Lactoferrin-conjugated Biodegradable Nanoparticles For Brain Drug Delivery

Blood-brain barrier(BBB) protects the internal environment of brain fromoutside toxicities, but meanwhile, it restricts entry of most drugs for brain diseasetherapy. Therefore, the research of enhancement of brain drug delivery throughnoninvasive means, especially development of novel drug delivery system for braindrug targeting, is on focusing in pharmaceutics. Among them, the most successfulstrategy is nanoparticle delivery systems targeting to the brain through receptormediated transcytosis. A murine anti-rat transferrin receptor monoclonal antibody,OX26, was used as brain targeting molecule and coupled with liposome, the formedimmunoliposome was proved delivery successfully small molecule drugs, proteins, aswell as gene therapies into the brain via receptor-mediated transcytosis. Nevertheless,the OX26 is only effective in rats, and it also possesses strong immunogenicity. Inaddition to that, liposome has poor stability both in vitro and in vivo, if itdisintegrated before arriving at the brain microvessels, the brain delivery capacity ofthe system will be largely impaired. In view of these, there is an urgent need toexplore new brain targeting molecule as well as drug loading vehicle to constructmore reasonable and effective brain drug delivery systems.To solve this problem, two novel biodegradable brain drug targeting systems,lactoferrin conjugated polyethylene glycol-polylactide-polyglycolide nanoparticlesystem(Lf-NP_PLGA) and lactoferrin conjugated polyethylene glycol-polylactidenanoparticle system(Lf-NP_PLA) were developed. Lactoferrin is a mammalian cationiciron-binding glycoprotein which has been demonstrated to cross the BBB viareceptor-mediated transcytosis. Markedly high brain uptake was observed forlactoferrin relative to transferrin and OX26 in vivo. The surface modified lactoferrincan enhance the nanoparticles' brain entrance via receptor-mediated transcytosis; thebiodegradable nanoparticle served as drug carrier, which can endow the drug deliverysystem with good safety and increase its drug loading capacity; the double-emulsionand solvent evaporation preparation method facilitated encapsulation ofmacromolecules such as proteins, peptides and genes, and can mask theirphysico-chemical characteristics instead of nanoparticle properties; the stealthy pegylated nanoparticle can avoid the uptake by reticuloendothelial system, prolongtheir plasma half-life, and enhance area under the concentration-time curve(AUC).Parkinson's disease is a devastating degenerative neurological illness affecting1-2% of the 'over 50' population. There are no effective therapies for PD at presentand clinical available treatments are all for symptom relief. Nowadays, thedevelopment of neuroprotective agents throws some new light on PD therapy.Following intracerebral injection, a corticotrophin releasing hormone related peptide,urocortin(UCN) significantly improved the behavior of PD model rats. But thepeptide does not cross the BBB after intravenous injection, which greatly restrains itsutilization. Therefore, we encapsulated it into the novel biodegradable brain drugtargeting system Lf-NP_PLGA, in order to enhance its therapeutic effect for PD.The first part described the research of Lf-NP_PLGA brain delivery system. Thefirst chapter is construction and characterization of Lf-NP_PLGA. The pegylated polylactide-poly glycolide nanoparticle, whose surface was co-modified withmaleimide-polyethylene glycol(MAL-PEG) and methoxy-polyethylene glycol(MPEG), was made by double-emulsion and solvent evaporation method andcovalently conjugated with thiolated lactoferrin(Lf) via its maleimide functionalgroup. The average particle size of Lf-NP_PLGA was below 100 nm, and its zetapotential was around -20 mV. Immuno-gold staining result showed that bioactive Lfwas covalently coupled to the nanoparticle's surface. ELISA detected the surface Lfnumber per nanoparticle was around 42. The optimized preparation conditions are:total polymer amount is 25 mg; 2-IT: Lf when thiolation is 40: 1; and the outer waterphase is 1% sodium cholate solution.In the second chapter of part one, a lipophilic fluorescent dye, coumarin-6,serving as a nanoparticle probe, was incorporated in Lf-NP_PLGA to investigate its braindrug delivery characteristics in a bEnd.3 BBB model with unconjugated nanoparticle(NP_PLGA) as control. The uptake results demonstrated that the uptake amount ofLf-NP_PLGA was significantly higher than that of NP_PLGA at 15, 30 and 60 min. MTTresults illustrated that after incubation with Lf-NP_PLGA, NP_PLGA and Lf respectively,the cell viability was always above 80% even at a relative high concentration of 3mg/mL. The uptake inhibition experiment results showed that Lf-NP_PLGA wastransport into brain by an energy-dependent process involving LfR, caveolae, clathrin,golgi apparatus, lysosome and macropinocytosis. All the in vitro results showed thatLf-NP_PLGA is a good brain delivery system with very low toxicity. Besides, the release of coumarin-6 from Lf-NP_PLGA and NP_PLGA in pH 7.0 and pH 4.0 PBS after 24 h wereall below 3.5% which proved that coumarin-6 is an ideal nanoparticle probe that canindicate accurately the in vitro and in vivo behavior of Lf-NP_PLGA and NP_PLGA.In the third chapter, the brain delivery property of Lf-NP_PLGA was evaluated bypharmacokinetics studies with NP_PLGA as control. The biodistribution result in micerevealed the AUC of Lf-NP_PLGA in cerebrum was 2.49 times of that of NP_PLGA, andthe brain drug targeting index of Lf-NP_PLGA was 2.19 compared with NP_PLGA. The twokinds of nanoparticles were both mainly distributed to the liver and spleen. Whilecompared to NP_PLGA, Lf-NP_PLGA distributed more to heart and spleen but less tokidney. One hour after a dose of 60 mg/kg Lf-NP_PLGA or NP_PLGA injection in micecaudal vein, fluorescent microscopy of brain coronal sections revealed a higheraccumulation of Lf-NP_PLGA in the substantia nigra, striatum and cerebral cortex regionthan that of NP_PLGA. Immunostaining of monocyte-macrophage demonstrated thathigh dose of Lf-NP_PLGA cannot induce the increase amount of macrophage incerebrum, cerebellum, heart and lung in BALB/c mice, only had light toxicity to liver,spleen and kidney, and its acute toxicity was transient. These results proved the braindelivery property and safety of Lf-NP_PLGA in vivo.In the fourth chapter, the urocortin(UCN) peptide was encapsulated inLf-NP_PLGA and NP_PLGA and their pharmacodynamics studies were carried out inunilateral 6-OHDA lesioned acute rat Parkinson's disease(PD) model and chronicrotenone rat PD model with evaluation of behavioral, immunohistochemical andneurotransmitters' changes. The results illustrated that the high dose Lf-NP_PLGA-UCNimproved the behavior of the rats in both PD model with increase of their striatumtyrosine hydroxylase and domapine levels, which is much better than the same dose ofunconjugated NP_PLGA-UCN. The above results proved that Lf-NP_PLGA is an effectivebrain delivery system which is promise in delivery brain active drugs, especiallyprotein and peptides with poor stability and low BBB transport.The second part describes the research of Lf-NP_PLA brain delivery system. In thefirst chapter we constructed and characterized Lf-NP_PLA. The average particle size ofLf-NP_PLA was below 150 nm, and its zeta potential was around -20 mV. X-rayphotoelectron spectroscopy and immuno-gold staining result showed that bioactive Lfwas covalently coupled to the nanoparticle's surface. ELISA detected the surface Lfnumber per nanoparticle constructed with different ratios of MAL-PEG-PLA toMPEG-PLA and Lf-SH to NP_PLA. The optimized preparation conditions are: MAL-PEG-PLA: MPEG-PLA weight ratio is 1: 9; Lf-SH: MAL-PEG-PLA is 2: 3;2-IT: Lf when thiolation is 40: 1; and the reacting time is 9 h.In the second chapter of part two, the brain delivery property of Lf-NP_PLA wascompared with unconjugated nanoparticle(NP_PLA) in a bEnd.3 BBB model withcoumarin-6 incorporated in them as nanoparticle probe. The results showed that thecoumarin-6 release from Lf-NP_PLA and NP_PLA in pH 7.0 and pH 4.0 PBS after 24 hwere all below 6% which proved that coumarin-6 can indicate accurately the in vitroand in vivo behavior of Lf-NP_PLA and NP_PLA. The bEnd.3 cells uptake resultsdemonstrated that the uptake amount of Lf-NP_PLA was significantly higher than that ofNP_PLA at 30, 60 and 120 min. The uptake inhibition test showed that Lf-NP_PLGA wasuptake by an Lf mediated energy-dependent process involving clathrin, golgiapparatus and macropinocytosis. MTT results showed that none of Lf-NP_PLA, NP_PLAor Lf has significant toxicity on the cells. All the in vitro results showed that Lf-NP_PLAcan be delivery into the brain through Lf receptor mediated process and Lf-NP_PLA is agood brain delivery system with low toxicity.In the third chapter of part two, the brain delivery property of Lf-NP_PLA wasevaluated by pharmacokinetics studies with NP_PLA as control. One hour after a dose of60 mg/kg coumarin-6 loaded Lf-NP_PLA or NP_PLA injection in mice caudal vein,fluorescent microscopy of brain coronal sections revealed a higher accumulation ofLf-NP_PLA in third ventricle region, striatum and cerebral cortex region than that ofNP_PLA. The biodistribution result in mice revealed the AUC of Lf-NP_PLA in cerebrumwas 2.98 times of that of NP_PLA, and the brain drug targeting index of Lf-NP_PLA was3.22 compared with NP_PLA. The two kinds of nanoparticles were mainly distributed tothe liver and spleen. While compared to NP_PLA, Lf-NP_PLA distributed more to liver,spleen and kidney. Immunostaining of monocyte-macrophage demonstrated that highdose of Lf-NP_PLA cannot induce the increase amount of macrophage in cerebrum,cerebellum, heart kidney and lung in BALB/c mice, only had light toxicity to liver,and spleen, and its acute toxicity was transient.In the third part, we prepared BSA loaded Lf-NP_PLGA and Lf-NP_PLA, comparedthe releasing and degradation behavior of them as well as its brain delivery properties.The release results showed that, the two kinds of nanoparticles possess similar releasebehavior in PBS at 37℃, which all fit the Weibull distribution, with a 45% release ofBSA during the 15 days period. While in PBS containing 5% plasma, the release ofthe two kinds of nanoparticles was more complete, with an 88% release for Lf-NP_PLGA and 61% release for Lf-NP_PLA. Using Lf as a targeting molecule, bothLf-NP_PLGA and Lf-NP_PLA can enhance brain delivery. Lf-NP_PLA has a better braintargeting property but its uptake by the reticuloendothelial system was moreprominent. In comparison, Lf-NP_PLGA has a moderate brain targeting property withgood safety.