Super-porous Hydrogel Used To Improve Drug Efficacy Of Biological Macromolecules, Nanoparticles Of New Drug Delivery Vectors

With the rapid development of science and technology, multi-interdisciplinary cooperation and technological application have demonstrated an effective artifice in overcoming the difficulties that have long been laid unsolved by an individual subject or technique. The current thesis provides a systemic investigation on the development of novel delivery carriers for significantly improving the effectiveness of protein, peptide, DNA, and siRNA, and elucidation of the underlying mechanisms, taking full advantages of the theories and experimental strategies in biological sciences, material sciences, nano sciences, and pharmaceutical sciences.Protein, peptide and nucleic acid drugs have been exploited which possess potent pharmacological activity as well as high specificity, and have shown great potentials in the treatment of diseases such as cancer, diabetes, and infectious diseases. However, their clinical applications are mostly restricted to injection formulations, which lead to short half-life and undesired patient compliance. When parenterally delivered, especially orally delivered, these hydrophilic biomacromolecules are barely absorbed by the lipophilic biological membranes, and are readily degraded by various enzymes, resulting in poor bioavailability. Therefore, development of drug delivery systems (DDS) is attracting more attention, which aims at improving in vivo drug stability, promoting drug absorption, and enhancing in vivo drug efficacy. Hydrogel delivery carriers are distinguished for its bioadhesion, biocompatibility, biodegradability, and a manner for controlled drug release. Nanoparticulate delivery carriers show distinct beneficial attributes, including solubilization of insoluble drugs, controlled and sustained drug release and targeted drug delivery. The characteristics of delivery systems are largely dependent on the polymeric carriers used.Consequently, multi-functional carriers that exhibited mucoadhesion, enzymatic inhibition, and absorption enhancement would be an efficient strategy in facilitating oral absorption of protein and peptide drugs. In addition, multi-functional non-viral gene carriers to allow overcoming of the extracellular barrier (phagocyte system and nuclease) and intracellular barrier (cell membrane, endosome, lysosome, nucleus membrane) could deliver the genes into target cells and tissues in an efficient, sustained, and targeted manner. The current investigation aims at constructing novel superporous hydrogels containing interpenetrating polymer networks (SPH-IPN), investigating efficacy of SPH-IPN as oral delivery vehicles for protein drugs with insulin as a model drug, and elucidation the absorption mechanisms. Besides, thiolated trimethyl chitosan (TMC-Cys) as a novel multifunctional chitosan derivative was synthesized to combine the advantages of TMC and thiolated polymers. With insulin and pEGFP as model protein drug and model pDNA, TMC-Cys was subjected to formation of TMC-Cys/insulin nanoparticles (TMC-Cys NP) and TMC-Cys/pEGFP nanocomplexes (TMC-Cys NC) through self-assembly, which were evaluated as oral delivery vehicles for protein drugs and gene delivery carriers, respectively. Besides, the mechanisms underlying the drug absorption enhancement and gene transfection elevation were elucidated. TMC-Cys nanocarriers were further modified with mannose, and the resultant mannose-modified TMC-Cys (MTC) nanocarriers were investigated for its active targeting towards intestinal M cells as well as macrophages with TNF-a siRNA as the target gene. Besides, RNAi efficacy was evaluated via oral delivery along with elucidation of the underlying mechanisms.Superporous hydrogels of P(AA-co-AM) was synthesized through solution copolymerization of acrylic acid (AA) and acrylamide (AM) with APS/TEMED as initiators, NaHCO3 as foaming agent, and Bis as cross-linker. O-CMC was allowed to be well distributed in SPH along with the polymerization reaction, and was further cross-linked by glutaraldehyde (GA) after SPH was synthesized, thus achieving the SPH-IPN. The structures of the SPH-IPNs were characterized with FTIR,13C NMR, and DSC. SEM, CLSM and light images revealed that the SPH-IPNs possessed both the IPN network and large numbers of pores with diameters of 100-300μm, and the cross-linked O-CMC molecules were located on the peripheries of these pores. SPH-IPN quickly swelled in water with equilibrium swelling ratio of 30-80, and an increase in O-CMC content, GA amount and cross-linking time led to slower swelling behavior. Due to the cross-linked O-CMC network, compression and tensile modulus of SPH-IPN were greatly improved, and an increase in O-CMC content, GA amount and cross-linking time was favorable for enhanced mechanical strength.Swelling behaviors of SPH-IPN were measured at different pH, ionic strength, and temperature. With insulin as a model drug, loading capacity of the SPH-IPN was determined. Release profiles were also evaluated at different pH, ionic strength, and temperature. Stability of insulin following drug loading and polymer-drug interactions were also investigated. State of water in SPH-IPN and water retention capacities were examined.Swelling of SPH-IPN was sensitive towards of pH, ionic strength, and temperature. An increase in the ionic strength within the range of 0.001-1M yields a significant decrease in the swelling ratio, while it brought about an insignificant difference in the swelling behavior of the polymer when the ionic strength was no higher than 0.001M. A drastic increase in the swelling was observed within the pH range of 3.0-6.2, whereas at pH> 6.2 or pH< 3.0, the change in the swelling behavior was slight. SPH-IPN could rapidly respond to pulsatile alternation of pH values between 1.2 and 7.4, thereby exhibiting fast swelling and de-swelling. An increase in temperature could further facilitate swelling of SPH-IPN. Insulin loading capacity of SPH-IPN was 4%-7%, which was notably higher than CSPH, and an increase in O-CMC content yielded slightly deceased loading capacity. Insulin release also exhibited sensitivity towards pH, ionic strength, and temperature. After drug loading and release, the circular dichroism (CD) spectra revealed that conformation of insulin had no significant alteration and bioactivity of insulin was well preserved according to hypoglycaemic effect in mice. Great discrepancies were noted between the theoretical and experimental loading levels of SPH-IPN for insulin, and fast and complete drug release was observed in pH 7.4 PBS. Besides, FTIR spectra of blank SPH-IPN and washed SPH-IPN after drug release were similar, which suggested strong physical interactions rather than chemical linkage between the polymer and the drug. Freezing water was the majority of the imbibed water in the swollen SPH-IPN, and the ability of SPH-IPN to form hydrogen bonding with water molecules was weakened as the O-CMC content and insulin loading amount increased. SPH-IPN showed desired water retention capacities against compression and exposure at 37℃, which further improved as the amount of O-CMC network increased.Pharmacokinetics and pharmacokinetics were investigated following oral and ileal administration of insulin-loaded SPH-IPN in normal rats, and the hypoglycemic effect of insulin-loaded SPH-IPN was also monitored in diabetic rats following oral delivery. Mucoadhesion, enzymatic inhibition, insulin permeation enhancing effect, and intestinal retention of SPH-IPN were evaluated to provide an insight into the intestinal absorption mechanisms of insulin in the presence of SPH-IPN. Besides, the effect of polymer integrity on the enzymatic inhibition, permeation enhancement, intestinal retention, and oral absorption of insulin was investigated.Oral administration of insulin-loaded integral SPH-IPN (I-SPH-IPN) led to marked insulin absorption and hypoglycemic effect in normal rats with F and PA of 5.0% and 6.3%, respectively. In comparison, minimal insulin absorption and unappreciable hypoglycemic effect were observed following powdered SPH-IPN (P-SPH-IPN) delivery. When ileally administered, both I-SPH-IPN and P-SPH-IPN yielded a faster and more potent insulin absorption and blood glucose depression, with the minimal blood glucose level of 30% of initial values. Notable hypoglycemic effect was also observed following oral administration of insulin-loaded I-SPH-IPN in diabetic rats. SPH-IPN could adhere to the intestinal mucosa through non-specific binding and mechanical fixation, and an increase in O-CMC yielded a higher muadhesion capacity. SPH-IPN exhibited potent enzymatic inhibitory effect towards luminal proteolytic enzymes (trypsin andα-chymotrypsin) via enzyme entrapment and Ca2+ binding. A higher O-CMC content correlated to enhanced Ca2+ binding capacities, which accounted for enhanced enzymatic inhibitory effect. Although I-SPH-IPN and P-SPH-IPN possessed equivalent enzymatic inhibition capacity in vitro, I-SPH-IPN outperformed P-SPH-IPN in protecting insulin from proteolytic hydrolysis under the in vivo conditions, because I-SPH-IPN released more insulin in the mucus layer rather than in the intestinal lumen and thereby prevented degradation by luminally secreted proteases. SPH-IPN facilitated paracellular transport of insulin through reversible opening of epithelial tight junctions as a result of mechanical pressure, and I-SPH-IPN exhibited potent permeation enhancing effect than P-SPH-IPN in that I-SPH-IPN and P-SPH-IPN led to a depression in TEER values of Caco-2 cell monlayers to 25% and 50% of initial values, an increase in FITC-insulin transport in Caco-2 cell monlayers by 4.9 and 1.9 folds, and an enhancement in FITC-insulin transport in excised rat ileum by 4.2 and 1.8 folds, respectively. Through mechanical fixation onto the gut wall, I-SPH-IPN showed a prolonged intestinal retention of more than 8 h, while P-SPH-IPN was cleared from the intestinal within 4 h due to failure in mechanical fixation.Biocompatibility of SPH-IPN was explored at molecular, cellular, tissue, and animal levels in terms of cytotoxicity, genetoxicity, tissue toxicity, oral acute and sub-acute toxicity, and blood compatibility. Residual monomers and cross-linkers in SPH-IPN were quantified by HPLC to provide evidences for biocompatibility assessment.Results of FDA/PI double staining assay, LDH assay, neutral red assay, and protein assay in RBL-2H3 and Caco-2 cells revealed lack of cytotoxicity of SPH-IPN and SPH-IPN extract (10,1,0.1 mg·mL-1) following short-time exposure (24 h), and MTT assay further evidenced that SPH-IPN did not interfere with cell proliferation following long-time treatment (7 d). DNA ladder assay, cytometry assay and comet assay in the above two cell lines and in vivo micronucleus (MN) assay in mice showed that SPH-IPN did not induce cell apoptosis, DNA breakage, and MN formation, suggesting lack of genotoxicity. SPH-IPN did not induce impairment to the intestinal mucous, indicating good tissue compatibility. Oral administration of SPH-IPN extract (1000 mg·kg-1) resulted in unappreciable acute toxicity, and in the 28-day sub-acute toxicity study (500,200,100 mg·kg-1), body weight of I-SPH-IPN extract treated mice gradually increased with no appreciable difference to control animals, weights of mouse livers, kidneys, and spleens were found to be normal, and in the histological examination no necrosis, inflammation, edema or other pathological signs were detected. With regard to the hematological parameters and biochemical assays, no statistically significant difference was observed against control. LPO, GOP, GPT, ACP, and AKLP activities in mouse livers, kidneys, and spleens showed no abnormality, either. Hemolysis ratio of SPH-IPN was lower than 5%, the dynamic blood clotting ratio was lower than silicated glass, and platelet adsorption ratio was low, suggesting its desired anticoagulant capacities. Residual amount of AA, AM and GA in SPH-IPN was quantified to be 1.4±0.6,2.0±0.2, and<0.2 ppm, which was within the safety range.TMC-Cys was synthesized through trimethylation of chitosan and thiolation of TMC, and TMC-Cys NP was prepared using the PEC method. It was thereafter evaluated as an oral delivery vehicle for protein drugs, and the mechanisms of oral absorption of insulin was elucidated.Chitosan (30,200,500 kDa) was allowed to react with CH3I to achieve TMC with quaterization degree (DQ) of 15% and 30%, and TMC was subsequently modified with Cys via amide bond formation between the residual primary amino groups on TMC and carboxyl groups on Cys as mediated by EDAC/NHS. About 400-500 mmol·g-1 of sulphydryl was immobilized on TMC-Cys, with approximately one-third remaining the free thiol groups while two thirds being oxidized to the disulfide. FTIR and 13C NMR confirmed covalent conjugation between TMC and Cys via amide bonding, while DSC and TGA evidenced reduced crystallinity and decreased thermal stability of the polymer following trimethylation and thiolation. TMC-Cys exhibited comparable antimicrobial effect to TMC while stronger scavenging effect against free radicals. TMC-Cys NP was prepared through electrostatic interactions between oppositely charged TMC-Cys and insulin, which demonstrated particle size of 100-170 nm, Zeta potential of+12-+18 mV, and high encapsulation efficiency of 90%. Particle size, Zeta potential, and insulin encapsulation efficiency were largely dependent on pH of the insulin solution, TMC-Cys/insulin weight ratio, and ionic strength. Besides, chitosan Mw, DQ of TMC, ionic strength and ion types of the dissolution medium also exerted appreciable effect on in vitro release profiles of insulin. TMC-Cys NP showed a 2.1-4.7-fold and a 1.5-2.2-fold increase in intestinal mucoadhesion and mucin adsorption compared to TMC NP, and higher Mw or DQ was favorable for mucin adsorption. DSC measurement evidenced disulfide formation between TMC-Cys and mucin. Compared to TMC NP, TMC-Cys NP induced increased insulin transport through rat intestine by 1.7-2.6 folds, promoted Caco-2 cell internalization by 1.7-3.0 folds, and augmented uptake in Peyer's patches by 1.7-5.0 folds, respectively, among which TMC-Cys(200,30) NP exhibited the optimal permeation enhancing effect. Such results were further confirmed by in vivo experiment. MTT assay in Caco-2 cells and LDH assay in rat intestine revealed lack of toxicity of TMC-Cys NP.With pEGFP as model pDNA, TMC-Cys NC was prepared using the PEC method and subjected to measurement of size and Zeta potential, and morphology observation using SEM and AFM. In vitro and in vivo transfection efficiency of TMC-Cys NC were monitored in HEK293 cells and mouse posterior tibialis muscles, respectively, and the transfection mechanisms were elucidated. TMC-Cys showed potent condensation capacity towards pDNA as evidenced by EB exclusion and gel retardation assays, and the resultant TMC-Cys NC demonstrated diameters of 150-400 nm and Zeta potentials of+14-+20 mV. TMC-Cys NC could also prevent degradation of pDNA by nucleases. Cell binding and mucin adsorption of TMC-Cys NC were enhanced 2.4-3.0 and 1.2-1.7 folds, respectively, compared to TMC NC, and cellular uptake of TMC-Cys NC was enhanced 1.4-3.0 fold and 1.6-4.4 fold compared to TMC NC and Lipofectamine2000. Lowering the temperature from 37 to 4℃substantially reduced uptake of TMC-Cys(100,30) NC by approximately 75%, and pretreatment of sodium azide or chlorpromazine yielded a depression in the complex internalization by approximately 30% and 70%, respectively, suggesting that an energy-dependent clathrin-mediated endocytic process was involved in the complex uptake. Comparatively, cytochalasin D and genistein exerted unappreciable effect on the cellular uptake of nanocomplexes, which indicated that complex uptake was not associated with cytoskeleton recognization and the caveolin-mediated pathway. pEGFP was slowly released from TMC-Cys NC at extracellular GSH concentrations, while was quickly released at the intracellular concentration and transported to the nuclei, leading to a 3.7-fold enhancement in nuclear accumulation of pEGFP compared to TMC NC. Besides, the relative amount of nuclear pEGFP increased with incubated time, which reached a plateau of 40% at 4 h. Consequently, TMC-Cys NC showed a 1.4 to 3.2-fold increase in the transfection efficiency in HEK293 cells as compared to TMC NC and the optimal TMC-Cys(100,30) NC showed a 1.5-fold enhancement than Lipofectamine2000. Such results were further confirmed by in vivo transfection with a 2.3-fold and 4.1-fold higher transfection efficiency of TMC-Cys(100,30) NC than TMC(100,30) NC and Lipofectamine2000, respectively.MTC was synthesized and MTC nanoparticles were prepared and characterized. With TNF-a siRNA as a target gene, MTC nanoparticles were evaluated for their capacity of actively targeting towards intestinal M cells and macrophages, improving oral RNAi effect and the underlying mechanisms.MTC with mannose modification degree of about 20% was synthesized through modification of TMC (Mw 200,500 kDa) with mannopyranosylphenylisothiocyanate and subsequent thiolation with cysteine. siRNA loaded MTC nanoparticles were prepared via the entrapment method, adsorption method, and self-assembly method. The nanoparticles were spherical or sub-spherical in shape, demonstrating diameters of 130-230 nm, positive Zeta potentials, and siRNA encapsulation efficiency of 70-80%. Serum stability of siRNA was significantly enhanced following nanoparticle encapsulation. Higher chitosan MW led to larger particle sizes; dilution led to augmentation of particle size and polydispersity of MTC-SA NP, and an increase in ionic strength resulted in notably decreased encapsulation efficiency of ad-MTC-TPP NP and MTC-SA NP. Comparatively, en-MTC-TPP NP was superior in resistance towards ionic strength. As compared to en-TC-TPP NP, en-MTC-TPP NP exhibited significantly higher cell binding capacity towards Caco-2 cells and Raw 264.7 cells, enhanced uptake level in Raw264.7 cells by 2.0-2.4 folds, increased Peyer's patch uptake by 1.9-2.4 folds, augmented Caco-2 cell monolayer transport of 1.2-2.1 folds, and elevated intestinal transport by 6.9-11.0 folds. Besides, transport in co-cultures of Caco-2 cell monolayers was higher than that in mono-cultures, and transport in excised rat intestine with Peyer's patches was higher than that in rat intestine without Peyer's patches, what suggested that mannose modification remarkably promoted active targeting of nanoparticles towards M cells and macrophages and thereby facilitating siRNA uptake and intestinal transport. With TNF-a siRNA as the target gene, en-MTC-TPP NP demonstrated potent silencing capacity in Raw 264.7 cells than en-TC-TPP NP and Lipofectamine2000, wherein en-MTC-TPP NP showed comparable silencing effect to Lipofectamine2000 at a reduced siRNA dose of 200 folds. en-MTC-TPP NP exhibited minimal cytoxicity in Raw264.7 cells and Caco-2 cells, suggesting lack of toxicity of the nanoparticles when exerting potent gene silencing effect. Oral delivery of en-MTC-TPP NP resulted in notable inhition of LPS-induced serum TNF-αproduction.