Construction And Characterization Of Protein-based Nano Delivery Systems For Blood-brain Barrier Penetration

The blood-brain barrier(BBB)stands for the strict barrier between the internal microenvironment and the surrounding brain microvasculature and limits the information exchange in between.Due to the existence of BBB,most of the small molecule and almost all macromolecular potential therapeutics cannot enter the brain,and this is the biggest obstacle for traditional means of chemotherapy for brain and central nervous system related diseases.Beyond that,due to the short in vivo circulation time and fast metabolism of most drug delivery systems,the drug cumulative concentration inside the brain is far from the therapeutic threshold,thus the treatment efficacy is low.In recent years,zwitterionic polymers with long in vivo circulation time are gradually applied in drug delivery systems.Among them,2-methacryloyloxyethyl phosphorylcholine(MPC),a kind of biomaterial with super hydrophilicity,great biocompatibility,high stability and low immune response in vivo has attracted lots of interests.The similar structure with the phospholipid bilayer end group of living cells' biological membranes endows the nano-systems encapsulated with PMPC much better biocompatibility and longer in vivo circulation time,providing an advantageous protection for the drug delivery systems during the BBB crossing process.In this thesis,based on different BBB-permeable shuttles,we have successfully set up a series of drug delivery systems with different structure and function.The nanoparticles possess the unique ability to cross the BBB and further to achieve their goals in the fields of central nervous system targeting,the treatment of brain tumors and Parkinson's disease.This thesis is divided into four parts,and the specific research contents and conclusions are summarized as follows: 1.Construction of BBB-permeable long circulating nano-delivery systemTo date,the effective treatments for central nervous system(CNS)diseases are impeded mostly by the existence of blood-brain barrier(BBB),a highly restrictive barrier separating the circulating blood and the brain parenchyma.The accumulation of therapeutic drugs inside the brain is far from the therapy threshold,which is closely related to the transient circulation time of drugs.In order to further promote the improvement in the treatment of CNS diseases,we built a protein based nano-delivery system encapsulated with a kind of zwitterionic polymer PMPC which possesses a similar structure with the lipids in the outer membrane of living cells to effectively prolong the circulation time of the nanoparticles in vivo.Besides,with the modification of TAT peptide on the surface of the nanoparticles,we can endow the BBB-permeability to the nanoparticles and effectively reduce the pumping out effect of the multi-resistant system on the cell membranes of BBB.Studies on the single cell model,in vitro BBB model and health mice model proved that the long circulating nanoparticles showed excellent cellular internalization ability and the ability to cross the BBB.In general,the surface modification of zwitterionic polymer PMPC changed the circulation mode of the nanoparticles in vivo,provided an effective way of crossing the BBB,and a potential platform for brain targeting drug delivery and the treatment of CNS related diseases.2.Construction of drug loaded nanoparticles for Parkinson's disease treatmentAs a typical pathological physiological character of Parkinson's disease(PD),the abnormal iron accumulation in substantia nigra compacta in the brain can not only lead to the apoptosis of dopaminergic neurons,the increasement of active oxygen and further lead to the loss of body movement control.Iron chelation therapy has been proved to be an effective treatment for PD.However,the treatment effect is greatly hindered by the poor BBB permeability,low affinity with iron and lack of adequate protection during the circulation in blood which can lead to saturation before reaching the lesion sites in brain.In this study,based on our previous work,we designed and constructed iron chelation therapeutic nanoparticles protected by a zwitterionic polymer PMPC to delay the saturation of iron chelators in blood circulation,and TAT served as a shuttle to enhance the BBB permeability for the treatment of PD.With the protection of zwitterionic polymer PMPC over the iron chelators,this new drug delivery system could reverse functional deficits in Parkinsonian mice not only physiologically but also behaviorally.We believe that the protection of zwitterionic polymer for iron chelators can endow the nanoparticles delayed saturation state in the blood circulation,and also provide a great treatment platform for PD phenotype reversal.3.BBB-permeability exploration of sinapic acid extracted from mustardThe past twenty years have witnessed the development of various approached for the treatment of brain diseases,from the invasion of physical methods to the use of delivery shuttles,including chemical delivery system,Trojan horses,peptides mediated delivery system.However,the application of existing shuttles are limited by their poor BBB permeability,lack of brain targeting and direct neurotoxicity caused by their irreversible damage to the normal physiological structure of BBB.Kinds of shuttle molecules extracted from plants provide a great platform to optimize the BBB-permeable delivery systems.In this study,inspired by the rapid brain stimulation after eating mustard,we used the main component of mustard sinapic acid(SA)to improve the cellular internalization and BBB permeability of the modified long circulating nanoparticles.From the studies on single cell model and healthy mice model,we proved that SA can penetrate through the BBB by only transient interference with barely any neurotoxicity.All the experimental results showed that SA is a kind of effective BBB-permeable small molecule and can also deliver different cargoes even nanoparticles into the brain regions.We believe that SA can be used for the construction of various drug delivery systems for the treatment of brain diseases with complete BBB.4.Drug delivery system for glioma treatment based on electrostatic interactionTemozolomide(TMZ)is a kind of effective chemotherapeutic agent for the treatment of glioma.However,due to its poor water solubility and poor aqueous solution stability,TMZ is often orally administered.At the same time,most of polymeric nanoparticles reach the tumor sites by passive enhanced penetration and retention(EPR)effect,and can not effectively or quickly release the loaded drug molecules inside cells,hence,the therapeutic effect is greatly weakened and a lot of side effects can be induced.Consequently,the development of controlled stimuli-responsive drug delivery system which can maintain its stable structure under normal physiological conditions(p H 7.4)and release the drugs in tumor sites(p H 6.0),will be of great significance.Thus,the focus of this chapter is to study how to enhance TMZ target capacity,prolong the circulation time in vivo,improve its water solubility and stability,thus effectively reduce the side effects and lead to an enhanced treatment efficacy.Based on the previous work of building the BBB-permeable nano delivery system modified with SA,we introduce the acrylic acid(AA)monomer to load TMZ through electrostatic interaction between carboxyl groups and TMZ.Besides,with the protection of the super hydrophilic zwitterionic polymer PMPC,the half-life and stability of TMZ under physiological conditions is able to be greatly improved.Therefore,the therapeutic effect can be greatly improved and the side effects on normal tissues and organs can be greatly reduced at the same time.In addition,because of the protonation of TMZ under acid condition to increase the solublility,the electrostatic interaction between AA and TMZ will be weakened and TMZ can then be released.Therefore,the nanoparticles maintain their stability under the neutral condition and release drugs under weak acid condition.The treatment on orthotropic tumor-bearing mice model proved that the drug delivery system can lead to a strong apoptosis on the tumor site,long median survival time of glioma-bearing mice and inhibitory effects on tumor metastasis to a certain extent can be achieved as well.This kind of drug loaded nanoparticles not only possessed great BBB permeability,greatly improved the stability and solubility of TMZ under physiological conditions but also significantly improved its antitumor activity in vivo.We believe that this work can further expand the application field of the BBB-permeable shuttle molecule SA.