Solanum Tuberosum Lectin-conjugated PLGA Nanoparticles For Nose To Brain Delivery

While enormous progress has been made regarding our understanding of the pathogenic mechanisms of central nervous system (CNS) disorders, there are few effective drugs for treating these diseases. A key obstacle for developing drugs for treating CNS diseases is the blockage of exogenous substances entrance into the brain by the blood-brain barrier (BBB). Many studies have indicated that intranasal administration could offer noninvasive delivery of large molecules to directly enter into the CNS through olfactory pathways or the trigeminal nerve pathway, effectively bypassing the BBB. However, after administrated nasally, the total amount of these macromolecules accessing the brain was reported to be very low, because of the poor capacity of penetration through nasal mucosa, the enzymatic degradation, limited absorption area and the rapid mucociliary clearance. The encapsulation of peptides and proteins into nanometer-sized particles has been shown to protect them from degradation and facilitate their transport across the mucosal barrier. In spite of various advantages, there remains the following "nose-brain barrier" for nanoparticles:first of all, the penetration of nanoparticles through the tight junctions between the nasal epithelium cells is negligible and the endocytosis of the nanoparticles is limited; secondly, the residence time of nanoparticles in nasal cavity is short (within15to20min) because of the mucociliary clearance, which seemed to be not long enough for complete absorption of the formulations.Lectins are proteins or glycoproteins possessing at least one noncatalytic domain which binds reversibly to specific mono-or oligosaccharides. They specifically recognize and bind with carbohydrate residues on cell surface to initiate vesicular transport processes in cells. Thus, a novel Solanum tuberosum L. modified PLGA nanoparticles (STL-NP) were constructed to enhance the nose-to-brain delivery and decrease the potential peripheral toxicity,.In the first part, STL-conjugated PLGA nanoparticles (STL-NPs) were prepared and characterized. Cellular uptake of STL-NP was quantitatively and qualitatively evaluated using Calu-3cell line as an in vitro nasal epithelial model. The ability of STL-NP to mediate drug delivery to the brain after intranasal administration was subsequently evaluated by near infrared fluorescence probe based ex vivo image as well as quantitative determination of the blood and brain tissue concentrations of coumarin-6, a lipophilic fluorescent probe, associated to STL-NP and NP. At last, the safety of STL-NP was investigated by in vitro cytotoxicity and in situ ciliotoxicity experiments.To achieve this goal, maleimide-PEG-PLGA was blended with MePEG-PLGA to prepare nanoparticles by emulsion/solvent evaporation method. The resulting nanoparticles were surface-modified with STL through covalent coupling reaction of maleimide group with thiol group from thiolated STL. The nanoparticles prepared had an average volume weighted diameter of about110nm, and the Zeta potential were about-30mV. The results of X-ray photoelectron spectroscopy (XPS) analysis showed that the content of main elements on the surface of nanoparticles changed, which indicated that STL had been conjugated to the surface of NPs. Haemagglutination test showed that STL, treated through the covalent coupling procedure, still remain their carbohydrates binding bioactivity on the surface of nanoparticles, which could be specifically inhibited by chitin hydrolysates.Uptake of STL-NP by the Calu-3cells was time-, concentration-and concentration-dependent. STL induced strong mucoadhesion on the surface of Calu-3cells, and then triggered or facilitated the internalization of STL-NP. The preincubation with excess chitin hydrolysates heavily suppressed the uptake of STL-NP at37℃, but have little influence on NP uptake, implying that STL-mediated specific adhesion of STL-NP to cell surface glycoproteins was the requisite first step in the cellular uptake. The results of endocytosis inhibition experiment demonstrated that STL-NPs were endocytosed via an energy-dependent process, involving clathrin, caveolae, macropinocytosis, lysosome and golgi apparatus. NP and STL-NP presented no toxicity on Calu-3cells at the concentration below25mg/mL, while showed mild cytotoxicity after24-hour incubation when the concentration increased to50mg/mL. The STL-NP safety experiments showed mild cytotoxicity and negligible cilia irritation. These intriguing in vitro and in vivo results suggest that STL-NP might serve as a promising and safe drug delivery system for intranasal administration.Following intranasal administration, coumarin-6carried by STL-NP was rapidly absorbed into blood and brain. The AUC(0â†'12h) of coumarin-6in blood, olfactory bulb, cerebrum and cerebellum were about0.77-,1.48-,1.89-and1.45-fold of those of NP, respectively (p<0.05). STL-NP demonstrated1.89-2.45times (p<0.01) higher brain targeting efficiency in different brain tissues than unmodified NP. Enhanced accumulation of STL-NP in the brain was also observed by near infrared fluorescence probe image following intranasal administration. The kidney, liver, spleen and lung were the main distribution organs of STL-NP after intranasal administration. The fluorescence signal of STL-NP appeared in olfactory bulb, cerebrum and brainstem early at0.25h. The signal in olfactory bulb decreased gradually after2h, while the obvious signal in brainstem, cerebrum and cerebellum lasted for more than8h, which indicated that the trigeminal nervous transport was more persistent.In the second part, the basic fibroblast growth factor (bFGF) was selected as a model drug and incorporated into the STL-NPs. The in vivo distribution and brain delivery characteristics of bFGF were investigated by isotopic labeling method. The AD rat model was established by bilateral injection of Aβ25-35and Ibotenic acid (IBO) into rat hippocampus. The protective effects of STL-bFGF-NP treatment for spatial memory deficits in AD rats was evaluated by Morris water maze experiment. After a3-week continuously intranasal administration of STL-bFGF-NPs, the influence on the morphology and cells differentiation of the nasal mucosa, as well as main organs (heart, liver, spleen, lung, kidney) were examined to evaluate the short-term toxicity.The bFGF-NPs were prepared by double emulsion/solvent evaporation method, followed by surface modification with STL. The mean size of the resulting nanoparticles was about110nm and the zeta potential was-30mV. The encapsulation efficiency and drug loading capacity of STL-bFGF-NP slightly decreased compared with bFGF-NP. The stability of bFGF-NP in rat plasma and rabbit nasal mucosa tissue homogenate was increased obviously. A slight burst of bFGF from bFGF-NP was observed in both plasma and nasal washing fluid (11.40%and6.32%, respectively) in vitro release experiment, while a sustained release was followed (73.09%and41.91%, respectively) after incubation in plasma and nasal washing fluid for24h.bFGF was radioiodinated by iodogen method to study the in vivo distribution of the drug after absorption as well as its ability to be delivered to the brain. The results showed the AUC0-24h of125I-bFGF incorporated in bFGF-NP in olfactory bulb, cerebrum and cerebellum was1.20-1.55times of that treated with bFGF solution after intranasal administration, and the AUC0-24h of125I-bFGF carried by STL-bFGF-NP was increased to1.61-3.21times of that from bFGF solution. STL-bFGF-NP demonstrated1.33-2.69times and1.18-2.06times higher brain targeting efficiency in different brain tissue than bFGF solution and bFGF-NP. bFGF was distributed mainly in the kidney, liver and spleen absorbed into the circulatory system following intranasal administration, and radioactive counts in these organs were significantly decreased in comparison with the intravenous group. The distribution of125I-bFGF in both liver and spleen from bFGF-NP and STL-bFGF-NP showed no significant increase when compared with bFGF solution.The results of Morris water maze experiment indicated that STL-bFGF-NPs (20μg/kg/d, in) treatment showed more better spatial learning ability than bFGF solution (40μg/kg/d, in and iv) and bFGF-NPs (20μg/kg/d and40μg/kg/d, in) in AD rats. And the STL-bFGF-NPs (20μg/kg/d, in) and bFGF-NPs (40μg/kg/d, in) treatment showed significantly improved ability in both short-term and long-term memory. The decreased activity of AChE and improved level of ChAT in the hippocampus of AD rats could be observed by treatment with bFGF. The ChAT level in rats treated with STL-bFGF-NP (20μg/kg/d, in) was2.04-fold of that in AD model control rats. In combination with the immunohistochemisty results in hippocampal sections, bFGF treatment demonstrated a protective effect on the hippocampal cells in AD rats by decreasing the apoptosis of the nerve cells and lowering the deposition of Aβ plaque. The trophic action of bFGF on the central cholinergic system, including elevating the ChAT activity and maintaining cholinergic nerve function, could weaken the cholinergic system dysfunction and cognitive function impairment caused by AD.The short-term toxicity of STL-bFGF-NP was investigated following repeated intranasal administration for three weeks. No obvious histopathological changes was found in the nasal mucosa as well as the main organs (heart, liver, spleen, lung). For the kidney is the target organ for in vivo toxicity of bFGF, less vacuolar degeneration was observed in renal tubular epithelial cells, compared to that of the intravenous administraton of bFGF solution group. These results indicated that the renal toxicity could be obviously alleviated by intranasal administration.