RVG29Modified Brain-targeted Nanoscale Drug Delivery System

Neurodegenerative diseases are usually characterized by the progressive loss of neurons in brain. The typical clinical symptoms include movement disorder or memory loss. It is mainly due to remarkable loss of neurons or myelin sheath which are in charge of relative functions. Parkinson’s disease (PD) and Alzheimer’s disease (AD) are common neurodegenerative diseases. The main characteristics of neurodegenerative diseases include long course of disease, progressive pathology, nonrenewable neuron death and irreversible disease development. Thus, it is notably important to achieve early diagnosis and therapy of neurodegenerative diseases.In the early stage of the diseases, the change of brain structure and functions is unobservable. As a result, the diagnosis needs to depend on monitoring the change of disease related molecular markers. Recently, fluorescent molecular imaging has been widely applied in diagnosis of cancers. The significant advantage of fluorescence imaging comparing with other strategies is its innately high sensitivity. Therefore, fluorescent molecular imaging is chosen as the diagnostic strategy for neurodegenerative diseases in this study. Gene drugs especially the recently widely used RNA interfering (RNAi) therapy, which shown highly specific and therapeutic effects, hold great promise in further clinical practice. As a result, gene therapy is applied in this study as main therapeutic strategy. Optimizing drug delivery system to achieve successful delivery of diagnostic and therapeutic drugs into the brain is what we focus and commit to.Blood brain barrier (BBB) is the main barrier structure existed between peripheral blood circulation and central nervous system (CNS). The main components of BBB are brain capillary endothelial cells (BCECs) with tightly intercellular conjunctions. The existence of BBB play an importance role in neuron protection, ensure the CNS away from being interfered by extraneous substances, and sustain the inner homeostasis of CNS. The BBB is also in charge of brain nutrition supplement by transporting ingredients from blood to the brain. Meanwhile, the super-compact structure of BBB hinders the successful delivery of diagnostic and therapeutic drugs through non- invasive approaches into the brain.RVG29peptide, a29-amino-acid peptide, which derived from rabies virus glycoprotein, is chosen as brain-targeted ligand modified on drug delivery system. Drug delivery system could get across BBB facilitating specific binding between RVG29peptide and its specific receptor in BBB as well as transcytosis transportation in BCECs after binding. Therefore, brain-targeted drug delivery could be achieved.Besides adopting effective brain-targeted strategy, the selection and optimization of drug delivery vector are also important for achieving successful drug delivery into the brain. Dendrigraft L-lysines (DGLs) are synthetic polymers with branched structure consisted of lysine monomer. The amido linkage within lysine monomers could be easily degraded by enzymes after in vivo administration. DGLs possess low immunogenicity and high biocompatibility due to lysines being one of the main components consisted of protein in vivo. The abundant amino groups on the surface of DGLs offer a lot of reaction sites so that functional groups could be modified on DGLs by covalence linkage. In addition, the rich amino groups could encapsulate negatively charged gene drugs through electrostatic interactions yielding nanoparticles. As a result, DGLs could also be applied as gene vectors. All the above mentioned advantages make DGLs be an ideal drug delivery platform. Thus, DGLs are used in this study as drug delivery vector.In the first chapter, researches are focused on the evaluation of brain-targeted efficiency of RVG29peptide. RVG29peptide is modified on previously well-investigated polyamidoamine dendritic polymers through bi-functionalized polyethyleneplycol (PEG) linker. The resultant brain-targeted drug delivery vector is further complexed with plasmid DNA as model drugs yielding RVG29peptide modified brain-targeted drug delivery system. The emphasis is placed on evaluations of in vitro and in vivo brain-targeted efficiencies. The gene delivery ability into the brain after intravenous administration is investigated intensively.The research in the second chapter is aimed to develop apoptosis detection system at the early stage of Parkinson’s disease. Numerous neuron apoptosis induced by the activation of caspase-3occurs at this stage of PD. To improve imaging contrast, the fluorescence resonance energy transfer (FRET) technology is introduced. The FRET pairs (fluorescent donor and quencher) are modified on DGLs, respectively. The activated caspase-3could recognize and cleave specific peptide sequence---DEVD. The sequence DEVD is used as linker so that the status of FRET effect "ON" or "OFF" could be controlled by the occurrence of activation of caspase-3. The above mentioned elements (DGLs, DEVD and FRET pairs) as well as RVG29brain-targeted peptide are combined into brain-targeted nano-device (DGLs-RVG29-FRET). The brain-targeted nano-device could be applied to in vivo fluorescence imaging in response to the activation of casapase-3in brain. Thereby, early apoptosis diagnosis in PD could be achieve.In the third chapter, DGLs-PEG-RVG29/DNA brain-targeted gene delivery system is constructed. The RVG29modified brain-targeted gene delivery system based on DGLs inherited excellent brain-targeted efficiency of RVG29modified polyamidoamine based system. Meanwhile, the biological safety of the system is highly improved.DGLs-PEG-RVG29/DNA brain-targeted gene delivery system is applied to gene therapy of PD in the fourth chapter. The reduction of caspase-3level in brain is confirmed as therapeutic target. The activation of caspase-3in brain could not only induce neuron apoptosis directly, but also exert neuron cytotoxicity by up-regulating the levels of inflammatory factors in glial cells. In rotenone chronic exposure induced PD model rats, the therapeutic gene drugs which express caspase-3specific RNA interfering sequence are delivered into brain efficiently using the RVG29modified gene delivery system based on DGLs. By a multi-dosing regimen at the early stage of PD by intravenous injection, the level of caspase-3is reduced, while the activation of caspase-3is decreased. The pharmacodynamics is evaluated on PD model rats by behavioral test and immunohistochemical staining with the administration of DGLs-PEG-RVG29loaded therapeutic gene. Based on the cellular apoptosis analysis as well as measuring the inflammatory factor levels, the dual therapeutic mechanism is demonstrated and helps to explain the therapeutic effects.In the last chapter, two typical pathologic features in Alzheimer’s disease are studied as therapeutic targets. One typical pathologic feature is extracellular amyloid Aβ plaque induced by abnormal adjustment of beta-site APP-cleaving enzyme1(BACE1) which is in charge of Aβ formation. The other characteristic of AD pathology is intracellular neurofibrillary tangles (NFTs). The level of BACE1could be regulated by RNAi using gene drugs containing specific RNA interfering sequence. A six-amine-acid peptide with D configuration is developed facilitating computer-based structure design. This D-peptide shows great impact on inhibition of NFTs formation. The branched structure of DGLs offer extra modification sites for D-peptide as a drug after loading therapeutic gene drugs and bearing RVG29brain-targeted modification. Thus, the co-delivery of gene drugs and peptide drugs is accomplished by a uniform drug delivery platform. The therapy of AD can be achieved aiming to interfering two main targets in AD pathology according to multi-dosing regimen. The pharmacodynamics is evaluated on APP/PS1double transgenetic AD model mice by evaluation of the BACE1level and Morris water maze behavioral performance. The therapeutic mechanism is further explored by Aβ plaque and phosphorylated tau protein specific staining.