Phage Displayed Peptide-conjugated Biodegradable Nanoparticles For Brain Drug Delivery

Alzheimer’s disease (AD) is one of the four common neurodegenerative diseases. About10%of people over65have AD. The harm of this disease is increasingly prominent with the aging of the human society. In addition, AD also presents the youth oriented tendency in recent years. AD has brought a great influence and pressure to the patient and their family and also the whole society. The treatment of AD has become an important problem to be resolved.The neuroprotective effect of many peptide and protein drugs (e.g., nerve growth factor (NGF), brainderived neurotrophic factor, basic fibroblast growth factor (bFGF)) has been reported to bring hope for neurodegenerative diseases such as AD. However, due to the poor stability, enzymatic degradation, short blood half-life in vivo and the presence of the blood-brain barrier (BBB), it’s hard for these protein drugs to be delivered into the brain for the treatment of brain diseases. The brain delivery of these protein drugs is of great value for clinical research and application.Among the strategies to enhance drug delivery across the BBB, the most successful and promising strategy is to deliver drugs into the brain by receptor mediated transcytosis. Motifs like transferrin, insulin or OX26can mediate drug delivery into the brain by interacting with their corresponding receptors on BBB. However, these brain-targeting motifs such as lactoferrin, insulin and OX26are endogenous ligands. To solve the problem of endogenous saturation of these existing brain-targeting motifs, a high-throughput screening method called phage display technology was employed for new brain-targeting motif searching in this study. A novel brain drug targeting system was developed by conjugating the screened phage-displayed peptides (the new brain-targeting motif) with polyethylene glycol-polylactide-polyglycolide (PEG-PLGA) nanoparticles. The protein drugs can be protected from enzymatic degradation when loaded in the PEG-PLGA nanoparticles while the phage-displayed peptide can mediate the drug-loading nanoparticles entrancing the brain thus improve the treatment of brain diseases.The first part of this study described the screening of brain-targeting peptide by phage display technology. Two phage-display peptide libraries were employed. Five monoclonal bacteriophages were obtained from the first library (Ph.D.-C7CTM) after optimization of screening methods and conditions.3out of the5bacteriophage displayed the same sequence:CTSTSAPYC and the repeatability is7.14%. Other two bacteriophages displayed another sequence:CPMKSHTNC at a repeatability of4.76%. The bacteriophage displaying CTSTSAPYC was named as Clone7-19and the sequence it displayed was named as CTY. From a second phage display library (Ph.D.-12TM), a peptide named as TGN (sequence:TGNYKALHPHNG) was obtained at a repeatability of60%. Also, the bacteriophage displaying TGN was named:Clone12-8. Both CTY and TGN showed brain target effect except for the quick excretion of the two pep tides by the kidneys.In order to extend interacting time of the phage-displayed peptides in vivo to better mediate drug delivery into the brain, in the second part of this study, the phage-displayed peptides were conjugated with a long-circulating PEG-PLGA nanoparticle (NP) to construct a novel brain drug delivery system with high efficiency. In the first chapter of part two, the pegylated nanoparticle was made by emulsion and solvent evaporation method and covalently conjugated with TGN via its Maleimide functional group. The optimized preparation conditions of TGN modified NP (TGN-NP) include:total polymer amount is25mg; MePEG-PLGA: Maleimide-PEG-PLGA is4:1(mol:mol); Maleimide:thiol:3:1(mol:mol); the reaction time of Maleimide and thiol is:6-8h. The average particle size of TGN-NP was around89nm, and its Zeta potential was around-24mV. The surface TGN number per nanoparticle was around195. After the entrapment of a near infrared dye DiR, different TGN-NP with different surface motif modification density including: TGN-NP (3:1)(low surface density), TGN-NP (1:1) and CTY-NP (1:1) were administrated into tail vein of nude mice and the brain targeting efficiency in vivo was evaluated by small animals in vivo imaging system. TGN-NP (1:1) showed best brain targeting ability and smaller accumulation in liver and kidney compared with normal nanoparticle. Thus TGN is a promising brain-targeting peptide for brain drug delivery.In the second chapter of part two, the brain drug delivery characteristics of TGN-NP was studied in vitro. Coumarin-6was serving as a nanoparticle probe. Coumarin-6loaded NP and TGN-NP own average particle size of87nm and91nm respectively. The Zeta potential of these two nanoparticles was both around-24mV. The release of coumarin-6from NP and TGN-NP in pH4.0and pH7.4PBS were low enough to ignore which proved that coumarin-6is an ideal nanoparticle probe for nanoparticle behavior tracing in vitro. The uptake of TGN-NP by bEnd.3cells was significantly higher than that of NP at30,60and120min. The cellular uptake of TGN-NP was a time, concentration and temperature dependent process. The uptake inhibition test showed that TGN-NP was uptake by a TGN mediated energy-dependent process involving caveolae and macropinocytosis. CCK-8results demonstrated the safety of NP, TGN-NP and TGN in0.1-10mg/mL at0-1level of RGR. TGN-NP is a good delivery system with low toxicity.In the third chapter of part two, the brain delivery property of TGN-NP was explored in vivo. Measuring method of Coumarin-6in whole blood sample and tissue sample were established and were proved to be specific and reproducible. One hour after coumarin-6loaded TGN-NP injection (a dose of50μg/kg coumarin-6), fluorescent microscopy of brain coronal sections revealed a higher accumulation of TGN-NP in third ventricle region, cerebral cortex region and hippocampus region compared with NP. In pharmacokinetics study, the brain targeting index of TGN-NP (3:1) and TGN-NP (1:1) were2.83and3.78compared with NP respectively. The distribution of NP and TGN-NP were mainly in spleen, liver and lung. Immunostaining of monocyte-macrophage demonstrated that high dose of TGN-NP cannot induce the increase amount of macrophage in cerebrum, cerebellum, heart and lung in Balb/c mice, but only had light and transient toxicity to liver, spleen and kidney. These results proved the brain delivery property and safety of TGN-NP in vivo.In the fourth chapter of part two, TGN-NP was used to deliver a protein drug NAP into the CNS. The NAP-loaded TGN-NP has an average particle size of150nm, Zeta potential of-19mV. The AD model was established by bilateral injection of Aβ1-40into mice hippocampus. The Morris water maze experiment was subsequently conducted to evaluate the effect of different NAP preparation on the mice spatial memory deficits. According to the behavioral research and biochemical indicators results, direct administration of NAP solution intravenously or subcutaneously had no improvement action on Aβ1-40induced AD model mice. The improvement of memory and biochemical index is still limited even the dose was five times higher (10μg/kg) via subcutaneous administration. The entrapment of NAP in the normal PEG-PLGA nanoparticle can only improve the stability of the NAP in vivo and release property but cannot effectively improve the mice memory deficits at the dose of4μg/kg of NAP. Administration of NAP-loaded TGN-NP at the dose of1μg/kg of NAP had shown better improvement than NAP solution intravenous or subcutaneous administration. When the dose of TGN-NP was increased to2μg/kg or4μg/kg, the latency was shortened significantly in behavior test and the acetylcholinesterase and cholineacetyltransferase activity was recovery to normal level compared to sham mice. These results demonstrated that TGN-NP is a promising brain drug delivery system for protein drugs which cannot cross the BBB to play their therapeutic role in the CNS.