Targeted Delivery of Nanotherapeutics to the Placenta A thesis submitted to The University of Manchester for the degree of Doctor of Philosophy (PhD) in the Faculty of Medical and Human Sciences. 2016 Anna King School of Medicine Chapter 1 : Introduction .............................................................................................. 21 1.1 Overview ............................................................................................................ 22 1.2 The placenta ....................................................................................................... 25 1.2.1 Human placentation .................................................................................... 26 1.2.2 Placental perfusion ...................................................................................... 30 1.2.3 Placental transport ....................................................................................... 31 1.2.4 Placental endocrinology .............................................................................. 33 1.3 Fetal growth restriction (FGR) ........................................................................... 34 1.3.1 Aetiology of FGR ........................................................................................ 35 1.3.2 Detection of FGR ........................................................................................ 37 1.3.3 Potential therapeutics for FGR .................................................................... 38 1.3.3.1 Low Dose Aspirin (LDA) .................................................................... 38 1.3.3.2 Low molecular weight heparin (LMWH) ............................................ 39 1.3.3.3 Vasodilators ......................................................................................... 40 1.3.3.4 Antioxidants ......................................................................................... 41 1.3.3.5 Insulin-like growth factors (IGFs) ....................................................... 43 1.3.4 The future of therapeutics for FGR ............................................................. 50 1.4 Nanoparticles for drug delivery ......................................................................... 51 1.4.1 Active nanoparticle drug targeting .............................................................. 54 1.4.2 Nanoparticles for use in pregnancy ............................................................. 55 1.5 Tissue-specific homing peptides ........................................................................ 57 1.5.1 Active targeting of tumours using homing peptides ................................... 58 1.5.2 Active targeting of the placenta using homing peptides ............................. 61 1.5.2.1 Possible placental receptors for homing peptide binding .................... 65 2 1.5.3 Active targeting of the placenta using homing peptide decorated nanoparticles ........................................................................................................... 66 1.5.4 Nanoparticle interaction with the placenta .................................................. 68 1.5.5 Liposome interaction with the placenta ...................................................... 69 1.5.5.1 Mechanisms of placental cellular uptake of liposomes ........................ 75 1.5.6 Nanoparticle clearance ................................................................................ 76 1.6 Mouse models of pregnancy .............................................................................. 78 1.6.1 Mouse placentation ..................................................................................... 79 1.6.2 Mouse placental perfusion .......................................................................... 81 1.6.3 Mouse placental endocrinology .................................................................. 83 1.6.4 Mouse models of fetal growth restriction ................................................... 85 1.6.4.1 Placental specific IGF-II transcript knock out mouse model (P0-/+) .... 86 1.7 Summary of the literature................................................................................... 88 1.8 Aims and objectives of the project ..................................................................... 89 Chapter 2 : Methods ..................................................................................................... 91 2.1 Liposome preparation and characterisation ....................................................... 92 2.1.1 Liposome preparation.................................................................................. 92 2.1.2 Size distribution (SD), polydispersity index (PDI), zeta potential (ZP) and stability .................................................................................................................... 95 2.1.3 Quantification of peptide attachment to liposome surface .......................... 96 2.1.4 Quantification of drug encapsulation .......................................................... 97 2.2 Human tissue collection and culture .................................................................. 98 2.2.1 Liposome binding assay .............................................................................. 99 2.3 Animal housing and husbandry ........................................................................ 100 2.3.1 General husbandry .................................................................................... 100 3 2.3.2 Cardiac perfusion ...................................................................................... 102 2.3.3 C57 wild type mouse ................................................................................. 102 2.3.4 P0 mouse model ........................................................................................ 102 2.3.5 Genomic DNA extraction ......................................................................... 103 2.3.6 Polymerase chain reaction (PCR) ............................................................. 103 2.4 Administration of liposomes to pregnant mice ................................................ 105 2.4.1 Qualitative assessment of liposome homing ............................................. 105 2.4.2 Administration of liposomes to C57 mice ................................................ 105 2.4.3 Administration of liposomes to P0 mice ................................................... 106 2.4.4 Quantification of IGF-II in maternal and fetal serum ............................... 107 2.4.5 Examination of placental morphology ...................................................... 107 2.4.6 Quantifying proliferation in harvested placentas and organs .................... 108 2.4.7 Statistical analysis ..................................................................................... 109 2.5 Standard solutions ............................................................................................ 110 2.5.1 Phosphate buffered saline (PBS) ............................................................... 110 2.5.2 Tris-buffered saline (TBS) ........................................................................ 110 2.5.3 Neutral buffered formalin (NBF) .............................................................. 111 2.5.4 Paraformaldehyde (PFA) .......................................................................... 111 Chapter 3 : Targeted Nanotherapeutic Design, Development and Characterisation ....................................................................................................................................... 112 3.1 Introduction ...................................................................................................... 113 3.2 Liposome composition for placental targeting ................................................. 113 3.2.1 Homing peptide attachment ...................................................................... 119 3.2.2 Liposome preparation techniques ............................................................. 120 3.2.3 Liposomal drug encapsulation .................................................................. 122 4 3.3 Aims ................................................................................................................. 124 3.4 Results .............................................................................................................. 125 3.4.1 Characterisation of liposome size distribution (SD), polydispersity index (PDI), zeta potential (ZP) and stability ................................................................. 125 3.4.2 Determining the concentration of free thiol groups on iRGD and CGKRK peptides using Ellman’s reagent............................................................................ 131 3.4.3 Quantifying peptide-liposome conjugation ............................................... 132 3.4.4 Determining the encapsulation efficiency of IGF-II ................................. 135 3.5 Discussion ........................................................................................................ 137 3.5.1 Liposome characterisation ........................................................................ 138 3.5.2 Liposome-peptide conjugation .................................................................. 141 3.5.3 Drug encapsulation.................................................................................... 143 3.6 Summary .......................................................................................................... 144 Chapter 4 : Validation