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

ϯ

ƚŽŵŝĐ&ŽƌĐĞDŝĐƌŽƐĐŽƉLJDĞĂƐƵƌĞŵĞŶƚƐ

ŽĨŶŝŽŶŝĐ>ŝƉŽƐŽŵĞƐZĞǀĞĂůƚŚĞīĞĐƚ

ŽĨ>ŝƉŽƐŽŵĂůZŝŐŝĚŝƚLJŽŶŶƟŐĞŶͲ^ƉĞĐŝĮĐ

ZĞŐƵůĂƚŽƌLJdĞůůZĞƐƉŽŶƐĞƐ

ƵƚŚŽƌƐĂŶĚĂĸůŝĂƟŽŶƐ

/ŶƚƌŽĚƵĐƟŽŶ

3

ƉĞƉƟĚĞǁĂƐĞdžƚƌĂĐƚĞĚĨƌŽŵůŝƉŽƐŽŵĞƐƵƐŝŶŐĂŵŽĚŝĮĞĚůŝŐŚͲLJĞƌŵĞƚŚŽĚ͕ĂƐĚĞƐĐƌŝďĞĚ previously29_{͘ƌŝĞŇLJ͕ϭϬϬђ>ŽĨĂƋƵĞŽƵƐůŝƉŽƐŽŵĂůĚŝƐƉĞƌƐŝŽŶŽƌĂŬŶŽǁŶĐŽŶĐĞŶƚƌĂƟŽŶ} ŽĨ ĨƌĞĞ ƉĞƉƟĚĞ ĂƐ ĐŽŶƚƌŽů ǁĂƐ ŵŝdžĞĚ ǁŝƚŚ ϮϱϬ ђ> ŵĞƚŚĂŶŽů ĂŶĚ ϭϮϱ ђ> ĐŚůŽƌŽĨŽƌŵ ĂŶĚǀŽƌƚĞdžĞĚ͘dŚĞŶ͕ϮϱϬђ>ŽĨϬ͘ϭD,ůĂŶĚϭϮϱђ>ĐŚůŽƌŽĨŽƌŵǁĞƌĞĂĚĚĞĚĂŶĚƚŚĞ ŵŝdžƚƵƌĞǁĂƐǀŽƌƚĞdžĞĚĂŶĚƐƵďƐĞƋƵĞŶƚůLJĐĞŶƚƌŝĨƵŐĞĚĨŽƌϱŵŝŶĂƚϱϮϰŐƚŽƐĞƉĂƌĂƚĞƚŚĞ ƚǁŽƉŚĂƐĞƐ͘dŚĞƵƉƉĞƌƉŚĂƐĞǁĂƐĐŽůůĞĐƚĞĚĂŶĚĂŶĂůLJnjĞĚďLJZWͲhW>͘^ĂŵƉůĞŝŶũĞĐƟŽŶƐ ǁĞƌĞϭϬђ>ĂŶĚƚŚĞĐŽůƵŵŶƵƐĞĚǁĂƐĂϭ͘ϳђŵ,ϭϴĐŽůƵŵŶ;Ϯ͘ϭdžϱϬŵŵ͕tĂƚĞƌƐ Yh/dz hW>͕ tĂƚĞƌƐ͕ D͕ h^Ϳ͘ ŽůƵŵŶ ĂŶĚ ƐĂŵƉůĞ ƚĞŵƉĞƌĂƚƵƌĞƐ ǁĞƌĞ ϰϬΣ ĂŶĚ ϰΣ͕ƌĞƐƉĞĐƟǀĞůLJ͘dŚĞŵŽďŝůĞƉŚĂƐĞƐǁĞƌĞDŝůůŝͲYǁĂƚĞƌǁŝƚŚϬ͘ϭйd&;ƐŽůǀĞŶƚͿĂŶĚ ĂĐĞƚŽŶŝƚƌŝůĞǁŝƚŚϬ͘ϭйd&;ƐŽůǀĞŶƚͿ͘&ŽƌƐĞƉĂƌĂƟŽŶ͕ƚŚĞŵŽďŝůĞƉŚĂƐĞƐǁĞƌĞĂƉƉůŝĞĚ ŝŶĂůŝŶĞĂƌŐƌĂĚŝĞŶƚĨƌŽŵϱйƚŽϵϱйƐŽůǀĞŶƚŽǀĞƌϱŵŝŶƵƚĞƐĂƚĂŇŽǁƌĂƚĞŽĨϬ͘ϯϳϬŵ>ͬ ŵŝŶ͘ WĞƉƟĚĞƐ ǁĞƌĞ ĚĞƚĞĐƚĞĚ ďLJ ĂďƐŽƌďĂŶĐĞ Ăƚ Ϯϭϰ Ŷŵ ƵƐŝŶŐ ĂŶ Yh/dz hW> dhs ĚĞƚĞĐƚŽƌ;tĂƚĞƌƐYh/dzhW>͕tĂƚĞƌƐ͕D͕h^Ϳ͘ WƌĞƉĂƌĂƟŽŶŽĨWd^ͲŵŽĚŝĮĞĚƐŝůŝĐŽŶƉůĂƚĞƐ dŽ ĂůůŽǁ ĨŽƌ ŝŵĂŐŝŶŐ ŽĨ ĂŶŝŽŶŝĐ ůŝƉŽƐŽŵĞƐ͕ ;ŶĞŐĂƟǀĞůLJ ĐŚĂƌŐĞĚͿ ƐŝůŝĐŽŶ ƉůĂƚĞƐ ǁĞƌĞ ŵŽĚŝĮĞĚǁŝƚŚWd^͕ƚŽŽďƚĂŝŶĂƉŽƐŝƟǀĞůLJĐŚĂƌŐĞĚƐƵƌĨĂĐĞ͕ĂƐĚĞƐĐƌŝďĞĚƉƌĞǀŝŽƵƐůLJ30_. ƌŝĞŇLJ͕ƚŚĞƉůĂƚĞƐǁĞƌĞǁĂƐŚĞĚǁŝƚŚĂĐĞƚŽŶĞĂŶĚŵĞƚŚĂŶŽůĂŶĚĚƌŝĞĚŝŶĂǀĂĐƵƵŵŽǀĞŶ ;ŝŶĚĞƌ͕'ĞƌŵĂŶLJͿĨŽƌϯϬŵŝŶƵƚĞƐĂƚϱϬΣ͘dŚĞƉůĂƚĞƐǁĞƌĞŝŶĐƵďĂƚĞĚŝŶĂƐŽůƵƟŽŶŽĨ ϯͬϳǀͬǀ,₂O₂ͬ,₂SO₄ĂƚϭϮϬΣƚŽƌĞŵŽǀĞĂŶLJŽƌŐĂŶŝĐĐŽŶƚĂŵŝŶĂŶƚƐĂŶĚƚŽŚLJĚƌŽdžLJůŝnjĞƚŚĞ silicon surface. The plates were then washed with water and dried for 30 minutes in a ǀĂĐƵƵŵŽǀĞŶ͘dŚĞƉůĂƚĞƐǁĞƌĞŝŶĐƵďĂƚĞĚŽǀĞƌŶŝŐŚƚŝŶϮйǀͬǀWd^ŝŶƚŽůƵĞŶĞ͕ǁĂƐŚĞĚ ƚŚŽƌŽƵŐŚůLJǁŝƚŚƚŽůƵĞŶĞƚŽƌĞŵŽǀĞĂŶLJĞdžĐĞƐƐWd^͕ĂŶĚƐƵďƐĞƋƵĞŶƚůLJǁĂƐŚĞĚǁŝƚŚ ŵĞƚŚĂŶŽů͕ĂŶĚĚƌŝĞĚŝŶĂǀĂĐƵƵŵŽǀĞŶĂƚϭϳϱΣ͘>ĂƐƚůLJ͕ĂĐƵƌŝŶŐƐƚĞƉĨŽƌƚŚĞŚLJĚƌŽůLJƐŝƐŽĨ ƌĞƐŝĚƵĂůĞƚŚŽdžLJŐƌŽƵƉƐǁĂƐƉĞƌĨŽƌŵĞĚĂƚϭϮϬΣĨŽƌϯϬŵŝŶƵƚĞƐĨŽůůŽǁĞĚďLJŝŶĐƵďĂƟŽŶ ŝŶDŝůůŝͲYǁĂƚĞƌĂƚϰϬΣĨŽƌϮŚŽƵƌƐĂŌĞƌǁŚŝĐŚƚŚĞƉůĂƚĞƐǁĞƌĞǁĂƐŚĞĚǁŝƚŚŵĞƚŚĂŶŽů ĂŶĚƐƚŽƌĞĚŝŶĂǀĂĐƵƵŵŽǀĞŶĂƚϭϳϱΣƵŶƟůƵƐĞ͘dŚĞƐŵŽŽƚŚŶĞƐƐŽĨƚŚĞWd^ͲŵŽĚŝĮĞĚ ƉůĂƚĞƐǁĂƐĐŽŶĮƌŵĞĚƵƐŝŶŐ&DŝŶY/ŵŽĚĞ͘ƐŚĂƌƉĐĂŶƟůĞǀĞƌ;KůƚĞƐƉĂ͕KƉƵƐ͕ƵůŐĂƌŝĂͿ ǁŝƚŚĂŶŽŵŝŶĂůƐƉƌŝŶŐĐŽŶƐƚĂŶƚŽĨϮEͬŵ͕ĂŶŽŵŝŶĂůƌĞƐŽŶĂŶĐĞĨƌĞƋƵĞŶĐLJŽĨϳϬŬ,njĂŶĚ ĂƟƉƌĂĚŝƵƐŽĨфϳŶŵǁĂƐƵƐĞĚƚŽŝŵĂŐĞƚŚĞƐƵƌĨĂĐĞŽĨƚŚĞƉůĂƚĞƐ͘dŚĞƐĂŵƉůĞƟůƚǁĂƐ ĐŽƌƌĞĐƚĞĚ ƵƐŝŶŐ ƚŚĞ :W< ĂƚĂ WƌŽĐĞƐƐŝŶŐ ƐŽŌǁĂƌĞ ǀϲ͘ϭ͘ϳϵ ŇĂƩĞŶŝŶŐ ĨƵŶĐƟŽŶ͘ /ŵĂŐĞƐ ǁĞƌĞĞdžƚƌĂĐƚĞĚƵƐŝŶŐ'ǁLJĚĚŝŽŶǀϮ͘ϱϬĂŶĚƚŚĞŚĞŝŐŚƚƐŽĨƐƚƌƵĐƚƵƌĞƐŽŶƚŚĞƉůĂƚĞƐǁĞƌĞ ĚĞƚĞƌŵŝŶĞĚ͘dŚĞƌŽƵŐŚŶĞƐƐŽĨƚŚĞƉůĂƚĞƐǁĂƐĞdžƉƌĞƐƐĞĚĂƐƌŽŽƚŵĞĂŶƐƋƵĂƌĞ;ZD^Ϳ31_, and plates used had RMS values between 0.6 and 3 nm, which was deemed to have ŵŝŶŝŵĂůŝŶƚĞƌĨĞƌĞŶĐĞĂŶĚƚŽĨĂǀŽƌĂĚƐŽƌƉƟŽŶŽĨůŝƉŽƐŽŵĞƐ32_. ^ĂŵƉůĞƉƌĞƉĂƌĂƟŽŶĨŽƌ&DŵĞĂƐƵƌĞŵĞŶƚƐ Wd^ͲŵŽĚŝĮĞĚ ƉůĂƚĞƐ ǁĞƌĞ ĂƩĂĐŚĞĚ ƚŽ ŐůĂƐƐ ŵŝĐƌŽƐĐŽƉĞ ƐůŝĚĞƐ ƵƐŝŶŐ ZĞƉƌŽƌƵďďĞƌ® ;dŚŝŶWŽƵƌ<ŝƚ͕&ůĞdžďĂƌDĂĐŚŝŶĞŽƌƉŽƌĂƟŽŶ͕h^Ϳ͘ƐŵĂůůŐůĂƐƐƌŝŶŐǁŝƚŚĂĚŝĂŵĞƚĞƌ ŽĨϭϱŵŵĂŶĚĂŚĞŝŐŚƚŽĨϯŵŵǁĂƐĂƩĂĐŚĞĚŽŶƚŚĞƉůĂƚĞƚŽĨŽƌŵĂƐŵĂůůďĂƐŝŶ͘EĞdžƚ͕ ϮϬϬʅ>ŽĨůŝƉŽƐŽŵĂůĨŽƌŵƵůĂƟŽŶ;ƉĂƌƟĐůĞĐŽŶĐĞŶƚƌĂƟŽŶƐŽĨĂďŽƵƚϭϬ12_ʹϭϬ13_{ƉĂƌƟĐůĞƐͬ} ŵ>͕ĂƐŵĞĂƐƵƌĞĚďLJEdͿǁĂƐĂƉƉůŝĞĚƚŽƚŚĞƉůĂƚĞĂŶĚŝŶĐƵďĂƚĞĚĂƚƌŽŽŵƚĞŵƉĞƌĂƚƵƌĞ ĨŽƌϱŵŝŶ͘dŚĞƉůĂƚĞǁĂƐŐĞŶƚůLJǁĂƐŚĞĚǁŝƚŚWǁŝƚŚŽƵƚĞdžƉŽƐƵƌĞŽĨƚŚĞƉůĂƚĞƚŽĂŝƌƚŽ remove free liposomes.

^ĐĂŶŶŝŶŐĞůĞĐƚƌŽŶŵŝĐƌŽƐĐŽƉLJ;^DͿŽĨĐĂŶƟůĞǀĞƌƐ

3

ďLJ^DƵƐŝŶŐĂŶ&/EŽǀĂŶĂŶŽ^DϮϬϬ͘/ŵĂŐŝŶŐǁĂƐƉĞƌĨŽƌŵĞĚŝŶŚŝŐŚǀĂĐƵƵŵŵŽĚĞĂƚ ϭϱŬsĂŶĚĂƐƉŽƚŽĨϰ͘ϬĂƚĂƟůƚŽĨϰϱΣ͘/ŵĂŐĞƐǁĞƌĞĐĂƉƚƵƌĞĚĂƚϮϬϬ͕ϬϬϬͲĨŽůĚŵĂŐŶŝĮĐĂƟŽŶ ĂŶĚƚŚĞƌĂĚŝƵƐŽĨƚŚĞĐĂŶƟůĞǀĞƌƟƉǁĂƐĚĞƚĞƌŵŝŶĞĚƵƐŝŶŐƚŚĞ^DƐŽŌǁĂƌĞ͘ &DŵĞĂƐƵƌĞŵĞŶƚƐ dŽŝŵĂŐĞƚŚĞůŝƉŽƐŽŵĞƐĂ:W<EĂŶŽǁŝnjĂƌĚϯ;:W</ŶƐƚƌƵŵĞŶƚƐ'͕ĞƌůŝŶ͕'ĞƌŵĂŶLJͿǁĂƐ ƵƐĞĚ͘ DĞĂƐƵƌĞŵĞŶƚƐ ǁĞƌĞ ƉĞƌĨŽƌŵĞĚ Ăƚ Ϯϱ ц ϭΣ ŝŶ Y/ ŵŽĚĞ͘ dŚĞ &D ƉƌŽďĞ ƵƐĞĚ ǁĂƐĂdĞĂŵEĂŶŽƚĞĐ;'ĞƌŵĂŶLJͿ,^ͲϮϬŚĞŵŝƐƉŚĞƌŝĐĂůĐŽŶĞͲƐŚĂƉĞĚƟƉĐĂŶƟůĞǀĞƌǁŝƚŚ ŐŽůĚͲƌĞŇĞĐƟǀĞĐŽĂƟŶŐĂŶĚŵĞƚĂůĐĂƌďŝĚĞĐŽĂƟŶŐŽŶƚŚĞƟƉƐŝĚĞ͕ǁŝƚŚĂŶŽŵŝŶĂůƐƉƌŝŶŐ ĐŽŶƐƚĂŶƚŽĨϬ͘ϮEͬŵ͕ĂŶŽŵŝŶĂůƌĞƐŽŶĂŶĐĞĨƌĞƋƵĞŶĐLJŽĨϭϱŬ,njĂŶĚĂƟƉƌĂĚŝƵƐŽĨϯϱŶŵ͕ ĂƐ ĚĞƚĞƌŵŝŶĞĚ ďLJ ^D͘ dŚĞ ĐĂŶƟůĞǀĞƌ ǁĂƐ ĐĂůŝďƌĂƚĞĚ ƵƐŝŶŐ ĐŽŶƚĂĐƚͲďĂƐĞĚ ĐĂůŝďƌĂƟŽŶ ĂŶĚƚŚĞƌŵĂůƚƵŶŝŶŐ͘&ŽƌĐĂůŝďƌĂƟŽŶ͕ƚŚĞƐĞƚƉŽŝŶƚǁĂƐϬ͘ϭsŽŶĂƉƉƌŽĂĐŚǁŝƚŚĂĚLJŶĂŵŝĐ baseline. Imaging of the liposomes was performed with a z-length 200 nm and a set ƉŽŝŶƚďĞƚǁĞĞŶϬ͘ϱʹϬ͘ϴϱŶE͘dŚĞĂƌĞĂŽĨƚŚĞŝŵĂŐĞǁĂƐϮdžϮђŵĂƚĂƌĞƐŽůƵƟŽŶŽĨ ϭϮϴdžϭϮϴƉŝdžĞůƐǁŝƚŚĂƉŝdžĞůƌĂƚĞŽĨϮϬŵƐĞĐͬƉŝdžĞů͘^ĐĂŶůŝŶĞĂƌƟĨĂĐƚƐǁĞƌĞƌĞŵŽǀĞĚ͕ ůŝŶĞĮƫŶŐǁĂƐĂƉƉůŝĞĚƚŽƚŚĞŝŵĂŐĞƐ͕ĂŶĚƐĂŵƉůĞƟůƚǁĂƐĐŽƌƌĞĐƚĞĚ͘>ŝƉŽƐŽŵĞƐǁŝƚŚĂ ŚĞŝŐŚƚďĞůŽǁϰϬŶŵǁĞƌĞĞdžĐůƵĚĞĚĨƌŽŵƚŚĞĂŶĂůLJƐŝƐƚŽƉƌĞǀĞŶƚŝŶƚĞƌĨĞƌĞŶĐĞĨƌŽŵƚŚĞ ƐƵďƐƚƌĂƚĞ͘&ŽƌĐĞǀƐ͘ŚĞŝŐŚƚĐƵƌǀĞƐǁĞƌĞŵĞĂƐƵƌĞĚĨŽƌĞĂĐŚƉŝdžĞůŽĨƚŚĞ&DŝŵĂŐĞ͘ŌĞƌ ƐƵďƚƌĂĐƟŶŐƚŚĞĐĂŶƟůĞǀĞƌĚĞŇĞĐƟŽŶ͕ƚŚĞĨŽƌĐĞǀƐ͘ƟƉͲƐĂŵƉůĞƐĞƉĂƌĂƟŽŶ;ŽƌĚŝƐƚĂŶĐĞͿ ĐƵƌǀĞƐǁĞƌĞĮƩĞĚƵƐŝŶŐƚŚĞ,Ğƌƚnjͬ^ŶĞĚĚŽŶŵŽĚĞů33_{ĨŽƌĂŚĞŵŝƐƉŚĞƌŝĐĂůĐĂŶƟůĞǀĞƌƟƉ} ĂĐĐŽƌĚŝŶŐƚŽƚŚĞĨŽůůŽǁŝŶŐĞƋƵĂƟŽŶ͗ ǁŚĞƌĞ&ŝƐƚŚĞĨŽƌĐĞĂƐŵĞĂƐƵƌĞĚďLJ&D͕ZŝƐƚŚĞƟƉƌĂĚŝƵƐĂƐŵĞĂƐƵƌĞĚďLJ^D͕ŝƐ ƚŚĞzD͕ʆŝƐWŽŝƐƐŽŶ͛ƐƌĂƟŽ͕ƐĞƚƚŽϬ͘ϱĨŽƌƐŽŌŵĂƚĞƌŝĂůƐƐƵĐŚĂƐůŝƉŽƐŽŵĞƐ35_{͕ĂŶĚɷŝƐ} ƚŚĞŝŶĚĞŶƚĂƟŽŶŽĨƚŚĞƐĂŵƉůĞ͕ĂůƐŽŵĞĂƐƵƌĞĚďLJ&D͘dŚĞzDǁĂƐĚĞƚĞƌŵŝŶĞĚĨŽƌƚŚĞ center of the liposomes.

ůŝƉŽƐŽŵĞƐŽƌŵĞĚŝƵŵͿǁĞƌĞĂĚĚĞĚĂƚĂĐŽŶĐĞŶƚƌĂƟŽŶŽĨϮϬђŐͬŵ>ůŝƉŝĚƐŝŶƐƵƉƉůĞŵĞŶƚĞĚ /DD͘ dŽ ŵĞĂƐƵƌĞ Ks₃₂₃ ĂƐƐŽĐŝĂƟŽŶ ƚŽ Ɛ͕ ůŝƉŽƐŽŵĞƐ ĞŶĐĂƉƐƵůĂƟŶŐ ŇƵŽƌĞƐĐĞŶƚůLJ ůĂďĞůĞĚKs₃₂₃ ǁĞƌĞĂĚĚĞĚƚŽĂĐŽŶĐĞŶƚƌĂƟŽŶŽĨϬ͘ϭђŐͬŵ>Ks₃₂₃͘ĞůůƐǁĞƌĞŝŶĐƵďĂƚĞĚ ĨŽƌ ϰ ŚŽƵƌƐ Ăƚ ϯϳȗ ĂŶĚ ϱй K₂͘ ^ƵďƐĞƋƵĞŶƚůLJ͕ ĞdžĐĞƐƐ ůŝƉŽƐŽŵĞƐ ǁĞƌĞ ƌĞŵŽǀĞĚ ďLJ ǁĂƐŚŝŶŐƚŚĞĐĞůůƐƐĞǀĞƌĂůƟŵĞƐǁŝƚŚĐ/DD͕ĂŶĚĐĞůůƐǁĞƌĞŝŶĐƵďĂƚĞĚŽǀĞƌŶŝŐŚƚ͘ĞůůƐ ǁĞƌĞƐƚĂŝŶĞĚǁŝƚŚĂŇƵŽƌĞƐĐĞŶƚĂŶƟďŽĚLJĂŐĂŝŶƐƚϭϭĐͲWĞƌWͲLJϱ͘ϱ;EϰϭϴͿĂŶĚĮdžĂďůĞ ǀŝĂďŝůŝƚLJĚLJĞͲWͲĞ&ůƵŽƌϳϴϬĂŶĚƐƵďƐĞƋƵĞŶƚůLJĂŶĂůLJnjĞĚďLJŇŽǁĐLJƚŽŵĞƚƌLJ;LJƚŽ&>y^͕ ĞĐŬŵĂŶŽƵůƚĞƌ͕͕h^Ϳ͘dŚĞƉƌĞƐĞŶĐĞŽĨƚŚĞŇƵŽƌĞƐĐĞŶƚůĂďĞůŝŶƐŝŶĚŝĐĂƚĞĚƚŚĞ ĂƐƐŽĐŝĂƟŽŶŽĨĞŝƚŚĞƌůŝƉŽƐŽŵĞƐŽƌƉĞƉƟĚĞďLJDƐ͘ĂƚĂǁĞƌĞĂŶĂůLJnjĞĚǁŝƚŚ&ůŽǁ:Ž ƐŽŌǁĂƌĞsϭϬ;dƌĞĞƐƚĂƌ͕KZ͕h^Ϳ͘ In vitrodƌĞŐŝŶĚƵĐƟŽŶďLJůŝƉŽƐŽŵĞͲƉƵůƐĞĚDƐ DƐǁĞƌĞĐƵůƚƵƌĞĚĂƐĚĞƐĐƌŝďĞĚĂďŽǀĞ͕ƉůĂƚĞĚƚŽϱϬ͕ϬϬϬĐĞůůͬǁĞůůŝŶĂϵϲͲǁĞůů&ͲďŽƩŽŵ plate, and pulsed for 4 hours with liposomes or controls in cIMDM. Spleens from OT-//ŵŝĐĞǁĞƌĞƐƚƌĂŝŶĞĚƚŚƌŽƵŐŚĂϳϬђŵĐĞůůƐƚƌĂŝŶĞƌƚŽŽďƚĂŝŶĂƐŝŶŐůĞͲĐĞůůƐƵƐƉĞŶƐŝŽŶ͘ ƌLJƚŚƌŽĐLJƚĞƐǁĞƌĞůLJƐĞĚƵƐŝŶŐĂŵŵŽŶŝƵŵͲĐŚůŽƌŝĚĞͲƉŽƚĂƐƐŝƵŵ;<ͿůLJƐŝƐďƵīĞƌ;Ϭ͘ϭϱD NH₄ů͕ϭŵD<,K₃, 0.1 mM Na₂d͖Ɖ,ϳ͘ϯͿ͘ϰ+_{dĐĞůůƐǁĞƌĞŝƐŽůĂƚĞĚƵƐŝŶŐĂϰ}+ _{T cell} ŝƐŽůĂƟŽŶŬŝƚ;DŝůƚĞŶLJŝŝŽƚĞĐ͘s͕͘>ĞŝĚĞŶ͕EĞƚŚĞƌůĂŶĚƐͿĂĐĐŽƌĚŝŶŐƚŽƚŚĞŵĂŶƵĨĂĐƚƵƌĞƌ͛Ɛ ƉƌŽƚŽĐŽů͘ŌĞƌĞdžƉŽƐƵƌĞ͕DƐǁĞƌĞǁĂƐŚĞĚǁŝƚŚĐ/DDƚŽƌĞŵŽǀĞĨƌĞĞůŝƉŽƐŽŵĞƐ͕ ĂŶĚϭϬϬ͕ϬϬϬϰ+_{dĐĞůůƐͬǁĞůůǁĞƌĞĂĚĚĞĚƚŽƚŚĞDƐ͘ŽͲĐƵůƚƵƌĞƐǁĞƌĞŝŶĐƵďĂƚĞĚ} ĨŽƌϳϮŚŽƵƌƐŝŶZWD/ϭϲϰϬŵĞĚŝƵŵƐƵƉƉůĞŵĞŶƚĞĚǁŝƚŚϮŵD>ͲŐůƵƚĂŵŝŶĞ͕ϭϬйǀͬǀ&^͕ ϭϬϬhͬŵ>ƉĞŶŝĐŝůůŝŶͬƐƚƌĞƉƚŽŵLJĐŝŶ͕ĂŶĚϱϬђDɴͲŵĞƌĐĂƉƚŽĞƚŚĂŶŽů͘ĞůůƐǁĞƌĞƐƚĂŝŶĞĚĨŽƌ dŚLJϭ͘ϮͲWͲLJϳ͕ ϰͲsϱϬϬ͕ ǀŝĂďŝůŝƚLJͲWͲĞ&ůƵŽƌϳϴϬ͕ &KyWϯͲĞ&ůƵŽƌϰϱϬ͕ ĂŶĚ <ŝͲϲϳͲ&/d͕ ĂŶĚ ĂŶĂůLJnjĞĚ ďLJ ŇŽǁ ĐLJƚŽŵĞƚƌLJ ;LJƚŽ&>y ^͕ ĞĐŬŵĂŶ ŽƵůƚĞƌ͕ ͕ h^Ϳ͘ ĂƚĂ ǁĞƌĞ ĂŶĂůLJnjĞĚƵƐŝŶŐ&ůŽǁ:ŽƐŽŌǁĂƌĞsϭϬ;dƌĞĞƐƚĂƌ͕KZ͕h^Ϳ͘

ŶŝŵĂůƐ

ϱϳ>ͬϲ ĂŶĚ KdͲ// ƚƌĂŶƐŐĞŶŝĐ ŵŝĐĞ ŽŶ Ă ϱϳ>ͬϲ ďĂĐŬŐƌŽƵŶĚ ǁĞƌĞ ƉƵƌĐŚĂƐĞĚ ĨƌŽŵ :ĂĐŬƐŽŶ>ĂďŽƌĂƚŽƌLJ;͕h^Ϳ͕ďƌĞĚŝŶͲŚŽƵƐĞƵŶĚĞƌƐƚĂŶĚĂƌĚůĂďŽƌĂƚŽƌLJĐŽŶĚŝƟŽŶƐ͕ĂŶĚ provided with food and water ad libitum. All animal work was performed in compliance ǁŝƚŚƚŚĞƵƚĐŚŐŽǀĞƌŶŵĞŶƚŐƵŝĚĞůŝŶĞƐĂŶĚƚŚĞŝƌĞĐƟǀĞϮϬϭϬͬϲϯͬhŽĨƚŚĞƵƌŽƉĞĂŶ WĂƌůŝĂŵĞŶƚ͘džƉĞƌŝŵĞŶƚƐǁĞƌĞĂƉƉƌŽǀĞĚďLJƚŚĞƚŚŝĐƐŽŵŵŝƩĞĞĨŽƌŶŝŵĂůdžƉĞƌŝŵĞŶƚƐ of Leiden University.

ĚŽƉƟǀĞƚƌĂŶƐĨĞƌ

3

dŽ ĚĞƚĞƌŵŝŶĞ ǁŚĞƚŚĞƌ ƚŚĞ ůŝƉŽƐŽŵĂů ĐŽŵƉŽƐŝƟŽŶ ĂīĞĐƚĞĚ dƌĞŐ ƌĞƐƉŽŶƐĞƐ͕ DƐ ǁĞƌĞ ƉƵůƐĞĚ ǁŝƚŚ ůŝƉŽƐŽŵĞƐ ĨŽƌ ϰ ŚŽƵƌƐ͕ ĂŶĚ ƐƵďƐĞƋƵĞŶƚůLJ ĐŽͲĐƵůƚƵƌĞĚ ǁŝƚŚ KdͲ//ϰ+_{dĐĞůůƐ͘ŌĞƌƚŚƌĞĞĚĂLJƐŽĨŝŶĐƵďĂƟŽŶ͕ŝŶĚƵĐĞĚdƌĞŐƐǁĞƌĞŝĚĞŶƟĮĞĚƵƐŝŶŐ} ŇŽǁĐLJƚŽŵĞƚƌLJ;&ŝŐƵƌĞϯ͕͕ĂŶĚͿ͘ŵŽŶŐĂůůƚĞƐƚĞĚKs₃₂₃-containing liposomes, the ^W͗^W' ůŝƉŽƐŽŵĞƐ ŝŶĚƵĐĞĚ ƚŚĞ ƐƚƌŽŶŐĞƐƚ dƌĞŐ ƌĞƐƉŽŶƐĞƐ͕ ǁŝƚŚ Ă ϭ͘ϲͲĨŽůĚ ŝŶĐƌĞĂƐĞ ŝŶ й &KyWϯ+_{ ƉŽƉƵůĂƟŽŶ ŝŶ ůŝǀĞ ϰ}+_<ŝͲϲϳ+_{ d ĐĞůů ƉŽƉƵůĂƟŽŶ ĐŽŵƉĂƌĞĚ ƚŽ ĨƌĞĞ Ks} 323. ůƐŽ^W͗^W'͕^W͗^W'͗,K>͕^W͗WW'͗,K>͕ĂŶĚWW͗WW'͗,K>ůŝƉŽƐŽŵĞƐ ƐŚŽǁĞĚƐŝŐŶŝĮĐĂŶƚůLJŚŝŐŚĞƌdƌĞŐƌĞƐƉŽŶƐĞƐĐŽŵƉĂƌĞĚƚŽĨƌĞĞKs₃₂₃. Furthermore, we ŽďƐĞƌǀĞĚĂƐŝŐŶŝĮĐĂŶƚĐŽƌƌĞůĂƟŽŶďĞƚǁĞĞŶƚŚĞůŝƉŽƐŽŵĂůƌŝŐŝĚŝƚLJ;ĚĞƚĞƌŵŝŶĞĚďLJ&DͿ ĂŶĚdƌĞŐƌĞƐƉŽŶƐĞƐ;&ŝŐƵƌĞϯͿ͘/ŶƚĞƌĞƐƟŶŐůLJ͕ƚŚĞƌĞǁĂƐŶŽĐůĞĂƌƌĞůĂƟŽŶƐŚŝƉďĞƚǁĞĞŶ ƚŚĞĂƐƐŽĐŝĂƟŽŶŽĨůŝƉŽƐŽŵĞƐǁŝƚŚDƐĂŶĚƐƵďƐĞƋƵĞŶƚdƌĞŐƌĞƐƉŽŶƐĞƐ;&ŝŐƵƌĞϯͿ͘

&ŝŐƵƌĞ ϯ͗ /ŶĚƵĐƟŽŶ ŽĨ dƌĞŐƐ ďLJ Ks₃₂₃-loaded liposomes in vitro͘ DƐ ǁĞƌĞ ƉƵůƐĞĚ ĨŽƌ ϰ ŚŽƵƌƐǁŝƚŚůŝƉŽƐŽŵĞƐŽƌĐŽŶƚƌŽůƐĂŶĚƐƵďƐĞƋƵĞŶƚůLJĐŽͲĐƵůƚƵƌĞĚĨŽƌϳϮŚŽƵƌƐǁŝƚŚϰ+ _{T cells} ŝƐŽůĂƚĞĚĨƌŽŵKdͲ//ƐƉůĞŶŽĐLJƚĞƐ͘&ůŽǁĐLJƚŽŵĞƚƌLJǁĂƐƵƐĞĚƚŽŵĞĂƐƵƌĞ&KyWϯ+_<ŝͲϲϳ+_ϰ+ _{T cells.} ZĞƉƌĞƐĞŶƚĂƟǀĞŇŽǁĐLJƚŽŵĞƚƌLJƉůŽƚƐŽĨ&KyWϯ+_ϰ+_{dĐĞůůƐŝŶƚŚĞůŝǀĞ<ŝͲϲϳ}+_ϰ+_{dĐĞůůƉŽƉƵůĂƟŽŶ} ŽĨ KdͲ// d ĐĞůůƐ ĐƵůƚƵƌĞĚ ǁŝƚŚ ;Ϳ ĨƌĞĞ Ks₃₂₃ ;Ϭ͘ϭ ђŐͬŵ>Ϳ ĂŶĚ ;Ϳ Ks₃₂₃ͲůŽĂĚĞĚ ^W͗^W' ůŝƉŽƐŽŵĞƐ ;Ϭ͘ϭ ђŐͬŵ> Ks₃₂₃Ϳ͘ ;Ϳ ^ƵŵŵĂƌLJ dƌĞŐ ĚĂƚĂ ŽĨ Ăůů ůŝƉŽƐŽŵĂů ĨŽƌŵƵůĂƟŽŶƐ ƚĞƐƚĞĚ͘ ZĞƐƵůƚƐǁĞƌĞŶŽƌŵĂůŝnjĞĚƚŽй&KyWϯ+_<ŝͲϲϳ+_ϰ+_{dĐĞůůƐŝŶƚŚĞĨƌĞĞKs}

323 control. The dashed line

ŝƐĐƵƐƐŝŽŶ dŚĞ ŝŶĚƵĐƟŽŶ ŽĨ ĂŶƟŐĞŶͲƐƉĞĐŝĮĐ dƌĞŐƐ ƚŚƌŽƵŐŚ ǀĂĐĐŝŶĂƟŽŶ ŝƐ ĂŶ ĂƩƌĂĐƟǀĞ ĂƉƉƌŽĂĐŚ ĨŽƌ ƚŚĞ ƚƌĞĂƚŵĞŶƚ ŽĨ ĂƵƚŽŝŵŵƵŶĞ ĚŝƐĞĂƐĞƐ͘ tĞ ĂŶĚ ŽƚŚĞƌƐ ŚĂǀĞ ƉƌĞǀŝŽƵƐůLJ ƐŚŽǁŶ ƚŚĂƚ ŶĂŶŽƉĂƌƟĐůĞƐ͕ ƐƵĐŚ ĂƐ ůŝƉŽƐŽŵĞƐ͕ ĐĂŶ ďĞ ƵƐĞĚ ƚŽ ŝŶĚƵĐĞ ƚŚĞƐĞ dƌĞŐƐ10,13_. 'ĞŶĞƌĂůůLJ͕ĂƉƉƌŽĂĐŚĞƐƚŽŽƉƟŵŝnjĞƐƵĐŚŶĂŶŽƉĂƌƟĐůĞƐĂƌĞĨŽĐƵƐĞĚŽŶƐƚƵĚLJŝŶŐƚŚĞĞīĞĐƚ ŽĨƉĂƌƟĐůĞƐŝnjĞĂŶĚƐƵƌĨĂĐĞĐŚĂƌŐĞ͕ǁŚŝůĞĂŶŽƚŚĞƌŝŵƉŽƌƚĂŶƚƉĂƌĂŵĞƚĞƌ͕ƌŝŐŝĚŝƚLJ͕ŝƐŽŌĞŶ overlooked15_{͘ dŚŝƐ ƉĂƌĂŵĞƚĞƌ ŝƐ ƵƐƵĂůůLJ ĂƉƉƌŽdžŝŵĂƚĞĚ ďLJ ƚŚĞ ƉŚĂƐĞ ƉƌŽƉĞƌƟĞƐ ŽĨ ƚŚĞ} ůŝƉŝĚďŝůĂLJĞƌƐďLJĂŶĂůLJƟĐĂůƚĞĐŚŶŝƋƵĞƐƐƵĐŚĂƐ^ĂŶĚ>ĂƵƌĚĂŶĂŶŝƐŽƚƌŽƉLJ͘ĞƐŝĚĞƐƚŚĞ ĨĂĐƚƚŚĂƚƚŚĞƐĞƚĞĐŚŶŝƋƵĞƐŽŶůLJƉƌŽǀŝĚĞŝŶĨŽƌŵĂƟŽŶĂďŽƵƚƚŚĞďŝůĂLJĞƌŽĨůŝƉŽƐŽŵĞƐĂŶĚ ƚŚĞƌĞďLJ ĞdžĐůƵĚĞ ŽƚŚĞƌ ŝŶŇƵĞŶĐŝŶŐ ĨĂĐƚŽƌƐ͕ ƐƵĐŚ ĂƐ ƐŝnjĞ͕ ůĂŵĞůůĂƌŝƚLJ͕ ĂŶƟŐĞŶ ĐŽŶƚĞŶƚ͕ ĂŶĚ ĨŽƌŵƵůĂƟŽŶ ĞdžĐŝƉŝĞŶƚƐ͕ ƚŚĞƌĞ ĂƌĞ ŽƚŚĞƌ ŝƐƐƵĞƐ ĂƐƐŽĐŝĂƚĞĚ ǁŝƚŚ ƚŚĞƐĞ ƚĞĐŚŶŝƋƵĞƐ͘ &Žƌ ŝŶƐƚĂŶĐĞ͕ ŵĂŶLJ ůŝƉŽƐŽŵĂů ĨŽƌŵƵůĂƟŽŶƐ ĐŽŶƚĂŝŶ ,K> ŝŶ ƚŚĞ ůŝƉŝĚ ďŝůĂLJĞƌ͕ ǁŚŝĐŚ ĐŽŵƉƌŽŵŝƐĞƐ ƚŚĞ ƐƵŝƚĂďŝůŝƚLJ ŽĨ ƚĞĐŚŶŝƋƵĞƐ ƐƵĐŚ ĂƐ ^25_{, Fourier-transform infrared} ƐƉĞĐƚƌŽƐĐŽƉLJ;&d/ZͿ36_{, and Laurdan anisotropy}37_{. Furthermore, compounds encapsulated} ŝŶůŝƉŽƐŽŵĞƐĐĂŶĂůƐŽŝŶƚĞƌĨĞƌĞǁŝƚŚ^38,39 _{and FTIR measurements}40_{͘KƚŚĞƌĂŶĂůLJƟĐĂů} ƚĞĐŚŶŝƋƵĞƐ ƐƵĐŚ ĂƐ ŵĞĂƐƵƌŝŶŐ ƚŚĞ ĂďŝůŝƚLJ ŽĨ ƉĂƌƟĐůĞƐ ƚŽ ďĞ ĞdžƚƌƵĚĞĚ41,42 _{are indirect} ŵĞĂƐƵƌĞŵĞŶƚƐŽĨƌŝŐŝĚŝƚLJ͘/ŵƉŽƌƚĂŶƚůLJ͕ĂůůŽĨƚŚĞĂĨŽƌĞŵĞŶƟŽŶĞĚƚĞĐŚŶŝƋƵĞƐĐĂŶŽŶůLJ ŵĞĂƐƵƌĞƚŚĞĂǀĞƌĂŐĞƉƌŽƉĞƌƟĞƐŽĨĂďĂƚĐŚŽĨƉĂƌƟĐůĞƐ͘/ŶĐŽŶƚƌĂƐƚ͕&DŝƐĂŶĞdžĐĞůůĞŶƚ ƚĞĐŚŶŝƋƵĞƚŽĂĐĐƵƌĂƚĞůLJŵĞĂƐƵƌĞƚŚĞƌŝŐŝĚŝƚLJŽĨŝŶĚŝǀŝĚƵĂůƉĂƌƟĐůĞƐĂŶĚŝƐŶŽƚŚŝŶĚĞƌĞĚ ďLJ ƚŚĞ ƉƌĞƐĞŶĐĞ ŽĨ ŽƚŚĞƌ ĐŽŵƉŽƵŶĚƐ͕ ƐƵĐŚ ĂƐ ,K> ĂŶĚ ĂŶƟŐĞŶ͘ dŽ ĨƵƌƚŚĞƌ ĐŽŶĮƌŵ ƚŚŝƐ͕ƐƚƵĚŝĞƐƐŚŽƵůĚďĞƉĞƌĨŽƌŵĞĚƚŽĐŽŵƉĂƌĞ&DƚŽŽƚŚĞƌƚĞĐŚŶŝƋƵĞƐƚŚĂƚŵĞĂƐƵƌĞ liposomal rigidity. tĞ ŽďƐĞƌǀĞĚ ƚŚĂƚ ,K> ƌĞĚƵĐĞƐ ^W͗^W' ůŝƉŽƐŽŵĞ ƌŝŐŝĚŝƚLJ͕ ĂŶĚ ŝŶĐƌĞĂƐĞƐ KW͗KW' ůŝƉŽƐŽŵĞ ƌŝŐŝĚŝƚLJ͘ dŚĞ ůŝƉŝĚ ďŝůĂLJĞƌƐ ŽĨ ůŝƉŽƐŽŵĞƐ ĐĂŶ ŐĞŶĞƌĂůůLJ ďĞ ĐůĂƐƐŝĮĞĚŝŶƚŽƚŚƌĞĞƉŚLJƐŝĐĂůƐƚĂƚĞƐǁŝƚŚŝŶĐƌĞĂƐŝŶŐƌŝŐŝĚŝƚLJ͗ůŝƋƵŝĚĚŝƐŽƌĚĞƌĞĚ;ďŝůĂLJĞƌƐ composed of low-T_mƉŚŽƐƉŚŽůŝƉŝĚƐͿ͕ůŝƋƵŝĚŽƌĚĞƌĞĚ;ďŝůĂLJĞƌƐĐŽŵƉŽƐĞĚŽĨŚŝŐŚͲŽƌůŽǁͲ T_mƉŚŽƐƉŚŽůŝƉŝĚƐĐŽŶƚĂŝŶŝŶŐĂƚůĞĂƐƚϮϬŵŽůй,K>ͿĂŶĚŐĞůƐƚĂƚĞ;ďŝůĂLJĞƌƐĐŽŶƚĂŝŶŝŶŐ high-T_mƉŚŽƐƉŚŽůŝƉŝĚƐͿ͘/ŶƚŚĞŐĞůƐƚĂƚĞ͕,K>ĚŝƐƌƵƉƚƐƚŚĞƟŐŚƚƉĂĐŬŝŶŐŽĨƚŚĞůŝƉŝĚƐ͕ ǁŚŝůĞŝŶƚŚĞůŝƋƵŝĚĚŝƐŽƌĚĞƌĞĚƐƚĂƚĞ͕,K>ŽĐĐƵƉŝĞƐƐƉĂĐĞĂƚƚŚĞŝŶƚĞƌĨĂĐŝĂůƌĞŐŝŽŶŶĞĂƌ ƚŚĞŵĞŵďƌĂŶĞƐƵƌĨĂĐĞĂŶĚƌĞƉůĂĐĞƐƚŚĞŚLJĚƌĂƚĞĚ,₂ groups. As the acyl chains become ĚĞŚLJĚƌĂƚĞĚĂŶĚƐƚƌĂŝŐŚƚĞŶŽƵƚ͕ƚŚĞƉĂĐŬŝŶŐďĞĐŽŵĞƐƟŐŚƚĞƌĂŶĚƌŝŐŝĚŝƚLJŝƐ͕ƚŚĞƌĞĨŽƌĞ͕ increased43_{͘tŚŝůĞďŝůĂLJĞƌƐƚĂƚĞŝƐŶŽƚƚŚĞƐĂŵĞĂƐůŝƉŽƐŽŵĂůƌŝŐŝĚŝƚLJ͕ŝƚŝƐŵŽƐƚůŝŬĞůLJŽŶĞ} ŽĨƚŚĞŵŽƐƚŝŵƉŽƌƚĂŶƚƉĂƌĂŵĞƚĞƌƐƚŚĂƚŝŶŇƵĞŶĐĞƚŚĞƌŝŐŝĚŝƚLJŽĨƚŚĞůŝƉŽƐŽŵĞƐ͘dŚŝƐŝƐ also in line with results from Takechi-Haraya et al͘ǁŚŽƵƐĞĚ&DƚŽƐƚƵĚLJƚŚĞĞīĞĐƚŽĨ ,K>ŽŶůŝƉŽƐŽŵĞƌŝŐŝĚŝƚLJ44_.

&ƌŽŵŽƵƌƐƚƵĚLJ͕ŝƚďĞĐĂŵĞĐůĞĂƌƚŚĂƚƚŚĞŝŶƚƌŽĚƵĐƟŽŶŽĨůŝƉŝĚŽƌĚĞƌŵŝƐŵĂƚĐŚ ƐŝŐŶŝĮĐĂŶƚůLJĂīĞĐƚƐƚŚĞƌŝŐŝĚŝƚLJŽĨƚŚĞůŝƉŽƐŽŵĞƐ͘DŝdžŝŶŐŽĨůŝƉŝĚƐǁŝƚŚĚŝīĞƌĞŶƚƉŚĂƐĞ ƚƌĂŶƐŝƟŽŶƐ ŝŶ ƚŚĞ ĂďƐĞŶĐĞ ŽĨ ,K> ĐĂŶ ůĞĂĚ ƚŽ ƚŚĞ ĨŽƌŵĂƟŽŶ ŽĨ ůŝƉŝĚ ĚŽŵĂŝŶƐ ;e.g. ƌŝŐŝĚ ĚŽŵĂŝŶƐ ĂŶĚ ŇƵŝĚ ĚŽŵĂŝŶƐͿ͕ ǁŚŝĐŚ ĐŽƵůĚ ĚĞƐƚĂďŝůŝnjĞ ƚŚĞ ďŝůĂLJĞƌ ƉĂĐŬŝŶŐ͕ ĂŶĚ thereby the rigidity of the liposomes23_{͘tĞŽďƐĞƌǀĞĚĂƐŝŐŶŝĮĐĂŶƚĚĞĐƌĞĂƐĞŝŶƌŝŐŝĚŝƚLJŽĨ} liposomes in the presence of a small amount of lower-T_můŝƉŝĚ;e.g.^W͗^W'͗,K>ǀƐ͘ ^W͗WW'͗,K>͕dĂďůĞ^ϯͿ͕ǁŚŝĐŚĚŝĚŶŽƚĨƵƌƚŚĞƌĚĞĐƌĞĂƐĞƵƉŽŶĐŽŵƉůĞƚĞƐƵďƐƟƚƵƟŽŶ ;e.g.^W͗WW'͗,K>ǀƐ͘WW͗WW'͗,K>Ϳ͘dŚŝƐƐƵŐŐĞƐƚƐƚŚĂƚƚŚĞd_m values and the ŵŽůĂƌƌĂƟŽŽĨƚŚĞĐŽŶƐƟƚƵĞŶƚůŝƉŝĚƐĂƌĞƉŽŽƌƉƌĞĚŝĐƚŽƌƐŽĨƚŚĞƌŝŐŝĚŝƚLJŽĨƚŚĞůŝƉŽƐŽŵĞƐ ;&ŝŐƵƌĞ^ϭͿ͘

3

ĂůƐŽĂīĞĐƚƚŚĞŝƌƌŝŐŝĚŝƚLJ͘&ŽƌĞdžĂŵƉůĞ͕ƐŵĂůůĞƌůŝƉŽƐŽŵĞƐ͕ŵĞĂƐƵƌĞĚďLJ&D͕ĂƌĞŵŽƌĞ rigid than larger liposomes45_{͘dŚŝƐŵĂLJďĞĚƵĞƚŽƚŚĞĚŝīĞƌĞŶƚĐƵƌǀĂƚƵƌĞŽĨůŝƉŽƐŽŵĞƐ} ŽĨĚŝīĞƌĞŶƚƐŝnjĞƐ͕ůĞĂĚŝŶŐƚŽŵŽƌĞƌĞƐƚƌŝĐƚĞĚŵŽůĞĐƵůĂƌŝŶƚĞƌĂĐƟŽŶƐďĞƚǁĞĞŶƚŚĞůŝƉŝĚƐ of smaller liposomes46_{͘dŚĞĐŚĂƌŐĞŽĨůŝƉŽƐŽŵĞƐĐĂŶĂůƐŽĂīĞĐƚƌŝŐŝĚŝƚLJ͖ŶĞƵƚƌĂůKW} ůŝƉŽƐŽŵĞƐ ŵĞĂƐƵƌĞĚ ďLJ &D ǁĞƌĞ ƐŚŽǁŶ ƚŽ ďĞ ŵŽƌĞ ƌŝŐŝĚ ƚŚĂŶ ĂŶŝŽŶŝĐ KW͗KW' Žƌ ĐĂƟŽŶŝĐ KW͗KdW ůŝƉŽƐŽŵĞƐ͘ dŚŝƐ ǁĂƐ ŚLJƉŽƚŚĞƐŝnjĞĚ ƚŽ ďĞ ĚƵĞ ƚŽ ĞůĞĐƚƌŽƐƚĂƟĐ ƌĞƉƵůƐŝŽŶƐ ĐĂƵƐŝŶŐ ƐƚƌƵĐƚƵƌĂů ĚĞƐƚĂďŝůŝnjĂƟŽŶ ŽĨ ƚŚĞ ůŝƉŝĚ ďŝůĂLJĞƌ47_{. Due to technical} ůŝŵŝƚĂƟŽŶƐ͕ƚŚĞ&DŵĞĂƐƵƌĞŵĞŶƚƐǁĞƌĞƉĞƌĨŽƌŵĞĚĂƚϮϱΣ͘^ŝŶĐĞƚĞŵƉĞƌĂƚƵƌĞĂīĞĐƚƐ ƚŚĞƉŚĂƐĞƐƚĂƚĞŽĨƚŚĞůŝƉŝĚďŝůĂLJĞƌƐ͕ŝƚĂůƐŽŝŶŇƵĞŶĐĞƐƚŚĞƌŝŐŝĚŝƚLJŽĨůŝƉŽƐŽŵĞƐ͘,ŽǁĞǀĞƌ͕ ƚŚĞƉŚĂƐĞƐƚĂƚĞƐŽĨƚŚĞƵƐĞĚƉŚŽƐƉŚŽůŝƉŝĚƐĂƌĞƚŚĞƐĂŵĞĂƚϮϱΣĂŶĚϯϳΣ;dĂďůĞ^ϭͿ͕ƐŽ ƚŚĞƚƌĞŶĚƐŝŶzDĂƚƚŚŝƐƉŚLJƐŝŽůŽŐŝĐĂůůLJŵŽƌĞƌĞůĞǀĂŶƚƚĞŵƉĞƌĂƚƵƌĞƐŚŽƵůĚďĞǀĞƌLJƐŝŵŝůĂƌ ƚŽƚŚŽƐĞŽďƐĞƌǀĞĚĂƚϮϱΣ͘&ƵƌƚŚĞƌŵŽƌĞ͕ĂƌĞĐĞŶƚƐƚƵĚLJĨŽƵŶĚŶŽĚŝīĞƌĞŶĐĞŝŶĂŶŝŽŶŝĐ ůŝƉŽƐŽŵĞƐƟīŶĞƐƐŽǀĞƌƚŚĞƚĞŵƉĞƌĂƚƵƌĞƌĂŶŐĞŽĨϮϱͲϯϳΣ48_{͘&ŝŶĂůůLJ͕ĂŶƟŐĞŶĐŽŶƚĞŶƚŵĂLJ} ĂīĞĐƚůŝƉŽƐŽŵĂůƌŝŐŝĚŝƚLJ͘/ŶƚŚŝƐƐƚƵĚLJ͕ǁĞŚĂǀĞŐĞŶĞƌĂƚĞĚůŝƉŽƐŽŵĂůĨŽƌŵƵůĂƟŽŶƐǁŝƚŚ ƐŝŵŝůĂƌƐŝnjĞ͕ĂŶƟŐĞŶůŽĂĚŝŶŐ͕ĂŶĚɺͲƉŽƚĞŶƚŝĂů to allow an in vitro and in vivo assessment primarily based on liposome rigidity.

dŚĞůŝƉŽƐŽŵĞƐǁĞŵĞĂƐƵƌĞĚŚĂĚĂƌĂŶŐĞŽĨzDĨƌŽŵĂďŽƵƚϱϬϬŬWĂƚŽĂůŵŽƐƚ ϰ DWĂ ;&ŝŐƵƌĞ ϭͿ͘ &Žƌ ĐŽŵƉĂƌŝƐŽŶ͕ ƚŚĞ zD ŽĨ ŵŽƐƚ ŵĂŵŵĂůŝĂŶ ĐĞůůƐ ǁĂƐ ŵĞĂƐƵƌĞĚ ƚŽďĞďĞƚǁĞĞŶϬ͘ϬϮĂŶĚϰϬϬŬWĂ49_{ĂŶĚƚŚĂƚŽĨĐŽƌƟĐĂůďŽŶĞǁĂƐƌĞƉŽƌƚĞĚƚŽďĞĂďŽƵƚ} ϮϬ'WĂ50_{͘DŽƌĞŽǀĞƌ͕ǁĞŚĂǀĞƵƐĞĚŽƵƌ&DŵĞƚŚŽĚƚŽŵĞĂƐƵƌĞŽƚŚĞƌĂŶŝŽŶŝĐƉĂƌƟĐůĞ} ƚLJƉĞ͕W>'ŶĂŶŽƉĂƌƟĐůĞƐ;ͲĂǀĞƌĂŐĞĚŝĂŵĞƚĞƌŽĨϭϰϮ͘ϭцϭ͘ϭŶŵ͕W/ŽĨϬ͘ϬϳϯцϬ͘Ϭϭϲ͕ ɺͲƉŽƚĞŶƚŝĂů -49.7 цϰ͘ϮŵsͿ͕ǁŚŝĐŚŚĂĚĂzDŽĨϭϰ͘ϰDWĂцϭ͘ϴDWĂ͘dŚŝƐŝůůƵƐƚƌĂƚĞƐƚŚĂƚ the range of rigidities of the liposomes is relatively small. This is especially true for the ,K>ͲĐŽŶƚĂŝŶŝŶŐůŝƉŽƐŽŵĞƐ͕ƚŚĞƌŝŐŝĚŝƚLJŽĨǁŚŝĐŚƌĂŶŐĞĚĨƌŽŵĂďŽƵƚϴϬϬŬWĂƚŽϭϱϬϬ ŬWĂ;&ŝŐƵƌĞϭͿ͘dŚŝƐǁĂƐĞdžƉĞĐƚĞĚƐŝŶĐĞƚŚĞŝƌďŝůĂLJĞƌƐĂƌĞĂůůŝŶƚŚĞůŝƋƵŝĚĚŝƐŽƌĚĞƌĞĚ state. However, even such small differences in rigidity could be measured using our AFM ŵĞƚŚŽĚ͕ĂŶĚǁĞĐŽƵůĚŵĞĂƐƵƌĞƐŝŐŶŝĨŝĐĂŶƚĚŝĨĨĞƌĞŶĐĞƐďĞƚǁĞĞŶĨŽƌŵƵůĂƚŝŽŶƐ;dĂďůĞ^ϯͿ͕ ŝůůƵƐƚƌĂƚŝŶŐƚŚĞƐƚƌĞŶŐƚŚŽĨƚŚŝƐƚĞĐŚŶŝƋƵĞ͘

macrophages52_{͘ /Ŷ ŽƵƌ ƐƚƵĚLJ͕ ĂůŵŽƐƚ Ăůů ĨŽƌŵƵůĂƟŽŶƐ ƐŚŽǁ Ă ƉŽƐŝƟǀĞ ƚƌĞŶĚ ďĞƚǁĞĞŶ} ůŝƉŽƐŽŵĂů ƌŝŐŝĚŝƚLJ ĂŶĚ W ĂƐƐŽĐŝĂƟŽŶ͘ ,ŽǁĞǀĞƌ͕ KW͗KW' ůŝƉŽƐŽŵĞƐ ƐŚŽǁĞĚ ƚŚĞ ŚŝŐŚĞƐƚ ĂƐƐŽĐŝĂƟŽŶ ƚŽ DƐ ŽĨ Ăůů ƚĞƐƚĞĚ ĨŽƌŵƵůĂƟŽŶƐ ;&ŝŐƵƌĞ ϮͿ͕ ǁŚŝĐŚ ǁĂƐ ŶŽƚ ĞdžƉĞĐƚĞĚƐŝŶĐĞƚŚĞƐĞůŝƉŽƐŽŵĞƐƉƌĞƐĞŶƚĞĚƚŚĞůŽǁĞƐƚƌŝŐŝĚŝƚLJ;&ŝŐƵƌĞϭͿ͘dŚĞŝƌƌŽƵƚĞŽĨ ƵƉƚĂŬĞĐŽƵůĚďĞĚŝīĞƌĞŶƚĨƌŽŵƚŚĂƚŽĨƚŚĞŵŽƌĞƌŝŐŝĚĨŽƌŵƵůĂƟŽŶƐ͘&ŽƌŝŶƐƚĂŶĐĞ͕ƉƵƌĞ KWůŝƉŽƐŽŵĞƐ;zDŽĨϰϱŬWĂĂƐŵĞĂƐƵƌĞĚďLJ&DͿǁĞƌĞŚLJƉŽƚŚĞƐŝnjĞĚƚŽĨƵƐĞǁŝƚŚ cells53_{͘dŚĞKW͗KW'ůŝƉŽƐŽŵĞƐƐŚŽǁĞĚŚŝŐŚƉĂƐƐŝǀĞĂƐƐŽĐŝĂƟŽŶ;&ŝŐƵƌĞϮͿ͕ĂŶĚĚŝĚ} ŶŽƚĚĞůŝǀĞƌƚŚĞŝƌĐĂƌŐŽƚŽDƐ;&ŝŐƵƌĞϮͿ͕ĞdžƉůĂŝŶŝŶŐƚŚĞŝƌŝŶĂďŝůŝƚLJƚŽŝŶĚƵĐĞdĐĞůů ƌĞƐƉŽŶƐĞƐ;&ŝŐƵƌĞϯͿ͘dŚĞƌĞŝƐĞǀŝĚĞŶĐĞŽĨĂŶŝŶǀĞƌƐĞƌĞůĂƟŽŶƐŚŝƉďĞƚǁĞĞŶůŝƉŽƐŽŵĂů rigidity and membrane permeability, as measured by calcein leakage37_{. So, it may be that} ƚŚĞƉĞƉƟĚĞŚĂĚůĞĂŬĞĚŽƵƚŽĨƚŚĞKW͗KW'ĂŶĚKW͗KW'͗,K>ůŝƉŽƐŽŵĞƐƉƌŝŽƌ ƚŽDĂƐƐŽĐŝĂƟŽŶ͘dŚŝƐŚLJƉŽƚŚĞƐŝƐŝƐƐƵƉƉŽƌƚĞĚďLJŽƵƌŽďƐĞƌǀĂƟŽŶƚŚĂƚĂŵŝdžƚƵƌĞ ŽĨ ĨƌĞĞ Ks₃₂₃ ĂŶĚ ĞŵƉƚLJ KW͗KW' Žƌ KW͗KW'͗,K> ůŝƉŽƐŽŵĞƐ ƐŚŽǁĞĚ ƚŚĞ ƐĂŵĞĂŵŽƵŶƚŽĨKs₃₂₃ĂƐƐŽĐŝĂƟŽŶĂƐĞŶĐĂƉƐƵůĂƚĞĚƉĞƉƟĚĞ;ĚĂƚĂŶŽƚƐŚŽǁŶͿ͘^ŝŶĐĞǁĞ ƵƐĞĚŇƵŽƌĞƐĐĞŶƚůLJͲůĂďĞůĞĚůŝƉŝĚƐĂŶĚƉĞƉƟĚĞƐ͕ǁĞĐĂŶŶŽƚĞdžĐůƵĚĞƚŚĞŝŶŇƵĞŶĐĞŽĨƚŚĞ ŇƵŽƌŽƉŚŽƌĞŝƚƐĞůĨŽŶƚŚĞŵĞĂƐƵƌĞĚĂƐƐŽĐŝĂƟŽŶŝŶDƐ͘,ŽǁĞǀĞƌ͕ƐŝŶĐĞĂůůĨŽƌŵƵůĂƟŽŶƐ ;ĂƐŝĚĞĨƌŽŵKW͗KW'ůŝƉŽƐŽŵĞƐͿƐŚŽǁĂůŵŽƐƚŶŽĂƐƐŽĐŝĂƟŽŶŽĨŇƵŽƌŽƉŚŽƌĞǁŚĞŶ ŝŶĐƵďĂƚĞĚĂƚϰΣ;&ŝŐƵƌĞ^ϮͿ͕ƚŚĞŇƵŽƌŽƉŚŽƌĞŝƚƐĞůĨůŝŬĞůLJŚĂƐŶŽĞīĞĐƚŽŶĂƐƐŽĐŝĂƟŽŶ͘ tĞŽďƐĞƌǀĞĚĂĐůĞĂƌĐŽƌƌĞůĂƟŽŶďĞƚǁĞĞŶƚŚĞƌŝŐŝĚŝƚLJŽĨƚŚĞůŝƉŽƐŽŵĞƐĂŶĚƚŚĞ Treg responses they elicited in vitro and in vivo ;&ŝŐƵƌĞ ϯ͕ &ŝŐƵƌĞ ϰ&Ϳ͘ tŚŝůĞ ŽƚŚĞƌƐ ŚĂǀĞƐŚŽǁŶĂůŝŶŬďĞƚǁĞĞŶŝŶĐƌĞĂƐĞĚƌŝŐŝĚŝƚLJĂŶĚD,Ͳ//ƉƌĞƐĞŶƚĂƟŽŶ19_{, and enhanced} humoral and cellular responses54,55_{͕ǁĞĂƌĞƚŚĞĮƌƐƚƚŽƌĞƉŽƌƚƚŚĞĞīĞĐƚƐŽĨƌŝŐŝĚŝƚLJŽŶ} dƌĞŐƌĞƐƉŽŶƐĞƐ͘dŚŝƐŵĂLJŚĂǀĞŝŵƉůŝĐĂƟŽŶƐĨŽƌƚŚĞĚĞƐŝŐŶŽĨĚĞůŝǀĞƌLJƐLJƐƚĞŵƐĂŝŵĞĚƚŽ ĞŶŚĂŶĐĞĂŶƟŐĞŶͲƐƉĞĐŝĮĐdƌĞŐƌĞƐƉŽŶƐĞƐ͘

3

ZĞĨĞƌĞŶĐĞƐ

ϭ ^ĂŬĂŐƵĐŚŝ͕ ^͕͘ zĂŵĂŐƵĐŚŝ͕ d͕͘ EŽŵƵƌĂ͕ d͘ Θ KŶŽ͕ D͘ ZĞŐƵůĂƚŽƌLJ d ĐĞůůƐ ĂŶĚ immune tolerance. Cell ϭϯϯ͕ϳϳϱͲϳϴϳ͕ĚŽŝ͗ϭϬ͘ϭϬϭϲͬũ͘ĐĞůů͘ϮϬϬϴ͘Ϭϱ͘ϬϬϵ;ϮϬϬϴͿ͘

Ϯ DĂůůĂƚ͕͕͘ŝƚͲKƵĨĞůůĂ͕,͘ΘdĞĚŐƵŝ͕͘ZĞŐƵůĂƚŽƌLJdͲĐĞůůŝŵŵƵŶŝƚLJŝŶĂƚŚĞƌŽƐĐůĞƌŽƐŝƐ͘ Trends Cardiovasc Med ϭϳ͕ϭϭϯͲϭϭϴ͕ĚŽŝ͗ϭϬ͘ϭϬϭϲͬũ͘ƚĐŵ͘ϮϬϬϳ͘Ϭϯ͘ϬϬϭ;ϮϬϬϳͿ͘

ϯ <ĞŝũnjĞƌ͕͕͘ǀĂŶĚĞƌĞĞ͕Z͕͘ǀĂŶĚĞŶ͕t͘ΘƌŽĞƌĞ͕&͘dƌĞŐŝŶĚƵĐŝŶŐĂĚũƵǀĂŶƚƐĨŽƌ ƚŚĞƌĂƉĞƵƟĐǀĂĐĐŝŶĂƟŽŶĂŐĂŝŶƐƚĐŚƌŽŶŝĐŝŶŇĂŵŵĂƚŽƌLJĚŝƐĞĂƐĞƐ͘Front Immunol ϰ͕Ϯϰϱ͕ĚŽŝ͗ϭϬ͘ϯϯϴϵͬĮŵŵƵ͘ϮϬϭϯ͘ϬϬϮϰϱ;ϮϬϭϯͿ͘ ϰ ŽŵŝŶŐƵĞnjͲsŝůůĂƌ͕D͘Θ,ĂŇĞƌ͕͘͘ZĞŐƵůĂƚŽƌLJdĐĞůůƐŝŶĂƵƚŽŝŵŵƵŶĞĚŝƐĞĂƐĞ͘ Nat Immunol ϭϵ͕ϲϲϱͲϲϳϯ͕ĚŽŝ͗ϭϬ͘ϭϬϯϴͬƐϰϭϱϵϬͲϬϭϴͲϬϭϮϬͲϰ;ϮϬϭϴͿ͘ ϱ &ĂƌŝĂ͕͘D͘ΘtĞŝŶĞƌ͕,͘>͘KƌĂůƚŽůĞƌĂŶĐĞ͗ƚŚĞƌĂƉĞƵƟĐŝŵƉůŝĐĂƟŽŶƐĨŽƌĂƵƚŽŝŵŵƵŶĞ diseases. Clin Dev Immunol ϭϯ͕ ϭϰϯͲϭϱϳ͕ ĚŽŝ͗ϭϬ͘ϭϬϴϬͬϭϳϰϬϮϱϮϬϲϬϬϴϳϲϴϬϰ

;ϮϬϬϲͿ͘ ϲ <ŝƐŚŝŵŽƚŽ͕d͘<͘ΘDĂůĚŽŶĂĚŽ͕Z͘͘EĂŶŽƉĂƌƟĐůĞƐĨŽƌƚŚĞ/ŶĚƵĐƟŽŶŽĨŶƟŐĞŶͲ ^ƉĞĐŝĮĐ /ŵŵƵŶŽůŽŐŝĐĂů dŽůĞƌĂŶĐĞ͘ Front Immunol ϵ͕ ϮϯϬ͕ ĚŽŝ͗ϭϬ͘ϯϯϴϵͬ ĮŵŵƵ͘ϮϬϭϴ͘ϬϬϮϯϬ;ϮϬϭϴͿ͘ 7 Macauley, M. S. et al. ŶƟŐĞŶŝĐ ůŝƉŽƐŽŵĞƐ ĚŝƐƉůĂLJŝŶŐ ϮϮ ůŝŐĂŶĚƐ ŝŶĚƵĐĞ ĂŶƟŐĞŶͲƐƉĞĐŝĮĐ  ĐĞůů ĂƉŽƉƚŽƐŝƐ͘ J Clin Invest ϭϮϯ͕ ϯϬϳϰͲϯϬϴϯ͕ ĚŽŝ͗ϭϬ͘ϭϭϳϮͬ :/ϲϵϭϴϳ;ϮϬϭϯͿ͘ ϴ WĂŶŐ͕ >͕͘ DĂĐĂƵůĞLJ͕ D͘ ^͕͘ ƌůŝĂŶ͕ ͘ D͕͘ ELJĐŚŽůĂƚ͕ ͘ D͘ Θ WĂƵůƐŽŶ͕ :͘ ͘ ŶĐĂƉƐƵůĂƟŶŐĂŶ/ŵŵƵŶŽƐƵƉƉƌĞƐƐĂŶƚŶŚĂŶĐĞƐdŽůĞƌĂŶĐĞ/ŶĚƵĐƟŽŶďLJ^ŝŐůĞĐͲ ŶŐĂŐŝŶŐ dŽůĞƌŽŐĞŶŝĐ >ŝƉŽƐŽŵĞƐ͘ Chembiochem ϭϴ͕ ϭϮϮϲͲϭϮϯϯ͕ ĚŽŝ͗ϭϬ͘ϭϬϬϮͬ ĐďŝĐ͘ϮϬϭϲϬϬϳϬϮ;ϮϬϭϳͿ͘ 9 Maldonado, R. A. et al.WŽůLJŵĞƌŝĐƐLJŶƚŚĞƟĐŶĂŶŽƉĂƌƟĐůĞƐĨŽƌƚŚĞŝŶĚƵĐƟŽŶŽĨ ĂŶƟŐĞŶͲƐƉĞĐŝĮĐŝŵŵƵŶŽůŽŐŝĐĂůƚŽůĞƌĂŶĐĞ͘Proc Natl Acad Sci U S A ϭϭϮ͕ϭϱϲͲ ϭϲϱ͕ĚŽŝ͗ϭϬ͘ϭϬϳϯͬƉŶĂƐ͘ϭϰϬϴϲϴϲϭϭϭ;ϮϬϭϱͿ͘

10 Rodriguez-Fernandez, S. et al. WŚŽƐƉŚĂƟĚLJůƐĞƌŝŶĞͲ>ŝƉŽƐŽŵĞƐ WƌŽŵŽƚĞ dŽůĞƌŽŐĞŶŝĐ&ĞĂƚƵƌĞƐŽŶĞŶĚƌŝƟĐĞůůƐŝŶ,ƵŵĂŶdLJƉĞϭŝĂďĞƚĞƐďLJƉŽƉƚŽƟĐ Mimicry. Front Immunol ϵ͕Ϯϱϯ͕ĚŽŝ͗ϭϬ͘ϯϯϴϵͬĮŵŵƵ͘ϮϬϭϴ͘ϬϬϮϱϯ;ϮϬϭϴͿ͘

ϭϭ tĂƚƐŽŶ͕ ͘ ^͕͘ ŶĚƐůĞLJ͕ ͘ E͘ Θ ,ƵĂŶŐ͕ >͘ ĞƐŝŐŶ ĐŽŶƐŝĚĞƌĂƟŽŶƐ ĨŽƌ ůŝƉŽƐŽŵĂů ǀĂĐĐŝŶĞƐ͗ ŝŶŇƵĞŶĐĞ ŽĨ ĨŽƌŵƵůĂƟŽŶ ƉĂƌĂŵĞƚĞƌƐ ŽŶ ĂŶƟďŽĚLJ ĂŶĚ ĐĞůůͲŵĞĚŝĂƚĞĚ ŝŵŵƵŶĞ ƌĞƐƉŽŶƐĞƐ ƚŽ ůŝƉŽƐŽŵĞ ĂƐƐŽĐŝĂƚĞĚ ĂŶƟŐĞŶƐ͘ Vaccine ϯϬ, 2256-2272, ĚŽŝ͗ϭϬ͘ϭϬϭϲͬũ͘ǀĂĐĐŝŶĞ͘ϮϬϭϮ͘Ϭϭ͘ϬϳϬ;ϮϬϭϮͿ͘ ϭϮ ,ƵŶƚĞƌ͕ ͘ et al.  ďŝŽĚĞŐƌĂĚĂďůĞ ŶĂŶŽƉĂƌƟĐůĞ ƉůĂƞŽƌŵ ĨŽƌ ƚŚĞ ŝŶĚƵĐƟŽŶ ŽĨ ĂŶƟŐĞŶͲƐƉĞĐŝĮĐŝŵŵƵŶĞƚŽůĞƌĂŶĐĞĨŽƌƚƌĞĂƚŵĞŶƚŽĨĂƵƚŽŝŵŵƵŶĞĚŝƐĞĂƐĞ͘ACS Nano ϴ͕ϮϭϰϴͲϮϭϲϬ͕ĚŽŝ͗ϭϬ͘ϭϬϮϭͬŶŶϰϬϱϬϯϯƌ;ϮϬϭϰͿ͘ 13 Benne, N. et al. ŶŝŽŶŝĐ ϭ͕ϮͲĚŝƐƚĞĂƌŽLJůͲƐŶͲŐůLJĐĞƌŽͲϯͲƉŚŽƐƉŚŽŐůLJĐĞƌŽů ;^W'Ϳ ůŝƉŽƐŽŵĞƐŝŶĚƵĐĞĂŶƟŐĞŶͲƐƉĞĐŝĮĐƌĞŐƵůĂƚŽƌLJdĐĞůůƐĂŶĚƉƌĞǀĞŶƚĂƚŚĞƌŽƐĐůĞƌŽƐŝƐ in mice. J Control Release Ϯϵϭ͕ ϭϯϱͲϭϰϲ͕ ĚŽŝ͗ϭϬ͘ϭϬϭϲͬũ͘ũĐŽŶƌĞů͘ϮϬϭϴ͘ϭϬ͘ϬϮϴ

;ϮϬϭϴͿ͘

ϭϰ ĂĐŚŵĂŶŶ͕D͘&͘Θ:ĞŶŶŝŶŐƐ͕'͘d͘sĂĐĐŝŶĞĚĞůŝǀĞƌLJ͗ĂŵĂƩĞƌŽĨƐŝnjĞ͕ŐĞŽŵĞƚƌLJ͕ ŬŝŶĞƟĐƐ ĂŶĚ ŵŽůĞĐƵůĂƌ ƉĂƩĞƌŶƐ͘ Nat Rev Immunol ϭϬ͕ ϳϴϳͲϳϵϲ͕ ĚŽŝ͗ϭϬ͘ϭϬϯϴͬ ŶƌŝϮϴϲϴ;ϮϬϭϬͿ͘

vaccines. J Control Release Ϯϯϰ͕ ϭϮϰͲϭϯϰ͕ ĚŽŝ͗ϭϬ͘ϭϬϭϲͬũ͘ũĐŽŶƌĞů͘ϮϬϭϲ͘Ϭϱ͘Ϭϯϯ

;ϮϬϭϲͿ͘

ϭϲ ŶƐĞůŵŽ͕͘͘ΘDŝƚƌĂŐŽƚƌŝ͕^͘/ŵƉĂĐƚŽĨƉĂƌƟĐůĞĞůĂƐƟĐŝƚLJŽŶƉĂƌƟĐůĞͲďĂƐĞĚĚƌƵŐ delivery systems. Adv Drug Deliv Rev ϭϬϴ͕ϱϭͲϲϳ͕ĚŽŝ͗ϭϬ͘ϭϬϭϲͬũ͘ĂĚĚƌ͘ϮϬϭϲ͘Ϭϭ͘ϬϬϳ

;ϮϬϭϳͿ͘ ϭϳ ĞŶŝŶŐŽ͕<͘͘ΘtĂŶŐ͕z͘>͘&ĐͲƌĞĐĞƉƚŽƌͲŵĞĚŝĂƚĞĚƉŚĂŐŽĐLJƚŽƐŝƐŝƐƌĞŐƵůĂƚĞĚďLJ ŵĞĐŚĂŶŝĐĂůƉƌŽƉĞƌƟĞƐŽĨƚŚĞƚĂƌŐĞƚ͘J Cell Sci ϭϭϱ͕ϴϰϵͲϴϱϲ;ϮϬϬϮͿ͘ ϭϴ ŚƌŝƐƚĞŶƐĞŶ͕͘ et al.ĐĂƟŽŶŝĐǀĂĐĐŝŶĞĂĚũƵǀĂŶƚďĂƐĞĚŽŶĂƐĂƚƵƌĂƚĞĚƋƵĂƚĞƌŶĂƌLJ ĂŵŵŽŶŝƵŵůŝƉŝĚŚĂǀĞĚŝīĞƌĞŶƚŝŶǀŝǀŽĚŝƐƚƌŝďƵƟŽŶŬŝŶĞƟĐƐĂŶĚĚŝƐƉůĂLJĂĚŝƐƟŶĐƚ ϰ d ĐĞůůͲŝŶĚƵĐŝŶŐ ĐĂƉĂĐŝƚLJ ĐŽŵƉĂƌĞĚ ƚŽ ŝƚƐ ƵŶƐĂƚƵƌĂƚĞĚ ĂŶĂůŽŐ͘ J Control Release ϭϲϬ͕ϰϲϴͲϰϳϲ͕ĚŽŝ͗ϭϬ͘ϭϬϭϲͬũ͘ũĐŽŶƌĞů͘ϮϬϭϮ͘Ϭϯ͘Ϭϭϲ;ϮϬϭϮͿ͘ ϭϵ EŽƌůŝŶŐ͕ <͘ et al. 'Ğů WŚĂƐĞ ϭ͕ϮͲŝƐƚĞĂƌŽLJůͲƐŶͲŐůLJĐĞƌŽͲϯͲƉŚŽƐƉŚŽĐŚŽůŝŶĞͲĂƐĞĚ >ŝƉŽƐŽŵĞƐƌĞ^ƵƉĞƌŝŽƌƚŽ&ůƵŝĚWŚĂƐĞ>ŝƉŽƐŽŵĞƐĂƚƵŐŵĞŶƟŶŐŽƚŚŶƟŐĞŶ WƌĞƐĞŶƚĂƟŽŶ ŽŶ DĂũŽƌ ,ŝƐƚŽĐŽŵƉĂƟďŝůŝƚLJ ŽŵƉůĞdž ůĂƐƐ // ĂŶĚ ŽƐƟŵƵůĂƚŽƌLJ DŽůĞĐƵůĞ ŝƐƉůĂLJ ďLJ ĞŶĚƌŝƟĐ ĞůůƐ ŝŶ sŝƚƌŽ͘ ACS Infect Dis͕ ĚŽŝ͗ϭϬ͘ϭϬϮϭͬ ĂĐƐŝŶĨĞĐĚŝƐ͘ϵďϬϬϭϴϵ;ϮϬϭϵͿ͘

ϮϬ zĂƐƵĚĂ͕ d͕͘ ĂŶĐĞLJ͕ '͘ &͘ Θ <ŝŶƐŬLJ͕ ^͘ ͘ /ŵŵƵŶŽŐĞŶŝĐŝƚLJ ŽĨ ůŝƉŽƐŽŵĂů ŵŽĚĞů ŵĞŵďƌĂŶĞƐŝŶŵŝĐĞ͗ĚĞƉĞŶĚĞŶĐĞŽŶƉŚŽƐƉŚŽůŝƉŝĚĐŽŵƉŽƐŝƟŽŶ͘Proc Natl Acad Sci U S A ϳϰ͕ϭϮϯϰͲϭϮϯϲ͕ĚŽŝ͗ϭϬ͘ϭϬϳϯͬƉŶĂƐ͘ϳϰ͘ϯ͘ϭϮϯϰ;ϭϵϳϳͿ͘ Ϯϭ ĂŶĐĞLJ͕ '͘ &͕͘ zĂƐƵĚĂ͕ d͘ Θ <ŝŶƐŬLJ͕ ^͘ ͘ īĞĐƚ ŽĨ ůŝƉŽƐŽŵĂů ŵŽĚĞů ŵĞŵďƌĂŶĞ ĐŽŵƉŽƐŝƟŽŶŽŶŝŵŵƵŶŽŐĞŶŝĐŝƚLJ͘J Immunol ϭϮϬ͕ϭϭϬϵͲϭϭϭϯ;ϭϵϳϴͿ͘ ϮϮ ĂŬŽƵĐŚĞ͕ K͘ Θ 'ĞƌůŝĞƌ͕ ͘ ŶŚĂŶĐĞŵĞŶƚ ŽĨ ŝŵŵƵŶŽŐĞŶŝĐŝƚLJ ŽĨ ƚƵŵŽƵƌ ǀŝƌƵƐ ĂŶƟŐĞŶďLJůŝƉŽƐŽŵĞƐ͗ƚŚĞĞīĞĐƚŽĨůŝƉŝĚĐŽŵƉŽƐŝƟŽŶ͘Immunology ϱϴ, 507-513 ;ϭϵϴϲͿ͘ Ϯϯ DŽƵƌŝƚƐĞŶ͕K͘'͘Θ:ŽƌŐĞŶƐĞŶ͕<͘LJŶĂŵŝĐĂůŽƌĚĞƌĂŶĚĚŝƐŽƌĚĞƌŝŶůŝƉŝĚďŝůĂLJĞƌƐ͘ Chem Phys Lipids ϳϯ͕ϯͲϮϱ͕ĚŽŝ͗ϭϬ͘ϭϬϭϲͬϬϬϬϵͲϯϬϴϰ;ϵϰͿϵϬϭϳϭͲϲ;ϭϵϵϰͿ͘

Ϯϰ ĞŵĞƚnjŽƐ͕͘ŝīĞƌĞŶƟĂů^ĐĂŶŶŝŶŐĂůŽƌŝŵĞƚƌLJ;^Ϳ͗ĂƚŽŽůƚŽƐƚƵĚLJƚŚĞƚŚĞƌŵĂů behavior of lipid bilayers and liposomal stability. J Liposome Res ϭϴ, 159-173,

ĚŽŝ͗ϭϬ͘ϭϬϴϬͬϬϴϵϴϮϭϬϬϴϬϮϯϭϬϮϲϭ;ϮϬϬϴͿ͘

Ϯϱ DĂƚƐŝŶŐŽƵ͕͘ΘĞŵĞƚnjŽƐ͕͘ĂůŽƌŝŵĞƚƌŝĐƐƚƵĚLJŽŶƚŚĞŝŶĚƵĐƟŽŶŽĨŝŶƚĞƌĚŝŐŝƚĂƚĞĚ ƉŚĂƐĞŝŶŚLJĚƌĂƚĞĚWWďŝůĂLJĞƌƐďLJďŝŽĂĐƟǀĞůĂďĚĂŶĞƐĂŶĚĐŽƌƌĞůĂƟŽŶƚŽƚŚĞŝƌ liposome stability: The role of chemical structure. Chem Phys Lipids ϭϰϱ, 45-62,

ĚŽŝ͗ϭϬ͘ϭϬϭϲͬũ͘ĐŚĞŵƉŚLJƐůŝƉ͘ϮϬϬϲ͘ϭϬ͘ϬϬϰ;ϮϬϬϳͿ͘

Ϯϲ ^ƉLJƌĂƚŽƵ͕ ͕͘ DŽƵƌĞůĂƚŽƵ͕ ͘ ͕͘ DĂŬƌŽƉŽƵůŽƵ͕ D͘ Θ ĞŵĞƚnjŽƐ͕ ͘ ƚŽŵŝĐ force microscopy: a tool to study the structure, dynamics and stability of liposomal drug delivery systems. Expert Opin Drug Deliv ϲ, 305-317,

ĚŽŝ͗ϭϬ͘ϭϱϭϳͬϭϳϰϮϱϮϰϬϵϬϮϴϮϴϯϭϮ;ϮϬϬϵͿ͘

Ϯϳ sĂƌLJƉĂƚĂŬŝ͕͘D͕͘ĞŶŶĞ͕E͕͘ŽƵǁƐƚƌĂ͕:͕͘:ŝƐŬŽŽƚ͕t͘ΘKƐƐĞŶĚŽƌƉ͕&͘ĸĐŝĞŶƚ ƌĂĚŝĐĂƟŽŶŽĨƐƚĂďůŝƐŚĞĚdƵŵŽƌƐŝŶDŝĐĞǁŝƚŚĂƟŽŶŝĐ>ŝƉŽƐŽŵĞͲĂƐĞĚ^LJŶƚŚĞƟĐ >ŽŶŐͲWĞƉƟĚĞ sĂĐĐŝŶĞƐ͘ Cancer Immunol Res ϱ͕ ϮϮϮͲϮϯϯ͕ ĚŽŝ͗ϭϬ͘ϭϭϱϴͬϮϯϮϲͲ ϲϬϲϲ͘/ZͲϭϲͲϬϮϴϯ;ϮϬϭϳͿ͘

Ϯϴ &ŝůŝƉĞ͕ s͕͘ ,ĂǁĞ͕ ͘ Θ :ŝƐŬŽŽƚ͕ t͘ ƌŝƟĐĂů ĞǀĂůƵĂƟŽŶ ŽĨ EĂŶŽƉĂƌƟĐůĞ dƌĂĐŬŝŶŐ ŶĂůLJƐŝƐ;EdͿďLJEĂŶŽ^ŝŐŚƚĨŽƌƚŚĞŵĞĂƐƵƌĞŵĞŶƚŽĨŶĂŶŽƉĂƌƟĐůĞƐĂŶĚƉƌŽƚĞŝŶ aggregates. Pharm Res Ϯϳ͕ϳϵϲͲϴϭϬ͕ĚŽŝ͗ϭϬ͘ϭϬϬϳͬƐϭϭϬϵϱͲϬϭϬͲϬϬϳϯͲϮ;ϮϬϭϬͿ͘

3

ĂĚũƵǀĂŶƚĞĚǀĂĐĐŝŶĞĨŽƌŝŶĚƵĐƟŽŶŽĨĂŶƟŐĞŶͲƐƉĞĐŝĮĐdĐĞůůĐLJƚŽƚŽdžŝĐŝƚLJ͘AAPS J

ϭϳ͕ϮϭϲͲϮϮϲ͕ĚŽŝ͗ϭϬ͘ϭϮϬϴͬƐϭϮϮϰϴͲϬϭϰͲϵϲϴϲͲϰ;ϮϬϭϱͿ͘

ϯϬ ǀĂŶĚĞƌDĂĂĚĞŶ͕<͕͘^ůŝĞĚƌĞŐƚ͕<͕͘<ƌŽƐ͕͕͘:ŝƐŬŽŽƚ͕t͘ΘŽƵǁƐƚƌĂ͕:͘&ůƵŽƌĞƐĐĞŶƚ ŶĂŶŽƉĂƌƟĐůĞ ĂĚŚĞƐŝŽŶ ĂƐƐĂLJ͗ Ă ŶŽǀĞů ŵĞƚŚŽĚ ĨŽƌ ƐƵƌĨĂĐĞ Ɖ<Ă ĚĞƚĞƌŵŝŶĂƟŽŶ of self-assembled monolayers on silicon surfaces. Langmuir Ϯϴ, 3403-3411,

ĚŽŝ͗ϭϬ͘ϭϬϮϭͬůĂϮϬϯϱϲϬŬ;ϮϬϭϮͿ͘

ϯϭ ,ŽǁĂƌƚĞƌ͕ :͘ ͘ Θ zŽƵŶŐďůŽŽĚ͕ :͘ W͘ KƉƟŵŝnjĂƟŽŶ ŽĨ ƐŝůŝĐĂ ƐŝůĂŶŝnjĂƟŽŶ ďLJ ϯͲĂŵŝŶŽƉƌŽƉLJůƚƌŝĞƚŚŽdžLJƐŝůĂŶĞ͘ Langmuir ϮϮ͕ ϭϭϭϰϮͲϭϭϭϰϳ͕ ĚŽŝ͗ϭϬ͘ϭϬϮϭͬ ůĂϬϲϭϮϰϬŐ;ϮϬϬϲͿ͘

32 Duarte, A. A. et al.ĚƐŽƌƉƟŽŶŬŝŶĞƟĐƐŽĨWW'ůŝƉŽƐŽŵĞůĂLJĞƌƐ͗ĂƋƵĂŶƟƚĂƟǀĞ analysis of surface roughness. Microsc Microanal ϭϵ͕ ϴϲϳͲϴϳϱ͕ ĚŽŝ͗ϭϬ͘ϭϬϭϳͬ

^ϭϰϯϭϵϮϳϲϭϯϬϬϭϲϮϭ;ϮϬϭϯͿ͘

ϯϯ :W</ŶƐƚƌƵŵĞŶƚƐ'ƉƉůŝĐĂƟŽŶEŽƚĞ͘

ϯϰ >ŝŶ͕͕͘͘ŝŵŝƚƌŝĂĚŝƐ͕͘<͘Θ,ŽƌŬĂLJ͕&͘ZŽďƵƐƚƐƚƌĂƚĞŐŝĞƐĨŽƌĂƵƚŽŵĂƚĞĚ&D ĨŽƌĐĞ ĐƵƌǀĞ ĂŶĂůLJƐŝƐͲͲ/͘ EŽŶͲĂĚŚĞƐŝǀĞ ŝŶĚĞŶƚĂƟŽŶ ŽĨ ƐŽŌ͕ ŝŶŚŽŵŽŐĞŶĞŽƵƐ materials. J Biomech Eng ϭϮϵ͕ϰϯϬͲϰϰϬ͕ĚŽŝ͗ϭϬ͘ϭϭϭϱͬϭ͘ϮϳϮϬϵϮϰ;ϮϬϬϳͿ͘

ϯϱ >ŝĂŶŐ͕y͕͘DĂŽ͕'͘Θ^ŝŵŽŶEŐ͕<͘z͘WƌŽďŝŶŐƐŵĂůůƵŶŝůĂŵĞůůĂƌŐŐWǀĞƐŝĐůĞƐŽŶ mica surface by atomic force microscopy. Colloids Surf B Biointerfaces ϯϰ, 41-51,

ĚŽŝ͗ϭϬ͘ϭϬϭϲͬũ͘ĐŽůƐƵƌĩ͘ϮϬϬϯ͘ϭϬ͘Ϭϭϳ;ϮϬϬϰͿ͘ ϯϲ ůƚƵŶĂLJĂƌ͕ ͕͘ ^ĂŚŝŶ͕ /͘ Θ <ĂnjĂŶĐŝ͕ E͘  ĐŽŵƉĂƌĂƟǀĞ ƐƚƵĚLJ ŽĨ ƚŚĞ ĞīĞĐƚƐ ŽĨ ĐŚŽůĞƐƚĞƌŽů ĂŶĚ ĚĞƐŵŽƐƚĞƌŽů ŽŶ njǁŝƩĞƌŝŽŶŝĐ WW ŵŽĚĞů ŵĞŵďƌĂŶĞƐ͘ Chem Phys Lipids ϭϴϴ͕ϯϳͲϰϱ͕ĚŽŝ͗ϭϬ͘ϭϬϭϲͬũ͘ĐŚĞŵƉŚLJƐůŝƉ͘ϮϬϭϱ͘Ϭϯ͘ϬϬϲ;ϮϬϭϱͿ͘ ϯϳ dĂŬĞĐŚŝͲ,ĂƌĂLJĂ͕ z͕͘ ^ĂŬĂŝͲ<ĂƚŽ͕ <͘ Θ 'ŽĚĂ͕ z͘ DĞŵďƌĂŶĞ ZŝŐŝĚŝƚLJ ĞƚĞƌŵŝŶĞĚ ďLJ ƚŽŵŝĐ &ŽƌĐĞ DŝĐƌŽƐĐŽƉLJ /Ɛ Ă WĂƌĂŵĞƚĞƌ ŽĨ ƚŚĞ WĞƌŵĞĂďŝůŝƚLJ ŽĨ >ŝƉŽƐŽŵĂů DĞŵďƌĂŶĞƐ ƚŽ ƚŚĞ ,LJĚƌŽƉŚŝůŝĐ ŽŵƉŽƵŶĚ ĂůĐĞŝŶ͘ AAPS PharmSciTech ϭϴ, ϭϴϴϳͲϭϴϵϯ͕ĚŽŝ͗ϭϬ͘ϭϮϬϴͬƐϭϮϮϰϵͲϬϭϲͲϬϲϮϰͲdž;ϮϬϭϳͿ͘ ϯϴ ŚĂŽ͕>͕͘&ĞŶŐ͕^͘^͕͘<ŽĐŚĞƌŐŝŶƐŬLJ͕E͘Θ<ŽƐƚĞƚƐŬŝ͕/͘^ĂŶĚWZŝŶǀĞƐƟŐĂƟŽŶƐŽŶ ĞīĞĐƚƐŽĨĐŚŽůĞƐƚĞƌŽůĐŽŵƉŽŶĞŶƚŽŶŵŽůĞĐƵůĂƌŝŶƚĞƌĂĐƟŽŶƐďĞƚǁĞĞŶƉĂĐůŝƚĂdžĞů and phospholipid within lipid bilayer membrane. Int J Pharm ϯϯϴ, 258-266,

ĚŽŝ͗ϭϬ͘ϭϬϭϲͬũ͘ŝũƉŚĂƌŵ͘ϮϬϬϳ͘Ϭϭ͘Ϭϰϱ;ϮϬϬϳͿ͘

ϯϵ ůŽLJ͕ :͘ K͘ et al. ŽͲůŽĂĚĞĚ ƉĂĐůŝƚĂdžĞůͬƌĂƉĂŵLJĐŝŶ ůŝƉŽƐŽŵĞƐ͗ ĞǀĞůŽƉŵĞŶƚ͕ ĐŚĂƌĂĐƚĞƌŝnjĂƟŽŶĂŶĚŝŶǀŝƚƌŽĂŶĚŝŶǀŝǀŽĞǀĂůƵĂƟŽŶĨŽƌďƌĞĂƐƚĐĂŶĐĞƌƚŚĞƌĂƉLJ͘ Colloids Surf B Biointerfaces ϭϰϭ͕ ϳϰͲϴϮ͕ ĚŽŝ͗ϭϬ͘ϭϬϭϲͬũ͘ĐŽůƐƵƌĩ͘ϮϬϭϲ͘Ϭϭ͘ϬϯϮ

;ϮϬϭϲͿ͘

ϰϬ dŽLJƌĂŶ͕ E͘ Θ ^ĞǀĞƌĐĂŶ͕ &͘ /ŶƚĞƌĂĐƟŽŶ ďĞƚǁĞĞŶ ǀŝƚĂŵŝŶ Ϯ ĂŶĚ ŵĂŐŶĞƐŝƵŵ ŝŶ ůŝƉŽƐŽŵĞƐ͗ ŝīĞƌĞŶƟĂů ƐĐĂŶŶŝŶŐ ĐĂůŽƌŝŵĞƚƌLJ ĂŶĚ &d/Z ƐƉĞĐƚƌŽƐĐŽƉLJ ƐƚƵĚŝĞƐ͘ Journal of Molecular Structure ϴϯϵ͕ϭϵͲϮϳ͕ĚŽŝ͗ϭϬ͘ϭϬϭϲͬũ͘ŵŽůƐƚƌƵĐ͘ϮϬϬϲ͘ϭϭ͘ϬϬϱ

;ϮϬϬϳͿ͘

ϰϭ ǀĂŶĚĞŶĞƌŐŚ͕͕͘͘tĞƌƚnj͕W͘t͕͘:ƵŶŐŝŶŐĞƌ͕,͘͘ΘŽƵǁƐƚƌĂ͕:͘͘ůĂƐƟĐŝƚLJŽĨ ǀĞƐŝĐůĞƐĂƐƐĞƐƐĞĚďLJĞůĞĐƚƌŽŶƐƉŝŶƌĞƐŽŶĂŶĐĞ͕ĞůĞĐƚƌŽŶŵŝĐƌŽƐĐŽƉLJĂŶĚĞdžƚƌƵƐŝŽŶ measurements. Int J Pharm Ϯϭϳ͕ ϭϯͲϮϰ͕ ĚŽŝ͗ϭϬ͘ϭϬϭϲͬƐϬϯϳϴͲϱϭϳϯ;ϬϭͿϬϬϱϳϲͲϮ

;ϮϬϬϭͿ͘

ϰϮ DLJĞƌƐŽŶ͕ :͘ t͘ et al. &ůĞdžŝďůĞ EĂŶŽƉĂƌƟĐůĞƐ ZĞĂĐŚ ^ƚĞƌŝĐĂůůLJ KďƐĐƵƌĞĚ ŶĚŽƚŚĞůŝĂůdĂƌŐĞƚƐ/ŶĂĐĐĞƐƐŝďůĞƚŽZŝŐŝĚEĂŶŽƉĂƌƟĐůĞƐ͘Adv Mater ϯϬ, e1802373, ĚŽŝ͗ϭϬ͘ϭϬϬϮͬĂĚŵĂ͘ϮϬϭϴϬϮϯϳϯ;ϮϬϭϴͿ͘

ĨƌŽŵĐŽŶĚĞŶƐĞĚďŝůĂLJĞƌƐƚŽůŝƉŝĚƌĂŌƐ͘Acc Chem Res ϰϳ͕ϯϱϭϮͲϯϱϮϭ͕ĚŽŝ͗ϭϬ͘ϭϬϮϭͬ ĂƌϱϬϬϮϲϬƚ;ϮϬϭϰͿ͘

ϰϰ dĂŬĞĐŚŝͲ,ĂƌĂLJĂ͕ z͘ et al. ƚŽŵŝĐ &ŽƌĐĞ DŝĐƌŽƐĐŽƉŝĐ ŶĂůLJƐŝƐ ŽĨ ƚŚĞ īĞĐƚ ŽĨ >ŝƉŝĚ ŽŵƉŽƐŝƟŽŶ ŽŶ >ŝƉŽƐŽŵĞ DĞŵďƌĂŶĞ ZŝŐŝĚŝƚLJ͘ Langmuir ϯϮ, 6074-6082, ĚŽŝ͗ϭϬ͘ϭϬϮϭͬĂĐƐ͘ůĂŶŐŵƵŝƌ͘ϲďϬϬϳϰϭ;ϮϬϭϲͿ͘

45 Delorme, N. & Fery, A. Direct method to study membrane rigidity of small vesicles based on atomic force microscope force spectroscopy. Phys Rev E Stat EŽŶůŝŶ^ŽŌDĂƩĞƌWŚLJƐ ϳϰ͕ϬϯϬϵϬϭ͕ĚŽŝ͗ϭϬ͘ϭϭϬϯͬWŚLJƐZĞǀ͘ϳϰ͘ϬϯϬϵϬϭ;ϮϬϬϲͿ͘ ϰϲ EĂŬĂŶŽ͕ <͕͘ dŽnjƵŬĂ͕ z͕͘ zĂŵĂŵŽƚŽ͕ ,͕͘ <ĂǁĂƐŚŝŵĂ͕ z͘ Θ dĂŬĞƵĐŚŝ͕ ,͘  ŶŽǀĞů

method for measuring rigidity of submicron-size liposomes with atomic force microscopy. Int J Pharm ϯϱϱ͕ ϮϬϯͲϮϬϵ͕ ĚŽŝ͗ϭϬ͘ϭϬϭϲͬũ͘ŝũƉŚĂƌŵ͘ϮϬϬϳ͘ϭϮ͘Ϭϭϴ

;ϮϬϬϴͿ͘ ϰϳ dĂŬĞĐŚŝͲ,ĂƌĂLJĂ͕z͕͘'ŽĚĂ͕z͘Θ^ĂŬĂŝͲ<ĂƚŽ͕<͘ƚŽŵŝĐ&ŽƌĐĞDŝĐƌŽƐĐŽƉLJ^ƚƵĚLJŽŶ ƚŚĞ^ƟīŶĞƐƐŽĨEĂŶŽƐŝnjĞĚ>ŝƉŽƐŽŵĞƐŽŶƚĂŝŶŝŶŐŚĂƌŐĞĚ>ŝƉŝĚƐ͘Langmuir ϯϰ, ϳϴϬϱͲϳϴϭϮ͕ĚŽŝ͗ϭϬ͘ϭϬϮϭͬĂĐƐ͘ůĂŶŐŵƵŝƌ͘ϴďϬϭϭϮϭ;ϮϬϭϴͿ͘ ϰϴ dĂŬĞĐŚŝͲ,ĂƌĂLJĂ͕z͕͘'ŽĚĂ͕z͕͘/njƵƚƐƵ͕<͘Θ^ĂŬĂŝͲ<ĂƚŽ͕<͘/ŵƉƌŽǀĞĚƚŽŵŝĐ&ŽƌĐĞ DŝĐƌŽƐĐŽƉLJ ^ƟīŶĞƐƐ DĞĂƐƵƌĞŵĞŶƚƐ ŽĨ EĂŶŽƐĐĂůĞ >ŝƉŽƐŽŵĞƐ ďLJ ĂŶƟůĞǀĞƌ dŝƉ^ŚĂƉĞǀĂůƵĂƟŽŶ͘ŶĂůLJƟĐĂůŚĞŵŝƐƚƌLJ ϵϭ͕ϭϬϰϯϮͲϭϬϰϰϬ͕ĚŽŝ͗ϭϬ͘ϭϬϮϭͬĂĐƐ͘ ĂŶĂůĐŚĞŵ͘ϵďϬϬϮϱϬ;ϮϬϭϵͿ͘ ϰϵ <ƵnjŶĞƚƐŽǀĂ͕ d͘ '͕͘ ^ƚĂƌŽĚƵďƚƐĞǀĂ͕ D͘ E͕͘ zĞŐŽƌĞŶŬŽǀ͕ E͘ /͕͘ ŚŝnjŚŝŬ͕ ^͘ ͘ Θ ŚĚĂŶŽǀ͕Z͘/͘ƚŽŵŝĐĨŽƌĐĞŵŝĐƌŽƐĐŽƉLJƉƌŽďŝŶŐŽĨĐĞůůĞůĂƐƟĐŝƚLJ͘Micron ϯϴ, 824-ϴϯϯ͕ĚŽŝ͗ϭϬ͘ϭϬϭϲͬũ͘ŵŝĐƌŽŶ͘ϮϬϬϳ͘Ϭϲ͘Ϭϭϭ;ϮϬϬϳͿ͘ ϱϬ ZŚŽ͕ :͘ z͕͘ ƐŚŵĂŶ͕ Z͘ ͘ Θ dƵƌŶĞƌ͕ ͘ ,͘ zŽƵŶŐ͛Ɛ ŵŽĚƵůƵƐ ŽĨ ƚƌĂďĞĐƵůĂƌ ĂŶĚ ĐŽƌƟĐĂů ďŽŶĞ ŵĂƚĞƌŝĂů͗ ƵůƚƌĂƐŽŶŝĐ ĂŶĚ ŵŝĐƌŽƚĞŶƐŝůĞ ŵĞĂƐƵƌĞŵĞŶƚƐ͘ J Biomech Ϯϲ͕ϭϭϭͲϭϭϵ͕ĚŽŝ͗ϭϬ͘ϭϬϭϲͬϬϬϮϭͲϵϮϵϬ;ϵϯͿϵϬϬϰϮͲĚ;ϭϵϵϯͿ͘ ϱϭ zŝ͕y͕͘^Śŝ͕y͘Θ'ĂŽ͕,͘ĞůůƵůĂƌƵƉƚĂŬĞŽĨĞůĂƐƟĐŶĂŶŽƉĂƌƟĐůĞƐ͘WŚLJƐZĞǀ>ĞƩ ϭϬϳ, ϬϵϴϭϬϭ͕ĚŽŝ͗ϭϬ͘ϭϭϬϯͬWŚLJƐZĞǀ>ĞƩ͘ϭϬϳ͘ϬϵϴϭϬϭ;ϮϬϭϭͿ͘ ϱϮ <ĂƵƌ͕ Z͘ et al. īĞĐƚ ŽĨ ŝŶĐŽƌƉŽƌĂƟŶŐ ĐŚŽůĞƐƚĞƌŽů ŝŶƚŽ ͗d ůŝƉŽƐŽŵĂů ĂĚũƵǀĂŶƚƐ ŽŶ ďŝůĂLJĞƌ ƉƌŽƉĞƌƟĞƐ͕ ďŝŽĚŝƐƚƌŝďƵƟŽŶ͕ ĂŶĚ ŝŵŵƵŶĞ ƌĞƐƉŽŶƐĞƐ͘ Mol Pharm ϭϭ͕ϭϵϳͲϮϬϳ͕ĚŽŝ͗ϭϬ͘ϭϬϮϭͬŵƉϰϬϬϯϳϮũ;ϮϬϭϰͿ͘ ϱϯ 'ƵŽ͕W͘ et al.EĂŶŽƉĂƌƟĐůĞĞůĂƐƟĐŝƚLJĚŝƌĞĐƚƐƚƵŵŽƌƵƉƚĂŬĞ͘Nat Commun ϵ, 130, ĚŽŝ͗ϭϬ͘ϭϬϯϴͬƐϰϭϰϲϳͲϬϭϳͲϬϮϱϴϴͲϵ;ϮϬϭϴͿ͘ ϱϰ 'ĂƌŶŝĞƌ͕&͕͘&ŽƌƋƵĞƚ͕&͕͘ĞƌƚŽůŝŶŽ͕W͘Θ'ĞƌůŝĞƌ͕͘ŶŚĂŶĐĞŵĞŶƚŽĨŝŶǀŝǀŽĂŶĚŝŶ ǀŝƚƌŽdĐĞůůƌĞƐƉŽŶƐĞĂŐĂŝŶƐƚŵĞĂƐůĞƐǀŝƌƵƐŚĂĞŵĂŐŐůƵƟŶŝŶĂŌĞƌŝƚƐŝŶĐŽƌƉŽƌĂƟŽŶ ŝŶƚŽ ůŝƉŽƐŽŵĞƐ͗ ĞīĞĐƚ ŽĨ ƚŚĞ ƉŚŽƐƉŚŽůŝƉŝĚ ĐŽŵƉŽƐŝƟŽŶ͘ Vaccine ϵ, 340-345, ĚŽŝ͗ϭϬ͘ϭϬϭϲͬϬϮϲϰͲϰϭϬdž;ϵϭͿϵϬϬϲϭͲĂ;ϭϵϵϭͿ͘ ϱϱ DĂnjƵŵĚĂƌ͕ d͕͘ ŶĂŵ͕ <͘ Θ ůŝ͕ E͘ /ŶŇƵĞŶĐĞ ŽĨ ƉŚŽƐƉŚŽůŝƉŝĚ ĐŽŵƉŽƐŝƟŽŶ ŽŶ ƚŚĞĂĚũƵǀĂŶƟĐŝƚLJĂŶĚƉƌŽƚĞĐƟǀĞĞĸĐĂĐLJŽĨůŝƉŽƐŽŵĞͲĞŶĐĂƉƐƵůĂƚĞĚ>ĞŝƐŚŵĂŶŝĂ ĚŽŶŽǀĂŶŝĂŶƟŐĞŶƐ͘J Parasitol ϵϭ͕ϮϲϵͲϮϳϰ͕ĚŽŝ͗ϭϬ͘ϭϲϰϱͬ'ͲϯϱϲZϭ;ϮϬϬϱͿ͘ ϱϲ ŚĂŶŐ͕ z͘ W͕͘ >ĞǁŝƐ͕ Z͘ E͘ Θ DĐůŚĂŶĞLJ͕ Z͘ E͘ ĂůŽƌŝŵĞƚƌŝĐ ĂŶĚ ƐƉĞĐƚƌŽƐĐŽƉŝĐ

studies of the thermotropic phase behavior of the n-saturated ϭ͕ϮͲĚŝĂĐLJůƉŚŽƐƉŚĂƟĚLJůŐůLJĐĞƌŽůƐ͘ Biophys J ϳϮ͕ ϳϳϵͲϳϵϯ͕ ĚŽŝ͗ϭϬ͘ϭϬϭϲͬƐϬϬϬϲͲ ϯϰϵϱ;ϵϳͿϳϴϳϭϮͲϱ;ϭϵϵϳͿ͘

ϱϳ ŝůƚŽŶĞŶ͕Z͘>͘Θ>ŝĐŚƚĞŶďĞƌŐ͕͘dŚĞhƐĞŽĨŝīĞƌĞŶƟĂů^ĐĂŶŶŝŶŐĂůŽƌŝŵĞƚƌLJĂƐ ĂdŽŽůƚŽŚĂƌĂĐƚĞƌŝnjĞ>ŝƉŽƐŽŵĞWƌĞƉĂƌĂƟŽŶƐ͘Chemistry and Physics of Lipids

3

ϱϴ hůƌŝĐŚ͕ ͘ ^͕͘ ^Ăŵŝ͕ D͘ Θ tĂƩƐ͕ ͘ ,LJĚƌĂƟŽŶ ŽĨ KW ďŝůĂLJĞƌƐ ďLJ ĚŝīĞƌĞŶƟĂů scanning calorimetry. Biochim Biophys Acta ϭϭϵϭ͕ϮϮϱͲϮϯϬ͕ĚŽŝ͗ϭϬ͘ϭϬϭϲͬϬϬϬϱͲ

Ϯϳϯϲ;ϵϰͿϵϬϮϱϯͲϰ;ϭϵϵϰͿ͘

&ŝŐƵƌĞ^Ϯ͘>ŝƉŽƐŽŵĂůĂƐƐŽĐŝĂƟŽŶ;ƵƉƚĂŬĞĂŶĚͬŽƌĂĚƐŽƌƉƟŽŶͿďLJDƐin vitro͘Ks₃₂₃ -ĐŽŶƚĂŝŶŝŶŐŇƵŽƌĞƐĐĞŶƚůLJůĂďĞůĞĚůŝƉŽƐŽŵĞƐǁĞƌĞŝŶĐƵďĂƚĞĚĨŽƌϰŚŽƵƌƐǁŝƚŚDƐĂƚϰΣ and ϯϳΣ͘>ŝƉŽƐŽŵĞƐǁĞƌĞƐƵďƐĞƋƵĞŶƚůLJǁĂƐŚĞĚĂǁĂLJĂŶĚĐĞůůƐǁĞƌĞŝŶĐƵďĂƚĞĚŽǀĞƌŶŝŐŚƚďĞĨŽƌĞ being analyzed viaŇŽǁĐLJƚŽŵĞƚƌLJ͘йƐƐŽĐŝĂƟŽŶŝŶĚŝĐĂƚĞƐƚŚĞƉĞƌĐĞŶƚĂŐĞŽĨůŝǀĞDƐƚŚĂƚ ĂƌĞƉŽƐŝƟǀĞĨŽƌƚŚĞŇƵŽƌĞƐĐĞŶƚůĂďĞůŝŶƚŚĞůŝƉŽƐŽŵĞƐ͘'ƌĂƉŚƐŚŽǁƐŵĞĂŶц^͕Ŷсϯ͘ΎΎΎΎƉ фϬ͘ϬϬϬϭ͕ĐŽŵƉĂƌŝŶŐϰΣ to 37ΣĚĞƚĞƌŵŝŶĞĚďLJƚǁŽͲǁĂLJEKsĂŶĚŽŶĨĞƌƌŽŶŝ͛ƐŵƵůƟƉůĞ comparisons test.

&ŝŐƵƌĞ^ϯ͘dŚϭĂŶĚdŚϮƌĞƐƉŽŶƐĞƐŝŶƐƉůĞĞŶƐŽĨŵŝĐĞϴĚĂLJƐĂŌĞƌŝ͘ǀ͘ŝŶũĞĐƟŽŶŽĨKs₃₂₃-containing ůŝƉŽƐŽŵĞƐ͘;ͿD&/dďĞƚĂŶĚ;ͿD&/'dϯŽĨϰϱ͘ϭ+_ϰ+ _{T cells. Graphs show mean ± SD, n}

сϴ͘EŽƐŝŐŶŝĮĐĂŶƚĚŝīĞƌĞŶĐĞƐďĞƚǁĞĞŶŐƌŽƵƉƐĂƐĂŶĂůLJnjĞĚďLJŽŶĞͲǁĂLJEKsĂŶĚŽŶĨĞƌƌŽŶŝ͛Ɛ ŵƵůƟƉůĞĐŽŵƉĂƌŝƐŽŶƐƚĞƐƚ͘