Cover Page The handle

Cover Page

The handle

http://hdl.handle.net/1887/136519

holds various files of this Leiden University

dissertation.

Author: Benne, N.

Title: Vaccination and targeted therapy using liposomes : opportunities for treatment of

atherosclerosis and cancer

ϱ

ŽŵƉůĞŵĞŶƚZĞĐĞƉƚŽƌdĂƌŐĞƚĞĚ>ŝƉŽƐŽŵĞƐ

ŶĐĂƉƐƵůĂƟŶŐƚŚĞ>ŝǀĞƌyZĞĐĞƉƚŽƌŐŽŶŝƐƚ

'tϯϵϲϱĐĐƵŵƵůĂƚĞŝŶĂŶĚ^ƚĂďŝůŝnjĞ

ƚŚĞƌŽƐĐůĞƌŽƟĐWůĂƋƵĞƐ

ƵƚŚŽƌƐĂŶĚĂĸůŝĂƟŽŶƐ

5

'ƌĂƉŚŝĐĂůďƐƚƌĂĐƚ

ďƐƚƌĂĐƚ

/ŶƚƌŽĚƵĐƟŽŶ

Atherosclerosis is the predominant underlying pathology of cardiovascular disease and is one of the leading causes of death worldwide1_{. It is characterized by} ĐŚƌŽŶŝĐŝŶŇĂŵŵĂƟŽŶŝŶŵĞĚŝƵŵͲĂŶĚůĂƌŐĞͲƐŝnjĞĚĂƌƚĞƌŝĞƐĐĂƵƐĞĚďLJƚŚĞƐƵďĞŶĚŽƚŚĞůŝĂů ĂĐĐƵŵƵůĂƟŽŶ ŽĨ ŽdžŝĚŝnjĞĚ ůŽǁͲĚĞŶƐŝƚLJ ůŝƉŽƉƌŽƚĞŝŶ ;Ždž>>Ϳ2_{͘dŚŝƐ ĂƩƌĂĐƚƐ ŝŵŵƵŶĞ ĐĞůůƐ͕} ƐƵĐŚ ĂƐ ŵŽŶŽĐLJƚĞƐ͕ ǁŚŝĐŚ ƵƉŽŶ ĚŝīĞƌĞŶƟĂƟŽŶ ŝŶƚŽ ŵĂĐƌŽƉŚĂŐĞƐ ĐůĞĂƌ Ždž>> ǀŝĂ scavenger receptors and can transform into large lipid-laden foam cells3_{. These foam} cells are unable to migrate out of the vessel wall, leading to a build-up at the site of ŝŶŇĂŵŵĂƟŽŶĂŶĚƚŚĞĨŽƌŵĂƟŽŶŽĨĂƚŚĞƌŽƐĐůĞƌŽƟĐƉůĂƋƵĞƐψ_.

5

;ĐŚŽǁͿǁĂƐƌĂƚĂŶĚŵŽƵƐĞŶŽ͘ϯďƌĞĞĚŝŶŐĚŝĞƚ͘tdĐŽŶƚĂŝŶĞĚϬ͘ϮϱǁƚйĐŚŽůĞƐƚĞƌŽůĂŶĚ ϭϱǁƚйĐŽĐŽĂďƵƩĞƌ͘ŶŝŵĂůƐŚĂĚĂĐĐĞƐƐƚŽǁĂƚĞƌĂŶĚĨŽŽĚĂĚůŝďŝƚƵŵ͘ůůĂŶŝŵĂůǁŽƌŬ ǁĂƐƉĞƌĨŽƌŵĞĚŝŶĐŽŵƉůŝĂŶĐĞǁŝƚŚƚŚĞƵƚĐŚŐŽǀĞƌŶŵĞŶƚŐƵŝĚĞůŝŶĞƐĂŶĚƚŚĞŝƌĞĐƟǀĞ ϮϬϭϬͬϲϯͬh ŽĨ ƚŚĞ ƵƌŽƉĞĂŶ WĂƌůŝĂŵĞŶƚ͘ džƉĞƌŝŵĞŶƚƐ ǁĞƌĞ ĂƉƉƌŽǀĞĚ ďLJ ƚŚĞ ƚŚŝĐƐ ŽŵŵŝƩĞĞĨŽƌŶŝŵĂůdžƉĞƌŝŵĞŶƚƐŽĨ>ĞŝĚĞŶhŶŝǀĞƌƐŝƚLJ͘ ^WͲW'ϮϬϬϬͲ>LJƉͲϭƐLJŶƚŚĞƐŝƐ dŚĞ >LJƉͲϭ ƉĞƉƟĚĞ͕ ''E<ZdZ' ǁŝƚŚ LJƐ ƌĞƐŝĚƵĞƐ ƉƌŽƚĞĐƚĞĚ ďLJ ƚŚĞ ŶŽŶͲĂĐŝĚͲůĂďŝůĞ acetamidomethyl group, was synthesized using a Liberty Blue microwave-assisted ƉĞƉƟĚĞ ƐLJŶƚŚĞƐŝnjĞƌ͘ dŚĞ ƐLJŶƚŚĞƐŝƐ ǁĂƐ ƉĞƌĨŽƌŵĞĚ ŽŶ Ă Ϭ͘ϭ ŵŵŽů ƐĐĂůĞ ǁŝƚŚ Ă ůŽǁͲ ůŽĂĚŝŶŐ ;Ϭ͘ϭϴ ŵŵŽůͬŐͿ ƚĞŶƚĂŐĞů ZͲZD ƌĞƐŝŶ͘ ŵŝŶŽ ĂĐŝĚ ĂĐƟǀĂƟŽŶ ǁĂƐ ĂĐŚŝĞǀĞĚ ďLJ ƵƐŝŶŐE͕E͛ͲĚŝŝƐŽƉƌŽƉLJůĐĂƌďŽĚŝŝŵŝĚĞĂƐƚŚĞĂĐƟǀĂƚŽƌĂŶĚKdžLJŵĂĂƐƚŚĞďĂƐĞ͕ǁŚŝůĞ&ŵŽĐͲ ĚĞƉƌŽƚĞĐƟŽŶǁĂƐĂĐŚŝĞǀĞĚǁŝƚŚϮϬйƉŝƉĞƌŝĚŝŶĞŝŶD&͘KŶĐĞƚŚĞƐLJŶƚŚĞƐŝƐǁĂƐĐŽŵƉůĞƚĞ͕ ƚŚĞƌĞƐŝŶǁĂƐƚƌĞĂƚĞĚǁŝƚŚϭϬĞƋƵŝǀĂůĞŶƚƐŽĨŝŽĚŝŶĞĨŽƌŽŶĞŚŽƵƌ͕ƚŽĂĐĐŽŵŵŽĚĂƚĞƚŚĞ ĐŽŶĐŽŵŝƚĂŶƚĚĞƉƌŽƚĞĐƟŽŶĂŶĚĐLJĐůŝnjĂƟŽŶŽĨƚŚĞLJƐƌĞƐŝĚƵĞƐ͘dŚĞƌĞƐŝŶǁĂƐƐƵďƐĞƋƵĞŶƚůLJ ǁĂƐŚĞĚĮǀĞƟŵĞƐǁŝƚŚD&͕ĮǀĞƟŵĞƐǁŝƚŚϮйĂĐĞƟĐĂĐŝĚŝŶD&͕ĮǀĞƟŵĞƐǁŝƚŚD& ĂŶĚĮŶĂůůLJĮǀĞƟŵĞƐǁŝƚŚD͘dŚĞ^WͲW'ϮϬϬϬͲKK,ǁĂƐŵĂŶƵĂůůLJĐŽƵƉůĞĚ͕ŽŶ ƌĞƐŝŶ͕ ƚŽ ƚŚĞ EͲƚĞƌŵŝŶƵƐ ŽĨ ĂŶ ĞdžĐĞƐƐ ŽĨ ƚŚĞ ĐLJĐůŝnjĞĚ >LJƉͲϭ ďLJ ,dh ĂŶĚ /W͘ dŚĞ ĐŽƵƉůŝŶŐƌĞĂĐƟŽŶǁĂƐůĞŌŽǀĞƌŶŝŐŚƚ͕ĂŶĚƚŚĞƌĞƐŝŶǁĂƐƐƵďƐĞƋƵĞŶƚůLJǁĂƐŚĞĚǁŝƚŚD& ĂŶĚ D͘ dŚĞ ^WͲW'ϮϬϬϬͲ>LJƉϭ ǁĂƐ ĐůĞĂǀĞĚ ĨƌŽŵ ƚŚĞ ƌĞƐŝŶ ďLJ ƵƐŝŶŐ Ă ŵŝdžƚƵƌĞ ŽĨ ϵϳйd&ĂŶĚϯйƚƌŝŝƐŽƉƌŽƉLJůƐŝůĂŶĞ͘dŚĞĐůĞĂǀĂŐĞƌĞĂĐƟŽŶǁĂƐůĞŌƚŽƉƌŽĐĞĞĚĨŽƌŽŶĞ ŚŽƵƌ͕ĂŶĚƚŚĞƉĞƉƟĚĞǁĂƐƉƌĞĐŝƉŝƚĂƚĞĚŝŶƚŽŝĐĞͲĐŽůĚĚŝĞƚŚLJůĞƚŚĞƌ͘dŚĞƉƌĞĐŝƉŝƚĂƚĞǁĂƐ ĐŽůůĞĐƚĞĚďLJĐĞŶƚƌŝĨƵŐĂƟŽŶ͘>ĞǀĞůƐŽĨƉƌĞĐŝƉŝƚĂƚĞǁĞƌĞůŽǁ͕ůŝŬĞůLJĚƵĞƚŽƚŚĞŚLJĚƌŽƉŚŽďŝĐ ŶĂƚƵƌĞŽĨƚŚĞůŝƉŽƉĞƉƟĚĞ͕ƚŚĞƌĞĨŽƌĞĂŌĞƌĐĞŶƚƌŝĨƵŐĂƟŽŶ͕ƚŚĞĞƚŚĞƌǁĂƐƐĞƉĂƌĂƚĞĚĨƌŽŵ the precipitate and the ether was evaporated, leaving a white residue. This residue ǁĂƐĐŽŵďŝŶĞĚǁŝƚŚƚŚĞŽƌŝŐŝŶĂůƉƌĞĐŝƉŝƚĂƚĞ͕ĚŝƐƐŽůǀĞĚŝŶĂŵŝdžƚƵƌĞŽĨĂĐĞƚŽŶŝƚƌŝůĞĂŶĚ ǁĂƚĞƌ͕ĂŶĚĚŝĂůLJnjĞĚŝŶĂƐŽůƵƟŽŶŽĨϵϬйǁĂƚĞƌĂŶĚϭϬйĂĐĞƚŽŶŝƚƌŝůĞƚŽƌĞŵŽǀĞƚŚĞĞdžĐĞƐƐ ƵŶĐŽƵƉůĞĚƉĞƉƟĚĞĂŶĚĂŶLJƐĐĂǀĞŶŐĞƌƐŽƌŝŵƉƵƌŝƟĞƐƚŚĂƚƌĞŵĂŝŶĞĚ͘dŚĞƉƌŽĚƵĐƚǁĂƐ ƐƵďƐĞƋƵĞŶƚůLJĨƌĞĞnjĞͲĚƌŝĞĚĂŶĚĂŶĂůLJnjĞĚƵƐŝŶŐD>/ŵĂƐƐƐƉĞĐƚƌŽŵĞƚƌLJƚŽĐŽŶĮƌŵƚŚĞ desired product had been formed.

ĞdžƚƌƵƐŝŽŶ Ăƚ Zd ;>/Wy džƚƌƵĚĞƌ͕ EŽƌƚŚĞƌŶ >ŝƉŝĚƐ /ŶĐ͕͘ ĂŶĂĚĂͿ͘ dŚĞ ůŝƉŽƐŽŵĞ ŵŝdžƚƵƌĞ ǁĂƐƉĂƐƐĞĚĨŽƵƌƟŵĞƐƚŚƌŽƵŐŚƐƚĂĐŬĞĚϰϬϬŶŵĂŶĚϮϬϬŶŵƉŽůLJĐĂƌďŽŶĂƚĞƚƌĂĐŬͲĞƚĐŚĞĚ ƉŽƌĞƐŝnjĞŵĞŵďƌĂŶĞƐĂŶĚĂĨƵƌƚŚĞƌĞŝŐŚƚƟŵĞƐƚŚƌŽƵŐŚĂϱϬŶŵƉŽƌĞƐŝnjĞŵĞŵďƌĂŶĞ͘ dŽƉƌĞƉĂƌĞŇƵŽƌĞƐĐĞŶƚůLJůĂďĞůĞĚůŝƉŽƐŽŵĞƐ͕Ϭ͘ϭŵŽůйŽĨKWǁĂƐƌĞƉůĂĐĞĚǁŝƚŚKWͲ LJϱ͘>ŝƉŽƐŽŵĞƐǁĞƌĞƐƚŽƌĞĚĂƚϰΣĂŶĚƵƐĞĚĨŽƌĨƵƌƚŚĞƌĞdžƉĞƌŝŵĞŶƚƐǁŝƚŚŝŶϭǁĞĞŬ͘ >ŝƉŽƐŽŵĞĐŚĂƌĂĐƚĞƌŝnjĂƟŽŶ dŚĞͲĂǀĞƌĂŐĞĚŝĂŵĞƚĞƌĂŶĚƉŽůLJĚŝƐƉĞƌƐŝƚLJŝŶĚĞdž;W/ͿŽĨƚŚĞůŝƉŽƐŽŵĞƐǁĞƌĞŵĞĂƐƵƌĞĚ ďLJĚLJŶĂŵŝĐůŝŐŚƚƐĐĂƩĞƌŝŶŐ;>^ͿƵƐŝŶŐĂEĂŶŽ^ĞƚĂƐŝnjĞƌ;DĂůǀĞƌŶ>ƚĚ͕͘DĂůǀĞƌŶ͕h<Ϳ͘ ĞƚĂͲƉŽƚĞŶƟĂůŽĨƚŚĞůŝƉŽƐŽŵĞƐǁĂƐĚĞƚĞƌŵŝŶĞĚďLJůĂƐĞƌŽƉƉůĞƌĞůĞĐƚƌŽƉŚŽƌĞƐŝƐǁŝƚŚ ƚŚĞƐĂŵĞŝŶƐƚƌƵŵĞŶƚ͘>ŝƉŽƐŽŵĞƐǁĞƌĞĚŝůƵƚĞĚϭϬϬͲĨŽůĚŝŶWƚŽĂƚŽƚĂůǀŽůƵŵĞŽĨϭŵ> ƉƌŝŽƌƚŽŵĞĂƐƵƌŝŶŐ͘dŽĚĞƚĞƌŵŝŶĞƚŚĞĐŽŶĐĞŶƚƌĂƟŽŶŽĨĞŶĐĂƉƐƵůĂƚĞĚ'tϯϵϲϱĂŶĚ>LJƉͲϭ͕ ƐĂŵƉůĞƐǁĞƌĞĂŶĂůLJnjĞĚďLJƌĞǀĞƌƐĞĚͲƉŚĂƐĞhW>;tĂƚĞƌƐYh/dzhW>͕tĂƚĞƌƐ͕D͕ h^Ϳ͘ϮϬђ>ŽĨƚŚĞůŝƉŽƐŽŵĞĚŝƐƉĞƌƐŝŽŶǁĂƐĚŝƐƐŽůǀĞĚŝŶϭϴϬђ>ŵĞƚŚĂŶŽů͘ϭϬђ>ŽĨƚŚĞ ƐĂŵƉůĞ ǁĂƐ ŝŶũĞĐƚĞĚ ŝŶƚŽ Ă ϭ͘ϳ ђŵ , ϭϴ ĐŽůƵŵŶ ;Ϯ͘ϭ dž ϱϬ ŵŵ͕ tĂƚĞƌƐ Yh/dz hW>͕tĂƚĞƌƐ͕D͕h^Ϳ͘dŚĞĐŽůƵŵŶƚĞŵƉĞƌĂƚƵƌĞĂŶĚƚŚĞƚĞŵƉĞƌĂƚƵƌĞŽĨƚŚĞƐĂŵƉůĞ ǁĞƌĞƐĞƚĂƚϰϬΣĂŶĚϰΣ͕ƌĞƐƉĞĐƟǀĞůLJ͘dŚĞŵŽďŝůĞƉŚĂƐĞƐǁĞƌĞDŝůůŝͲYǁĂƚĞƌǁŝƚŚϬ͘ϭй d& ;ƐŽůǀĞŶƚ Ϳ ĂŶĚ ĂĐĞƚŽŶŝƚƌŝůĞ ǁŝƚŚ Ϭ͘ϭй d& ;ƐŽůǀĞŶƚ Ϳ͘ &Žƌ ĚĞƚĞĐƟŽŶ͕ ƚŚĞ ŵŽďŝůĞ ƉŚĂƐĞƐǁĞƌĞĂƉƉůŝĞĚŝŶĂůŝŶĞĂƌŐƌĂĚŝĞŶƚĨƌŽŵϱйƚŽϵϱйƐŽůǀĞŶƚŽǀĞƌϭϬ͘ϱŵŝŶƵƚĞƐ ĂƚĂŇŽǁƌĂƚĞŽĨϬ͘ϯϳϬŵ>ͬŵŝŶ͘>LJƉͲϭǁĂƐĚĞƚĞĐƚĞĚďLJĂďƐŽƌďĂŶĐĞĂƚϮϮϬŶŵƵƐŝŶŐĂŶ Yh/dzhW>dhsĚĞƚĞĐƚŽƌ;tĂƚĞƌƐYh/dzhW>͕tĂƚĞƌƐ͕D͕h^ͿĂŶĚ'tϯϵϲϱ was detected at 272 nm.

ŽŶĞŵĂƌƌŽǁͲĚĞƌŝǀĞĚŵĂĐƌŽƉŚĂŐĞ;DDͿĂŶĚĨŽĂŵĐĞůůĐƵůƚƵƌĞ

ǁĞƌĞ ĐĞŶƚƌŝĨƵŐĞĚ ;ϭϰϬϬϬ ƌƉŵͿ ƚŽ ƌĞŵŽǀĞ ŝŶƐŽůƵďůĞ ŵĂƚĞƌŝĂů ĂŶĚ ƚƌŝŐůLJĐĞƌŝĚĞƐ ǁĞƌĞ ŵĞĂƐƵƌĞĚďLJĂĐŽůŽƌŝŵĞƚƌŝĐĞŶnjLJŵĂƟĐĂƐƐĂLJ43_{. The Folch method}44_{ǁĂƐƵƐĞĚƚŽĞdžƚƌĂĐƚ} ĐŚŽůĞƐƚĞƌŽů ĨƌŽŵ ůŝǀĞƌ ƐĂŵƉůĞƐ ;ĐĂ͘ ϱϬ ŵŐ ƟƐƐƵĞͿ͘ ŚŽůĞƐƚĞƌŽů ǁĂƐ ƚŚĞŶ ƋƵĂŶƟĮĞĚ ďLJ ƵƐŝŶŐ Ă ĐŽůŽƌŝŵĞƚƌŝĐ ĞŶnjLJŵĂƟĐ ĂƐƐĂLJ43_{. Both triglyceride and cholesterol levels were} ĐŽƌƌĞĐƚĞĚĨŽƌƚŽƚĂůƉƌŽƚĞŝŶĐŽŶĐĞŶƚƌĂƟŽŶ͘WƌŽƚĞŝŶĐŽŶĐĞŶƚƌĂƟŽŶǁĂƐĚĞƚĞƌŵŝŶĞĚǁŝƚŚĂ WŝĞƌĐĞΡWƌŽƚĞŝŶƐƐĂLJ<ŝƚĂĐĐŽƌĚŝŶŐƚŽƚŚĞŵĂŶƵĨĂĐƚƵƌĞƌ͛ƐŝŶƐƚƌƵĐƟŽŶƐ͘EŽŶͲĨĂƐƚĞĚ ƉůĂƐŵĂůĞǀĞůƐŽĨĐŚŽůĞƐƚĞƌŽůĂŶĚƚƌŝŐůLJĐĞƌŝĚĞƐǁĞƌĞŵĞĂƐƵƌĞĚďLJĞŶnjLJŵĂƟĐĐŽůŽƌŝŵĞƚƌŝĐ assays, as previously described by Out et. al.43

^ƚĂƟƐƟĐĂůĂŶĂůLJƐŝƐ

5

ŝƐĐƵƐƐŝŽŶ

ĂŽƌƟĐůĞƐŝŽŶƌĞƚĞŶƟŽŶŽĨ>LJƉͲϭĚĞŶĚƌŝŵĞƌƐϯŚŽƵƌƐĂŌĞƌŝŶƚƌĂǀĞŶŽƵƐŝŶũĞĐƟŽŶŝŶƉŽ -ͬͲ _mice34_{. Remarkably, in our study, the presence of Lyp-1 on the liposomal surface leads} ƚŽĞŶŽƵŐŚƉĂƌƟĐůĞƌĞƚĞŶƟŽŶŝŶƚŚĞĂŽƌƚĂƚŽĂůůŽǁĂĐůĞĂƌƚŚĞƌĂƉĞƵƟĐĞīĞĐƚŽĨĂƌĞůĂƟǀĞůLJ ůŽǁĚŽƐĞŽĨ'tϯϵϲϱ;ĐĂ͘ϲ͘ϱŵŐͬŬŐͬŝŶũĞĐƟŽŶͿ͘&ŽƌƌĞĨĞƌĞŶĐĞ͕ƚŚĞĂĚŵŝŶŝƐƚƌĂƟŽŶŽĨĂ ŚŝŐŚĚŽƐĞŽĨĨƌĞĞ'tϯϵϲϱ;ϭϬŵŐͬŬŐͿŽƌĂůůLJĨŽƌϭϮǁĞĞŬƐƐŝŐŶŝĮĐĂŶƚůLJƌĞĚƵĐĞĚůĞƐŝŽŶ ƐŝnjĞ͕ďƵƚƚŚŝƐĞīĞĐƚǁĂƐĂĐĐŽŵƉĂŶŝĞĚďLJŚŝŐŚĞƌƐĞƌƵŵƚƌŝŐůLJĐĞƌŝĚĞƐůĞǀĞůƐ14_.

ĚƌƵŐĚĞůŝǀĞƌLJ͗ĚǀĂŶƚĂŐĞƐĂŶĚƉƌŽďůĞŵĂƟĐŝƐƐƵĞƐ͘ŝŽƉŽůLJŵĞƌƐϭϬϰ͕ϰϲϮͲϰϳϵ͕ ĚŽŝ͗ϭϬ͘ϭϬϬϮͬďŝƉ͘ϮϮϲϳϴ;ϮϬϭϱͿ͘ ϭϴ zƵ͕ D͘ Ğƚ Ăů͘ dĂƌŐĞƚĞĚ EĂŶŽƚŚĞƌĂƉĞƵƟĐƐ ŶĐĂƉƐƵůĂƟŶŐ >ŝǀĞƌ y ZĞĐĞƉƚŽƌ ŐŽŶŝƐƚ 'tϯϵϲϱ ŶŚĂŶĐĞ ŶƟĂƚŚĞƌŽŐĞŶŝĐ īĞĐƚƐ ǁŝƚŚŽƵƚ ĚǀĞƌƐĞ īĞĐƚƐ ŽŶ ,ĞƉĂƟĐ >ŝƉŝĚ DĞƚĂďŽůŝƐŵ ŝŶ >ĚůƌͲͬͲ DŝĐĞ͘ Ěǀ ,ĞĂůƚŚĐ DĂƚĞƌ͕ ĚŽŝ͗ϭϬ͘ϭϬϬϮͬ ĂĚŚŵ͘ϮϬϭϳϬϬϯϭϯ;ϮϬϭϳͿ͘ ϭϵ <ĞůůLJ͕<͕͘͘EĂŚƌĞŶĚŽƌĨ͕D͕͘zƵ͕͘D͕͘ZĞLJŶŽůĚƐ͕&͘ΘtĞŝƐƐůĞĚĞƌ͕Z͘/ŶsŝǀŽWŚĂŐĞ ŝƐƉůĂLJ^ĞůĞĐƟŽŶzŝĞůĚƐƚŚĞƌŽƐĐůĞƌŽƟĐWůĂƋƵĞdĂƌŐĞƚĞĚWĞƉƟĚĞƐĨŽƌ/ŵĂŐŝŶŐ͘ DŽůĞĐƵůĂƌ/ŵĂŐŝŶŐĂŶĚŝŽůŽŐLJϴ͕ϮϬϭ͕ĚŽŝ͗ϭϬ͘ϭϬϬϳͬƐϭϭϯϬϳͲϬϬϲͲϬϬϰϯͲϲ;ϮϬϬϲͿ͘ ϮϬ WĂƌŬ͕ <͘ Ğƚ Ăů͘  ŶĞǁ ĂƚŚĞƌŽƐĐůĞƌŽƟĐ ůĞƐŝŽŶ ƉƌŽďĞ ďĂƐĞĚ ŽŶ ŚLJĚƌŽƉŚŽďŝĐĂůůLJ ŵŽĚŝĮĞĚ ĐŚŝƚŽƐĂŶ ŶĂŶŽƉĂƌƟĐůĞƐ ĨƵŶĐƟŽŶĂůŝnjĞĚ ďLJ ƚŚĞ ĂƚŚĞƌŽƐĐůĞƌŽƟĐ ƉůĂƋƵĞ ƚĂƌŐĞƚĞĚƉĞƉƟĚĞƐ͘:ŽƵƌŶĂůŽĨŽŶƚƌŽůůĞĚZĞůĞĂƐĞϭϮϴ͕ϮϭϳͲϮϮϯ͕ĚŽŝ͗ŚƩƉƐ͗ͬͬĚŽŝ͘ ŽƌŐͬϭϬ͘ϭϬϭϲͬũ͘ũĐŽŶƌĞů͘ϮϬϬϴ͘Ϭϯ͘Ϭϭϵ;ϮϬϬϴͿ͘ Ϯϭ ĞŽƐĂƌŬĂƌ͕^͘W͘ĞƚĂů͘WŽůLJŵĞƌŝĐƉĂƌƟĐůĞƐĐŽŶũƵŐĂƚĞĚǁŝƚŚĂůŝŐĂŶĚƚŽsDͲϭ ĞdžŚŝďŝƚƐĞůĞĐƟǀĞ͕ĂǀŝĚ͕ĂŶĚĨŽĐĂůĂĚŚĞƐŝŽŶƚŽƐŝƚĞƐŽĨĂƚŚĞƌŽƐĐůĞƌŽƐŝƐ͘ŝŽƚĞĐŚŶŽů ŝŽĞŶŐϭϬϭ͕ϰϬϬͲϰϬϳ͕ĚŽŝ͗ϭϬ͘ϭϬϬϮͬďŝƚ͘Ϯϭϴϴϱ;ϮϬϬϴͿ͘ ϮϮ EĂŵĚĞĞ͕<͘ĞƚĂů͘/ŶǀŝǀŽĞǀĂůƵĂƟŽŶŽĨǀĂƐĐƵůĂƌͲƚĂƌŐĞƚĞĚƐƉŚĞƌŽŝĚĂůŵŝĐƌŽƉĂƌƟĐůĞƐ ĨŽƌŝŵĂŐŝŶŐĂŶĚĚƌƵŐĚĞůŝǀĞƌLJĂƉƉůŝĐĂƟŽŶŝŶĂƚŚĞƌŽƐĐůĞƌŽƐŝƐ͘ƚŚĞƌŽƐĐůĞƌŽƐŝƐϮϯϳ͕ ϮϳϵͲϮϴϲ͕ĚŽŝ͗ϭϬ͘ϭϬϭϲͬũ͘ĂƚŚĞƌŽƐĐůĞƌŽƐŝƐ͘ϮϬϭϰ͘Ϭϵ͘ϬϮϱ;ϮϬϭϰͿ͘ Ϯϯ WĞƚĞƌƐ͕ ͘ Ğƚ Ăů͘ dĂƌŐĞƟŶŐ ĂƚŚĞƌŽƐĐůĞƌŽƐŝƐ ďLJ ƵƐŝŶŐ ŵŽĚƵůĂƌ͕ ŵƵůƟĨƵŶĐƟŽŶĂů ŵŝĐĞůůĞƐ͘WƌŽĐEĂƚůĐĂĚ^Đŝh^ϭϬϲ͕ϵϴϭϱͲϵϴϭϵ͕ĚŽŝ͗ϭϬ͘ϭϬϳϯͬƉŶĂƐ͘ϬϵϬϯϯϲϵϭϬϲ ;ϮϬϬϵͿ͘ Ϯϰ ŽƌŵŽĚĞ͕͘W͘ĞƚĂů͘EĂŶŽĐƌLJƐƚĂůŽƌĞ,ŝŐŚͲĞŶƐŝƚLJ>ŝƉŽƉƌŽƚĞŝŶƐ͗DƵůƟŵŽĚĂůŝƚLJ ŽŶƚƌĂƐƚ ŐĞŶƚ WůĂƞŽƌŵ͘ EĂŶŽ >ĞƩĞƌƐ ϴ͕ ϯϳϭϱͲϯϳϮϯ͕ ĚŽŝ͗ϭϬ͘ϭϬϮϭͬŶůϴϬϭϵϱϴď ;ϮϬϬϴͿ͘

Ϯϱ ŚĂŶŐ͕ y͘ Y͘ Ğƚ Ăů͘ EĂŶŽƉĂƌƟĐůĞƐ ĐŽŶƚĂŝŶŝŶŐ Ă ůŝǀĞƌ y ƌĞĐĞƉƚŽƌ ĂŐŽŶŝƐƚ ŝŶŚŝďŝƚ ŝŶŇĂŵŵĂƟŽŶĂŶĚĂƚŚĞƌŽƐĐůĞƌŽƐŝƐ͘Ěǀ,ĞĂůƚŚĐDĂƚĞƌϰ͕ϮϮϴͲϮϯϲ͕ĚŽŝ͗ϭϬ͘ϭϬϬϮͬ ĂĚŚŵ͘ϮϬϭϰϬϬϯϯϳ;ϮϬϭϱͿ͘ Ϯϲ ŚĂŽƌŝŐĞƚƵ͕^͕͘ZŽĚƌŝŐƵĞnjͲŐƵĂLJŽ͕͕͘^ŽŽĚ͕͘<͕͘>ŽƉĞnjͲĞƌĞƐƚĞŝŶ͕'͘ΘtĂůƚŽŶ͕ ͘>͘ĞůŝǀĞƌLJŽĨŶĞŐĂƟǀĞůLJĐŚĂƌŐĞĚůŝƉŽƐŽŵĞƐŝŶƚŽƚŚĞĂƚŚĞƌŽƐĐůĞƌŽƟĐƉůĂƋƵĞŽĨ ĂƉŽůŝƉŽƉƌŽƚĞŝŶͲĚĞĮĐŝĞŶƚŵŽƵƐĞĂŽƌƟĐƟƐƐƵĞ͘:>ŝƉŽƐŽŵĞZĞƐϮϰ͕ϭϴϮͲϭϵϬ͕ĚŽŝ ͗ϭϬ͘ϯϭϬϵͬϬϴϵϴϮϭϬϰ͘ϮϬϭϯ͘ϴϲϯϮϬϴ;ϮϬϭϰͿ͘

5

ĚŽŝ͗ϭϬ͘ϭϬϯϴͬŶŵϳϮϬ;ϮϬϬϮͿ͘ ϯϮ WĞĞƌƐĐŚŬĞ͕ ͘ /͘ Ğƚ Ăů͘ džƉƌĞƐƐŝŽŶ ŽĨ ŐϭƋͲZͬƉϯϯ ĂŶĚ ŝƚƐ ŵĂũŽƌ ůŝŐĂŶĚƐ ŝŶ ŚƵŵĂŶ ĂƚŚĞƌŽƐĐůĞƌŽƟĐ ůĞƐŝŽŶƐ͘ DŽů /ŵŵƵŶŽů ϰϭ͕ ϳϱϵͲϳϲϲ͕ ĚŽŝ͗ϭϬ͘ϭϬϭϲͬũ͘ ŵŽůŝŵŵ͘ϮϬϬϰ͘Ϭϰ͘ϬϮϬ;ϮϬϬϰͿ͘ ϯϯ hĐŚŝĚĂ͕ D͘ Ğƚ Ăů͘ WƌŽƚĞŝŶ ĐĂŐĞ ŶĂŶŽƉĂƌƟĐůĞƐ ďĞĂƌŝŶŐ ƚŚĞ >LJWͲϭ ƉĞƉƟĚĞ ĨŽƌ ĞŶŚĂŶĐĞĚŝŵĂŐŝŶŐŽĨŵĂĐƌŽƉŚĂŐĞͲƌŝĐŚǀĂƐĐƵůĂƌůĞƐŝŽŶƐ͘^EĂŶŽϱ͕ϮϰϵϯͲϮϱϬϮ͕ ĚŽŝ͗ϭϬ͘ϭϬϮϭͬŶŶϭϬϮϴϲϯLJ;ϮϬϭϭͿ͘ ϯϰ ^ĞŽ͕ :͘ t͘ Ğƚ Ăů͘ ;ϲϰͿƵͲůĂďĞůĞĚ >LJWͲϭͲĚĞŶĚƌŝŵĞƌ ĨŽƌ WdͲd ŝŵĂŐŝŶŐ ŽĨ ĂƚŚĞƌŽƐĐůĞƌŽƟĐ ƉůĂƋƵĞ͘ ŝŽĐŽŶũƵŐ ŚĞŵ Ϯϱ͕ ϮϯϭͲϮϯϵ͕ ĚŽŝ͗ϭϬ͘ϭϬϮϭͬďĐϰϬϬϯϰϳƐ ;ϮϬϭϰͿ͘ ϯϱ ^ŽŶŐ͕E͕͘ŚĂŽ͕>͕͘ŚƵ͕D͘ΘŚĂŽ͕:͘ZĞĐĞŶƚƉƌŽŐƌĞƐƐŝŶ>LJWͲϭͲďĂƐĞĚƐƚƌĂƚĞŐŝĞƐ ĨŽƌƚĂƌŐĞƚĞĚŝŵĂŐŝŶŐĂŶĚƚŚĞƌĂƉLJ͘ƌƵŐĞůŝǀϮϲ͕ϯϲϯͲϯϳϱ͕ĚŽŝ͗ϭϬ͘ϭϬϴϬͬϭϬϳϭϳϱϰ ϰ͘ϮϬϭϵ͘ϭϱϴϳϬϰϳ;ϮϬϭϵͿ͘

ds,͘ϭϬϴ͘ϭϲϵϭϰϰ;ϮϬϬϴͿ͘ ϰϲ ZŽĚƌŝŐƵĞnjͲ&ĞƌŶĂŶĚĞnj͕ ^͘ Ğƚ Ăů͘ WŚŽƐƉŚĂƟĚLJůƐĞƌŝŶĞͲ>ŝƉŽƐŽŵĞƐ WƌŽŵŽƚĞ dŽůĞƌŽŐĞŶŝĐ&ĞĂƚƵƌĞƐŽŶĞŶĚƌŝƟĐĞůůƐŝŶ,ƵŵĂŶdLJƉĞϭŝĂďĞƚĞƐďLJƉŽƉƚŽƟĐ DŝŵŝĐƌLJ͘&ƌŽŶƚ/ŵŵƵŶŽůϵ͕Ϯϱϯ͕ĚŽŝ͗ϭϬ͘ϯϯϴϵͬĮŵŵƵ͘ϮϬϭϴ͘ϬϬϮϱϯ;ϮϬϭϴͿ͘ ϰϳ <ƌŝƐŚŶĂ͕^͘D͘ĞƚĂů͘ŶŝŽŶŝĐŶĂŶŽůŝƉŽƐŽŵĞƐƌĞĚƵĐĞĚĂƚŚĞƌŽƐĐůĞƌŽƐŝƐƉƌŽŐƌĞƐƐŝŽŶ ŝŶ>ŽǁĞŶƐŝƚLJ>ŝƉŽƉƌŽƚĞŝŶZĞĐĞƉƚŽƌ;>>ZͿĚĞĮĐŝĞŶƚŵŝĐĞĨĞĚĂŚŝŐŚĨĂƚĚŝĞƚ͘: ĞůůWŚLJƐŝŽů͕ĚŽŝ͗ϭϬ͘ϭϬϬϮͬũĐƉ͘ϮϲϲϭϬ;ϮϬϭϴͿ͘ ϰϴ ,ŽƐƐĞŝŶŝ͕ ,͘ Ğƚ Ăů͘ WŚŽƐƉŚĂƟĚLJůƐĞƌŝŶĞ ůŝƉŽƐŽŵĞƐ ŵŝŵŝĐ ĂƉŽƉƚŽƟĐ ĐĞůůƐ ƚŽ ĂƩĞŶƵĂƚĞ ĂƚŚĞƌŽƐĐůĞƌŽƐŝƐ ďLJ ĞdžƉĂŶĚŝŶŐ ƉŽůLJƌĞĂĐƟǀĞ /ŐD ƉƌŽĚƵĐŝŶŐ ϭĂ ůLJŵƉŚŽĐLJƚĞƐ͘ĂƌĚŝŽǀĂƐĐZĞƐϭϬϲ͕ϰϰϯͲϰϱϮ͕ĚŽŝ͗ϭϬ͘ϭϬϵϯͬĐǀƌͬĐǀǀϬϯϳ;ϮϬϭϱͿ͘ ϰϵ ĞŶŶĞ͕ E͘ Ğƚ Ăů͘ ŶŝŽŶŝĐ ϭ͕ϮͲĚŝƐƚĞĂƌŽLJůͲƐŶͲŐůLJĐĞƌŽͲϯͲƉŚŽƐƉŚŽŐůLJĐĞƌŽů ;^W'Ϳ ůŝƉŽƐŽŵĞƐŝŶĚƵĐĞĂŶƟŐĞŶͲƐƉĞĐŝĮĐƌĞŐƵůĂƚŽƌLJdĐĞůůƐĂŶĚƉƌĞǀĞŶƚĂƚŚĞƌŽƐĐůĞƌŽƐŝƐ ŝŶ ŵŝĐĞ͘ : ŽŶƚƌŽů ZĞůĞĂƐĞ Ϯϵϭ͕ ϭϯϱͲϭϰϲ͕ ĚŽŝ͗ϭϬ͘ϭϬϭϲͬũ͘ũĐŽŶƌĞů͘ϮϬϭϴ͘ϭϬ͘ϬϮϴ ;ϮϬϭϴͿ͘ ϱϬ WƵũŽůͲƵƚŽŶĞůů͕/͘ĞƚĂů͘hƐĞŽĨĂƵƚŽĂŶƟŐĞŶͲůŽĂĚĞĚƉŚŽƐƉŚĂƟĚLJůƐĞƌŝŶĞͲůŝƉŽƐŽŵĞƐ ƚŽĂƌƌĞƐƚĂƵƚŽŝŵŵƵŶŝƚLJŝŶƚLJƉĞϭĚŝĂďĞƚĞƐ͘W>Ž^KŶĞϭϬ͕ĞϬϭϮϳϬϱϳ͕ĚŽŝ͗ϭϬ͘ϭϯϳϭͬ ũŽƵƌŶĂů͘ƉŽŶĞ͘ϬϭϮϳϬϱϳ;ϮϬϭϱͿ͘