Blood protein coating of gold nanoparticles as potential tool for organ targeting Martin Schäffler, Fernanda Sousa, Alexander Wenk, Leopoldo Sitia, Stephanie Hirn, Carsten Schleh, Nadine Haberl, Martina Violatto, Mara Canovi, Patrizia Andreozzi, Mario Salmona, Paolo Bigini, Wolfgang G. Kreyling, Silke Krol Biomaterials, 2014, 35, Impact factor : Speaker : Ju-Yi Mao Adviser : Dr. Han-Jia Lin Date :

Abstract 2

Introduction 3

Gene therapy 4 Cancer cellNormal cell Specific ?? Biochemical Journal (2005) Volume 387, 1-15 NP Recognition molecular Nanoparticles (NP) specific targeting to cell by surface modification But…in vivo?! Traditional method Novel delivery system Ex : liposome More Stable !! More Specific !!

The problem of nanoparticles injected in blood 5 Expected Outcome True Outcome A C Recognition molecular Chem. Commun., 2015, 51, Molecular composition of whole blood Chem. Commun., 2015, 51, over 1100 unique proteins Nanoparticles exposed to the blood, rapidly attract many proteins to surface Off targeting

Protein corona formation  In biological fluid, proteins associate with NPs, and proteins on surface of the particles leads to an in vivo response  Corona variations depend on properties of nanomaterials and environment factors  size, shape, surface charge, surface functional groups, hydrophilicity / hydrophobicity  protein source, temperature variations, pH 6 Protein-Nanoparticle Interactions, Springer Series in Biophysics 15 PNAS, 2007, vol. 104, no. 7 Low affinity (high abundance) High affinity (low abundance) Dynamic equilibrium

Surface of NP to form protein corona upon exposure to biological environments 7  In dynamic environment  convection and cellular metabolism constantly change blood content  NP physicochemical, exposure time, the local environment drive this process  Human serum albumin and apolipoprotein E  frequently found in the protein corona of NP exposed to blood serum  supposed to influence the biodistribution PLoS One, 2010, 5, e10949 HeartBrainLungBlood J Am Chem Soc, 2011, 133, Nanotechnology, 2013, 24,

Human serum albumin (HSA)  Most abundant blood protein  Maitains osmotic pressure in blood and tissues  Transporter molecule for hydrophobic molecules  hormones and fatty acids  Albumin binding receptors distributed on endothelial cells of vasculature  liver, lungs, skeletal muscles, adipose tissue, brain, and heart  Means that alb-Au NP less phagocytized by macrophages of reticuloendothelial system (RES)  RES is a part of the immune system  consists of the phagocytic cells, located in reticular connective tissue 8 Negative charge ， pI 4.7 ， 67 kDa Br J Anaesth, 2000, 85, J Cell Biol, 1988, 107,

Apolipoprotein E (apoE)  Mainly transport fatty acid, cholesterol  Along with apoB100, as a ligand  high affinity binds to the low density lipoprotein (LDL) receptor  binds to the VLDL receptor and LDL receptor-related protein 1  VLDL receptor  Highly expressed : heart, muscle, adipose tissue, brain  Barely present : liver  LDL receptor  Highly expressed : liver 9 Negative charge ， pI 5.3 ， 34 kDa clathrin-coated pits

GAP  First time biokinetic and histology of stable protein-AuNP-conjugates  Demonstrates different accumulation and retention pattern in various organs 10 Protein-NP Where protein-NP go?!

Results 11

Scheme of successive steps of the in vivo experiments for quantitative biokinetic 12 Fig.1 Gamma ray 198 AuNP administration by in vivo Three time points  30 min 、 19 hr 、 48 hr Gamma-spectroscopic radio-analysis C57Bl/6 mice

Prepare of 15 nm Au NP and radioactively labeled by neutron irradiation 13 Anal. Chem., 1949, 21, 475 cit-AuNP Au HAuCl 4 citrate Au nuclear research reactor neutron irradiation 198 Au Au neutron

+ - Layer-by-layer (LbL)  LbL technique was established by Iler in 1966  Polyelectrolyte assembling on surface is induced by electrostatic interactions between the oppositely charged polyelectrolytes  More functionalize surfaces, stable, low cost and environmentally friendly 14 Nano Lett., Vol. 4, No. 10, 2004 Chem. Soc. Rev., 2012, 41, 7291–7321 sodium citrate only 19 layers Proc. Combust. Inst., 2011, 33, 3447– polystyrene sulfonate (PSS) poly-allylamine hydrochloride (PAH) PAH / PSS J. Colloid Interface Sci., 1966, 21, 569–594

Au Layer-by-layer functionalization of gold nanoparticles 15 Au 198 Au polystyrene sulfonate (PSS) Negative charge poly-allylamine hydrochloride (PAH) Positive charge PSS - Au dropper PAH + HSA - Au 198 Au dropper PSS apoE alb-AuNP (1p2s) apoE-AuNP (1s2p) Au PSS - PAH

Physicochemical parameters of the used Au NP 16 Table 2. HD = hydrodynamic diameter z = zeta potential PSS = polystyrene sulfonate PAH = polyallylamine hydrochloride PAH/PSS electrostatically bind to protein 5-fold increase HD of 15 nm AuNP after coating with protein/polyelectrolyte native form on 15 nm AuNP smaller size and curvature 80 nm AuNP increase in size is negligible protein denature Alb-AuNP (+20 mv)  PAH layer is not completely covered by protein ApoE-AuNP (-52 mv)  partial shielding of PSS charge by protein

Quantitative biokinetics assay ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ 17 Animal experiments per time point and particle type Table 1 Au Cy5.5-alb-co-FITC-PAH/PSS Au NP Histology experiment alb-AuNP (1s2p) apoE-AuNP (1p2s) cit-AuNP (1p2s / 1s2p) Au

19 hr Kinetics of translocation and retention of three kinds of Au NP (15 nm) in murine organs 1/3 18 Fig.2 Liver : cit-AuNP (0.95)  high retention ; alb-AuNP (0.47) and apoE-AuNP  low retention Liver  major accumulation for NP Lung : alb-AuNP  high retention ; cit-AuNP and apoE-AuNP  low retention Spleen : alb-AuNP and apoE-AuNP  high retention ; cit-AuNP  low retention LiverLungs Spleen 30 min 48 hr

Kinetics of translocation and retention of three kinds of Au NP (15 nm) in murine organs 2/3 19 Fig.2 Kidneys Heart Brain Kidney and Heart alb-AuNP  high retention ; apoE-AuNP  slightly higher retention ; cit-AuNP  low retention Brain : alb-AuNP  high retention ; apoE-AuNP  slightly higher retention ; cit-AuNP  low retention albumin modulates the transport across the blood-brain barrier (BBB) apoE enhances the uptake across the BBB to some extent  less effective than albumin

Fig.2 RemainderBloodFat Kinetics of translocation and retention of three kinds of Au NP (15 nm) in murine organs 3/3 20 Remainder and Fat : alb-AuNP  high retention ; apoE-AuNP  slightly higher retention ; cit-AuNP  low retention ApoE : as part of the lipid pathway Blood : alb-AuNP and apoE-AuNP  high retention ; cit-AuNP  low retention alb-AuNP and apoE-AuNP are less effectively cleared from the blood circulation compared to cit-AuNP No other organ is identified as preferred target for apoE-AuNP

Hepatobiliary clearance of three kinds of Au NP (15 nm) at different times after in vivo 21 Fig.3 alb-AuNP and apoE-AuNP indicate after initial clearance  No further clearance occurs during 48 hr The increase HBC of cit-AuNP over the entire time Easier Excretion than both protein conjugated NP Hepato-biliary clearance (HBC) clearance from the liver through the biliary duct into the duodenum the sum of the gastro-intestinal-tract and feces

Biodistribution of 15 nm and 80 nm core diameter of alb-Au NP with cit-Au NP after 19 hr 22 No higher accumulated fractions in lungs and brain of 80 nm alb-AuNP compared with cit-AuNP 15 nm and 80 nm alb-AuNP lower retained fraction in liver higher retained fraction in spleen and blood indicating a prolonged circulation time in blood compared to cit-AuNP Fig.5 Alb-AuNP : 15 nm 80 nm Cit-AuNP : 15 nm 80 nm

Biodistribution of polyelectrolyte coated 15 nm Au NP without protein pre-conjugation 23 Fig.6 Positively charged 1s2p-AuNP and alb-AuNP show the same trend Formed corona on the positively charged polyelectrolyte coated AuNP (1s2p) contains a significant amount Negatively charged polyelectrolyte coated 1p2s-AuNP show no changes in liver, spleen, kidneys and blood 1p2s-AuNP show decrease in lungs and brain retention compared to apoE-AuNP less effective spontaneous apoE binding in blood 0.5% - 1% apoE on silica NP Polyelectrolyte coated AuNP 1s2p (PSS+PAH) 1p2s (PAH+PSS) Protein AuNP : Alb ApoE ACS Nano 2011;5:

Biodistribution of 15 and 80 nm apoE-Au NP with cit-Au NP after 19 hr 24 Fig.7 The influence of particle size on the biodistribution of apoE-AuNP is lower than for alb-AuNP 7.4 fold lower 80 nm apoE-AuNP fraction than 15 nm apoE-AuNP in blood circulation after 19 hr partial denaturation of the protein on the NP surface apoE-AuNP : 15 nm 80 nm Cit-AuNP : 15 nm 80 nm

Images showing the localization of Cy5.5 labeled alb-Au NP in brain and lungs 25 Fig.4 ControlAlb-AuNP 30 minAlb-AuNP 19 hr Brain Lungs Cy5.5-alb-co-FITC-PAH/PSS AuNP 30 min to 19 hr : a robust migration to brain parenchyma (hippocampus) After 19 hr, the signal is still localized in the regions corresponding to the alveoli and the vascular zone Brain : sections of lateral ventricles nuclear dye : Hoechst Lung : circular structures alveoli ventricle layer parenchyma choroid plexus ventricle layer choroid plexus parenchyma circular structures alveoli hippocampus alveoli

Images showing the localization of Cy5.5 labeled alb-Au NP in kidney and liver 26 Fig.4 ControlAlb-AuNP 30 minAlb-AuNP 19 hr Kidney Liver Alb-AuNP accumulation in the vasculature especially in filter organs Signal localized cortical nephrons and more diffused to the cortical tubules after 19 hr in kidneys Signal is found more distributed over the liver parenchyma after 19 hr Kidney : cortical nephrons cortical tubules Liver : Vessels Periphery parenchyma cortical nephrons cortical tubules vessels parenchyma vessels M periphery parenchyma

Conclusion 27

Increases blood circulation time Shifts NP accumulation from liver to spleen Alb-AuNP increases translocation into brain NP targeting of the lungs 28 apoE-AuNPalb-AuNP Au liverlungspleenbrainheartadipose tissueKidney Albumin ✔✔✔✔✔ Apo E ✔✔✔✔ Alb-AuNPMedium (M)HHHHHH ApoE-AuNPMLow (L)HMMMM Cit-AuNPHigh (H)LLLLLL

Discussion 29

30  Histology for 80 nm protein-NP  apoE-AuNP not enhance retention in organ  LDL receptor binding site  HLRKLRKRLLR (pI = )  shielded  Albumin and apo E  only around 70% similarity with the mouse proteins  Attempt to use mouse protein to observe biokinetic in organ  Attempt to try more polyelectrolyte by use of layer-by-layer  No Cy5.5 signal in spleen  Lacking albumin-binding receptors  fluorescence label digested by phagolysosomes of spleen macrophages

Thanks For Your Attention 31 The End

Supplementary 32

Figure S1: Translocation and accumulation from blood circulation to various organs and tissues of three different 15 nm 198 AuNP conjugates 30 min after intravenous injection: cit- 198 AuNP, alb- 198 AuNP and apoE- 198 AuNP. Mean and standard deviation are given; n≥4. (* = p<0.05 apoE-AuNP versus cit-AuNP; + = p<0.05 alb-AuNP versus cit-AuNP) 33

Figure S2: Translocation and accumulation from blood circulation to various organs and tissues of three different 15 nm AuNP conjugates 19 h after intravenous injection: cit- 198 AuNP, alb- 198 AuNP and apoE- 198 AuNP. Mean and standard deviation are given; n≥4. (* = p<0.05 apoE-AuNP versus cit-AuNP; + = p<0.05 alb-AuNP versus cit-AuNP) 34

Figure S3: Translocation and accumulation from blood circulation to various organs and tissues of three different 15 nm 198 AuNP conjugates 48 h after intravenous injection: cit- 198 AuNP, alb- 198 AuNP and apoE- 198 AuNP. Mean and standard deviation are given; n≥4. (* = p<0.05 apoE-AuNP versus cit-AuNP; + = p<0.05 alb-AuNP versus cit-AuNP) 35

Figure S4: Translocation and accumulation from blood circulation to various organs and tissues of 15 nm cit- 198 AuNP 30 min, 19 h and 48 h after intravenous injection. Mean and standard deviation are given; n≥4. 36

Figure S5: Translocation and accumulation from blood circulation to various organs and tissues of 15 nm alb- 198 AuNP 30 min, 19 h and 48 h after intravenous injection. Mean and standard deviation are given; n≥4. 37

Figure S6: Translocation and accumulation from blood circulation to various organs and tissues of 15 nm apoE- 198 AuNP 30 min, 19 h and 48 h after intravenous injection. Mean and standard deviation are given; n≥4. (* = p< min versus 19 h; + = p< min versus 48 h) 38

Figure S7: Translocation and accumulation from blood circulation to various organs and tissues of 15 nm polyelectrolyte coated- 198 AuNP 19 h after intravenous injection. Mean and standard deviation are given; n≥4. (* = p<0.05 1s2p-AuNP versus 1p2s-AuNP) 39

Figure S8: Translocation and accumulation from blood circulation to various organs and tissues of three different 80 nm 198 AuNP conjugates 48 h after intravenous injection: cit- 198 AuNP, alb- 198 AuNP and apoE- 198 AuNP. Mean and standard deviation are given; n≥4. (* = p<0.05 apoE-AuNP versus cit-AuNP; + = p<0.05 alb-AuNP versus cit-AuNP) 40

Protein corona formation 41 Size and surface properties take an influence on the cytotoxicity Nature of the proteins adsorbed onto NP influence uptake and traffic throughout the body After NP injection, low tissue or tumor penetration, and specificity to receptors dramatically limit the success of targeted delivery Advanced Drug Delivery Reviews, 2009, 61, 428–437 Biomaterials, 2010, 31, corona Au-Phos NP 5 nm Au-PEG750 NP Au-PEG10k NP ACS Nano, 2010, 4, ; J Am Chem Soc, 2011, 133, ; ACS Nano, 2011, 5, Provides for the first time biokinetic and histological data of stable protein-AuNP- conjugates

Material and methods 42

An issue when NP administrated into blood vein  Aggregation  unstable  Easy phagocytosis by macrophages  Immune System  Non specific targeting  Protein 、 lipid  Protein corona 43 Chem. Commun., 2015, 51,

Specific binding to target cell  Past  Famous chemical molecule  Polyethylenimine (PEI)  Potential toxicity  Future  Nucleic acid  Advantages : Biocompatibility 、 specificity (aptamer)  Disadvantages : Unstable 、 Hard enter cell and target tissue  Protein  Specificity and more application  Protein corona formation 44 Proc Natl Acad Sci USA, 1995, 92, Adv. Healthcare Mater. 2012, 1, 337–341 Receptor recognition molecular Specific protein More application

Protein corona formation  In biological fluid, proteins associate with NPs, and proteins on surface of the particles leads to an in vivo response  Corona variations depend on properties of nanomaterials and environment factors  size, shape, surface charge, surface functional groups, hydrophilicity / hydrophobicity  protein source, slight temperature variations, pH 45 Protein-Nanoparticle Interactions, Springer Series in Biophysics 15 PNAS, 2007, vol. 104, no. 7 ACS Nano, 2010, 4, 3623–3632 Physiological Response Chem. Soc. Rev., 2012, 41, 2780–2799 Biological Identity distribution Low affinity protein Hard corona soft corona  hard corona replaced by high a ffi nity proteins

Biodistribution of Nanoparticles 46  In dynamic environment  convection and cellular metabolism constantly change blood content  prevent protein corona from reaching equilibrium  Protein corona forms is related to the site of administration and local environment  Human serum albumin (HSA) and apolipoprotein E (apoE)  frequently found in the protein corona of NP exposed to blood serum  supposed to influence the biodistribution PLoS One, 2010, 5, e10949 HeartBrainLungBlood J Am Chem Soc, 2011, 133, Nanotechnology, 2013, 24,

Fluorescein isothiocyanate 47 FITC contributes partial negative charges at normal pH

Cy5.5 dye near-infrared fluorophore labeling of amino-groups Red fluorescent (675 nm/694 nm) Increase resolution NHS group