg) inhibited TNF-α production in macrophages in vivo, which protected mice with acute hepatic injury from inflammation-induced liver damage and lethality. This study could provide broad insights into the rational design of oral siRNA vehicles for the treatment of inflammatory diseases.

Polymer integrity related absorption mechanism of superporous hydrogel containing interpenetrating polymer networks for oral delivery of insulin

Superporous hydrogel containing poly(acrylic acid-co-acrylamide)/O-carboxymethyl chitosan interpenetrating polymer networks (SPH-IPN) was evaluated as the oral delivery vehicle for insulin, emphasizing on the effect of polymer integrity on insulin absorption mechanisms. The integral SPH-IPN (I-SPH-IPN) and powdered SPH-IPN (P-SPH-IPN) exhibited potent and equivalent in vitro enzymatic inhibition capacities, which were attributed to both enzyme incorporation and Ca(2+) deprivation. Nevertheless, I-SPH-IPN showed marked superiority to P-SPH-IPN in in vivo enzymatic inhibition. Through reversible opening of epithelial tight junctions, I-SPH-IPN notably enhanced paracellular permeability of insulin in Caco-2 cell monolayers and excised rat intestine by 4.9 and 4.2 folds, respectively, wherein I-SPH-IPN outperformed P-SPH-IPN by 2.5 and 2.3 folds, respectively. Besides, orally delivered I-SPH-IPN could retain in rat intestine for more than 8 h while P-SPH-IPN was quickly eliminated, suggesting better retentive properties of I-SPH-IPN. Such results were further confirmed by in vivo assessment in that oral administration of insulin-loaded I-SPH-IPN yielded notable insulin absorption and hypoglycemic effect, while P-SPH-IPN was ineffective. Finally, an oral acute and sub-acute toxicity study in mice confirmed biocompatibility of SPH-IPN. Therefore, the detailed mechanism assessment confirmed that I-SPH-IPN was an effective and safe peroral carrier for protein drugs.

Preparation and evaluation of chitosan- ethylenediaminetetraacetic acid hydrogel films for the mucoadhesive transbuccal delivery of insulin

This manuscript describes the development of a new porous, flexible bilaminated film for buccal protein administration by a simple and mild casting procedure. It consists of a mucoadhesive layer (chitosan-ethylenediaminetetraacetic acid hydrogel film) containing protein drugs and an impermeable protective layer made of ethylcellose. The obtained mucoadhesive layer was characterized in terms of Fourier transform infrared spectroscopy, rheology, swelling, and mucoadhesion. Rheology results showed that chitosan-ethylenediaminetetraacetic acid hydrogel (10:2) possessed the greatest degree of viscoelasticity and was well-structured compared with other hydrogels. The in vitro mucoadhesion studies also showed that the mucoadhesive force of the hydrogel remained over 17,000 N/m2 during 4 h in the simulated oral cavity. The insulin loaded bilaminated film showed a pronounced hypoglycemic effect following buccal administration to healthy rats, achieving a 17% pharmacological availability compared with subcutaneous insulin injection. According to these results, the bilaminated film would be a promising delivery carrier for protein drugs via the buccal route.

Ternary Polymeric Nanoparticles for Oral siRNA Delivery

ABSTRACTPurposePoor stability and inefficient absorption in the intestinal tract are major barriers confronting oral delivery of siRNA. We aimed to uncover if ternary polymeric nanoparticles (cationic polymer/siRNA/anionic component) can overcome these obstacles through changing the formulation-related parameters.MethodsTernary polymeric nanoparticles were prepared by ionic gelation of chitosan, N-trimethyl chitosan (TMC), or thiolated trimethyl chitosan (TTMC) with tripolyphosphate (TPP) or hyaluronic acid (HA), and siRNA was simultaneously encapsulated. Structural stabilities and siRNA protection of these nanoparticles were assessed in simulated intestinal milieu. Their transport across ex vivo rat ileum, macrophage uptake, in vitro gene silencing, and in vivo biodistribution after oral administration were investigated.ResultsTernary polymeric nanoparticles formed by TTMC, siRNA, and TPP (TTMC/siRNA/TPP nanoparticles) showed suitable structural stability and siRNA protection in the intestinal tract, good permeability across ex vivo rat ileum, superior cellular uptake and gene silencing efficiency in Raw 264.7 cells, and high systemic biodistribution after oral administration.ConclusionsTTMC/siRNA/TPP nanoparticles demonstrated efficient gene silencing in vitro and systemic biodistribution in vivo, therefore, they were expected to be potential vehicles for oral siRNA delivery.

Optimization of multifunctional chitosan-siRNA nanoparticles for oral delivery applications, targeting TNF-α silencing in rats.

Secretion of tumor necrosis factor-α (TNF-α) by macrophages plays a predominant role in the development and progression of various inflammatory diseases. In the current contribution, multifunctional nanoparticles (NPs) containing TNF-α siRNA targeting macrophages via oral administration were developed to knockdown TNF-α expression against acute hepatic injury in rats. Mannose-modified trimethyl chitosan-cysteine (MTC) NPs were prepared by self-assembly method (sa-MTC NPs), ionic gelation and siRNA entrapment method (en-MTC NPs), and ionic gelation and siRNA adsorption method (ad-MTC NPs). Among them, en-MTC NPs demonstrated the best stability against ionic challenges with desired siRNA integrity against nucleases. By targeting normal enterocytes and M cells that express mannose receptors, en-MTC NPs notably promoted intestinal absorption of siRNA in rats. They further facilitated siRNA internalization by rat peritoneal exudate cells (PECs) via lipid-raft involved endocytosis and macropinocytosis, thus inducing effective in vitro TNF-α knockdown. Orally delivered en-MTC NPs at a low siRNA dose of 50 μg/kg inhibited systemic TNF-α production and decreased TNF-α mRNA levels in macrophage-enriched liver, spleen, and lung tissues, which consequently protected rats from acute hepatic injury. Therefore, the en-MTC NPs would provide an effective approach to orally deliver TNF-α siRNA for the anti-inflammatory therapy.

Trimethyl Chitosan-Cysteine Nanoparticles for Systemic Delivery of TNF-α siRNA via Oral and Intraperitoneal Routes

PurposeThe lack of effective delivery vehicles impedes in vivo applications of siRNA. The trimethyl chitosan-cysteine (TC) nanoparticles (NPs) were developed for in vivo delivery of tumor necrosis factor α (TNF-α) siRNA via oral gavage and intraperitoneal injection.MethodsThe nanoparticles formulated from TC conjugate of 100, 200, and 500xa0kDa were prepared through ionic gelation with sodium tripolyphosphate, termed as TC100 NPs, TC200 NPs, and TC500 NPs, respectively. They were evaluated in terms of stability, siRNA protection, cellular uptake and TNF-α knockdown in peritoneal exudates macrophage cells (PECs), and in vivo TNF-α silencing in acute hepatic injury mice.ResultsTC100 NPs exhibited poor stability in simulated physiological environment compared to TC200 NPs and TC500 NPs. Compared to TC500 NPs, TC200 NPs could significantly enhance in vitro and in vivo cellular uptake by PECs and facilitate cytoplasmic siRNA release, resulting in high in vitro and in vivo TNF-α knockdown. Superior TNF-α suppressing level was obtained with TC200 NPs via oral gavage rather than intraperitoneal injection.ConclusionsThe efficacies of in vivo TNF-α silencing were related to the molecular weight of TC conjugate and the administration route, which would assist in the rational design of siRNA vehicles.