Nanoparticle-mediated brain drug delivery for the treatment of Alzheimer's disease Gyeong Won Lee Supervised by Prof. Seung Yun Yang Functional Soft Nanomaterials Lab.

What is dementia? Dementia? Cause of dementia Caused by brain damage Continuous and overall decline of cognitive function Increased incidence rate due to increasing the aging population Alzheimer’s disease 69 % Vascular dementia 21 % Parkinson’s disease dementia 4% Alcoholism Injury

Alzheimer’s disease (AD) Symptom of AD Cause of AD Difficult to learn new things and to remember new information Normal concentration Neurofibrillary tangles, senile plaques Reduction of acetylcholine function Deposition of the senile plaques mainly composed of amyloid plaque in brain cells Formation of neurofibrillary tangle based on hyperphosphorylated tau protein

Target of AD therapies Acetylcholine Amyloid plaque Tau protein Neurotransmitters secreted from the nerve terminal Decreased acetylcholine secretion causes Alzheimer's disease Deposition of insoluble amyloid plaques on brain cells Necrocytosis of brain cells by deposited insoluble amyloid plaques Microtubule-associated protein in neuron cells Formation of neurofibrillary tangle by hyperphosphorylated tau protein

Blood-brain barrier; gate keeper of brain Blood-brain barrier (BBB) A gateway that limits the transfer of moleculars from blood to brain tissue Interfering with the transport of intercellular molecules by tight junction Permeable material Liposoluble material Non Permeable material Polar material, strong electrolyte, polymer, water soluble molecule The upper limit of pore size in the BBB that enables passive flow of molecules across : <1 nm particles Diameter of several nanometers can also cross the BBB by mediated transport.  Need to develop targeted drug delivery to BBB and AD region for penetrating into BBB and minimizing side effect

Nanoparticles for drug delivery across the BBB Nanoparticles (NPs) Particulate dispersions or solid particles with sizes ranging from 1 to 1,000 nm. Advantage of NPs Permeable into BBB without damaging Sustained release Improving permeability to BBB The transport mechanism of NPs across the BBB An increased retention of the nanoparticles in the brain blood capillaries. Inhibit the efflux system. especially P-glycoprotein A general surfactant effect characterised by the solubilisation of the endothelial cell membrane lipids. Endocytosis by the endothelial cells.

Type of NPs for drug delivery Polymer nanoparticle Thin membrane trapping oil core with drug or polymeric chains trapping drug Liposome Consisting of one or more phospholipid bilayers Around an aqueous compartment that serve as carriers of lipophilic or hydrophilic drugs Solid lipid nanoparticle Solid lipid core matrix with lipophilic molecules or solid liquid trapping oil core with drug 나노파티클 제조 방법 Bruno Fonseca-Santos, et al., Int. J. Nanomedicine, 10, (2015) Bruno Fonseca-Santos, et al., Int. J. Nanomedicine, 10, (2015)

Coated NPs for improving permeability Surface modification Coating with specific material for penetration of BBB Requires low toxicity and biocompatibility Coating material Surfactant Lead to membrane fluidisation and to an enhanced drug permeability across the blood–brain barrier Inhibit the efflux system, especially P-glycoprotein (ex. polysorbate 80) Ligand Targeted drug delivery to disease region Combination with specific receptor on BBB surface A. Zensi, et al., J. Control. Release, 137, (2009) The nanoparticles (dark spheres indicated by arrows) were observed inside the endothelium cells

Improve permeability of water-soluble drug (doxorubicin) Doxorubicin brain concentration after intravenous injection of one of the following preparations. ●, doxorubicin [5 mg/kg] solution in saline ■, doxorubicin [5 mg/kg] solution in saline plus 1% polysorbate 80 ▲, doxorubicin [5 mg/kg] bound to poly(butyl cyanoacrylate) nanoparticles ◇, doxorubicin [5 mg/kg] bound to poly(butyl cyanoacrylate) nanoparticles + 1% polysorbate 80. A.E. Gulyaev, et al., Pharm. Res., 16 (1999) Viability of in vitro cultured bovine brain blood vessel endothelial cells in percent of untreated control cells by the MTT test after addition of poly(butyl cyanoacrylate) nanoparticle (NP) preparations or of polysorbate 80 alone. 100 oP=100 μg/ml NP without polysorbate 80 100 mP=100 μg/ml NP with polysorbate 80 20 oP=20 μg/ml NP without polysorbate 80 20 mP=20 μg/ml NP with polysorbate 80 10 oP=10 μg/ml NP without polysorbate 80 10 mP=10 μg/ml NP with polysorbate 80 P 1000=1000 μg/ml polysorbate 80 P 100=100 μg/ml polysorbate 80 P 10=10 μg/ml polysorbate 80 A.E. Gulyaev, et al., Pharm. Res., 16 (1999)

Ligand-conjugated NPs for Targeted drug delivery Coating with specific ligand which has good affinity with disease region Delivering drug specifically to disease regions Minimizing side effects from taking medication Maximizing drug efficiency Receptor-mediated endocytosis Process by which cells absorb metabolites, hormones, other proteins by the inward budding of plasma membrane vesicles containing proteins with receptor sites specific to the molecules being absorbed

Maleimide groups on NPs can conjugate with thiol groups on ligand Ligand-conjugated NPs for BBB targeting Phage display for find specific ligand Fusion of peace of DNA and bacteriophage coat protein gene for displaying specific peptide on phage surface Selection of phage passed through BBB after injecting the phage displaying the peptide into mouse Ligand-conjugated NPs Select ligand specifically binding to BBB Covalently coupled onto the surface of NPs Increasing selective permeability to BBB Ligand 붙이는 방법 Karsten Ulbrich, et al., Eur. J. Pharm. Biopharm., 72, (2009) Maleimide groups on NPs can conjugate with thiol groups on ligand

Distribution of ligand-conjugated NPs Concentrations-time curves of coumarin 6 in cerebrum In vivo and in vitro test Enhanced brain accumulation efficiency Covalently coupled onto the surface of NPs Lower accumulation in liver and spleen Does not increase cytotoxicity Ligand 붙이는 방법 Jingwei Li, et al., Biomaterials, 32, (2011)  In vitro cytotoxicity In vivo distribution Jingwei Li, et al., Biomaterials, 32, (2011) In vivo distribution of TGN-NP. (I) blank nude mice as blank control. (II) NP; (III) TGN-NP (1:1); (IV) TGN-NP (1:3) Jingwei Li, et al., Biomaterials, 32, (2011)

Targeted drug delivery to AD region Concentration vs. time curves of coumarin-6 in the cerebrum Dual-functional NPs Conjugated with BBB targeting ligand and AD region targeting ligand Direct drug delivery to AD region is possible Minimizing side effects (ex. NGF) 이중 리간드 The ex vivo imaging and fluorescent intensity of the brains from nude mice treated with NP Cerebellum Hippocampus Hippocampus of the AD model mice

Summary Drug delivery using NPs can overcome the limitations of drugs that could not penetrate the BBB, thus broadening the range of AD medication and enabling sustained release drug delivery system NPs coated with a ligand targeting the BBB and AD regions can minimize drug side effects by delivering drugs specifically to the BBB and AD regions NPs conjugated with targeting ligand can be used to treat a wide range of diseases because they can be used for various CNS treatments besides AD therapy.

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