Cha ra c t e r i z i n g   t he   f o r ce s   t ha t  nanopar1cles  exert  on  cell  membranes  is  a n   i m p o r t a n t   c o m p o n e n t   o f  understanding   their   interac1ons   with  biological   systems,   examining   both   ease  of  penetra1on  and  poten1al  disrup1on  of  healthy   membrane   func1on.   The   DIB  technique   is   one   method   used   to  generate   lipid  bilayers   that   resemble  cell  membranes1,2.  Here,  we  specifically  form  a   DIB   at   a   droplet/hydrogel   interface  (DHB)   using   the   “lipid-‐out”   method,  meaning   the   lipids   are   dissolved   in   the  surrounding   oil   rather   than   within   the  aqueous   droplet1,2.   A   lipid-‐out   DHB   is  

Characteriza1on  of  Nanopar1cle-‐Membrane  Interac1ons  Nicole  Gay1,  Eric  Freeman2,  Xianqiao  Wang2  

1College  of  Engineering,  University  of  Connec1cut,  Storrs,  CT  2College  of  Engineering,  University  of  Georgia,  Athens,  GA  

Abstract  

Results  

Future  Work  Methods  

Background  

Freeman  Lab,  College  of  Engineering,  University  of  Georgia,  Athens,  GA  30602,  USA.  Nanotechnology  and  Biomedicine  REU:  An  Interdisciplinary  NSF  REU  Site  @  UGA.  21  July  2015.  Project  website:  hbp://reu.engr.uga.edu/?p=184  

References  (1)   Bayley,   H.;   Cronin,   B.;   Heron,   A.;   Holden,   M.A.;   Hwang,  W.;  Syeda,  R.;  Thompson,  J.;  Wallace,  M.  Mol.  Biosyst.,  2008,  4,  1191-‐1208.    (2)  Holden,  M.  Methods  in  Cell  Bio.,  2015,  128,  1-‐22.    (3)   Villar,   G.;   Graham,   A.D.;   Bayley,   H.   Science,   2013,   340,  48-‐52.  (4)  Lep1hn,  S.;  Castell,  O.K.;  Cronin,  B.;  Lee,  E.;  Gross,  L.C.M.;  Marshall,   D.P.;   Thompson,   J.R.;   Holden,   M.;   Wallace,   M.I.  Nature  Protocols,  2013,  8,  1048  –  1057.    (5)   Neuberger,   T.;  Schopf,   B.;   Hofmann,   H.;   Hofmann,   M.;  von  Rechenberg,   B.  Jour.   Magne9sm   and   Mag.   Mat.,  2005, 293,  483-‐496.     (6)   Zhang,   L.;   Becton,  M.;  Wang,   X.   J.   Phys.   Chem.   B,   2015,  119,  3786-‐3794.  (7)  Shevkoplyas,  S.S.;  Siegel,  A.C.;  Westervelt,  R.M.;  Pren1ss,  M.G.;  Whitesides,  G.M.  Lab  Chip,  2007,  7,  1294  –  1302.  

The   rapid   development   of   nanotechnology   is   revolu1onizing   the   future   of   medicine.  Nanopar1cles   and   their   interac1ons   with   biological   systems   must   be   comprehensively  characterized  before  employing   them  extensively   in   clinical   applica1ons.  One   component  of  this   characteriza1on   is   understanding   the   forces   they   exert   on   cell   membranes.   Here,   the  droplet   interface   bilayer   (DIB)   technique   is   used   to   form   a   double   lipid   membrane   at   the  interface   between   a   SPION-‐containing   ferrofluid   droplet   and   a   cured   hydrogel.   Ag/AgCl  electrodes  are  inserted  into  the  media  on  either  side  of  the  membrane  to  observe  the  current  signal   generated   when   an   alterna1ng   voltage   is   applied.   Simultaneous   visual   analysis   and  electrical  measurements   are   used   to   characterize   the   forces   exerted   by   superparamagne1c  iron  oxide  nanopar1cles  (SPIONs)  on  the  robust  DIB  within  an  adjustable  magne1c  field.  As  the  strength   of   the   magne1c   field   is   increased,   we   observe   an   increase   in   the   membrane   size  through   a   magne1cally-‐driven   weqng   effect.     At   the   maximum   magne1c   field   strength,  liposome   forma1on   events   are   measured   as   the   magne1c   fluid   is   pulled   through   the  membrane.  Membrane  recovery  is  observed  when  it  is  distanced  from  the  magnet.    

Figure  9.  Coarse-‐grained  molecular  dynamics   simula1ons  of   nanopar1cles   with   four   different   copolymer   ligand  coa1ngs  passing  through  a  cell  membrane6.    

Figure  4.  Geometry  of  a  SPION  coated  with  a  surfactant.  

Poten.al  Applica.ons  of  DIBs   Poten.al  Applica.ons  of  SPIONs  Energy  produc1on  as  bio-‐baberies1,2   Contrast  agent  for  magne1c  resonance  

imaging5  Environmentally  responsive  networks  (e.g.  light-‐sensing  networks)  1,2  

Magne1c  fluid  hyperthermia  to  irreversibly  damage  unhealthy  cells  or  coagulate  small  blood  vessels5  

Complex  circuit  models2   Non-‐viral  gene  vector  for  gene  therapy5  Stable  3D  networks  for  1ssue  engineering  substrates3  

Localized  drug  delivery  to  reduce  dosages  and  side  effects5  

Biomime1c  1ssues  capable  of  communica1on1,3   Immunomagne1c  separa1on  to  isolate  cells5  

Figure  2.  DIB  between  two  droplets  on  an  “egg-‐crate”  PDMS  substrate.  

Figure   3.   Schema1c   and   results   of   3D   prin1ng  networks  of  droplets  into  drops  of  oil,  which  can  then   be   stabilized   in   bulk   aqueous   solu1ons3.  Adapted  from  Science,  2013,  340,  48-‐52.  

Figure   1.   An   illustra1on   of   one   varia1on   of   the   forma1on   of   droplet-‐hydrogel   interface   bilayers   (DHBs)4.   Adapted   from   Nature   Protocols,  2013,  8,  1048  –  1057.    

formed  by  injec1ng  an  aqueous  droplet  into  an  oil  and  lipid  mixture  above  a  cured  hydrogel1.  The   hydrophilic   heads   of   the   lipids   spontaneously   coat   the   hydrogel   and   droplet   in   a   lipid  monolayer.  The  exposed  hydrophobic   tails  of   the  monolayers  connect  when   they  come   into  contact,  forming  a  robust  lipid  bilayer  at  the  junc1on  between  the  droplet  and  the  hydrogel1.  DIBs  have  poten1al  applica1ons  ranging   from  energy  storage  to  1ssue  engineering,  many  of  which  rely  on  the  ability  to  incorporate  membrane  proteins  in  the  DIB1-‐3.  

SPIONs  are  the  par1cular  nanopar1cle  we  examine  here  because  of   their  array  of  promising  clinical   applica1ons.   They  are   synthe1cally   fabricated  with   ferrous   salts   to  produce  par1cles  with  a  core  size  of  less  than  10  nm,  which  are  then  coated  in  a  surfactant  and  suspended  in  a  carrier   liquid.   These   superparamagne1c   fluids   are   called   ferrofluids.   Superparamagne1c  substances  are  defined  by  several  fundamental  characteris1cs,  detailed  in  Table  1.  The  binary  nature  of   their  magne1za1on  allows   for   easy  magne1c  manipula1on  of   SPIONs.  Given   their  myriad   of   poten1al   biomedical   applica1ons,   it   is   vital   to   comprehensively   characterize   the  interac1ons  of  SPIONs  with  biological  systems.  

Table  1.  Defining  characteris.cs  of  superparamagne.c  fluids  High  magne1c  suscep1bility;  strongly  magne1zed  within  an  applied  magne1c  field5  Cons1tuents  remain  in  suspension  even  within  a  strong  magne1c  field  due  to  the  negligible  difference  in  densi1es  between  the  par1cles  and  their  carrier  liquid5  Do  not  retain  residual  magne1za1on  when  an  applied  magne1c  field  is  removed  because  the  magne1c  poles  of  the  individual  nanopar1cles  are  randomized  by  thermal  mo1on5  

Acknowledgements  This   material   is   based   upon   work   supported   by   a  Na1onal   Science   Founda1on   Research   Experiences  for  Undergraduates  (REU)  site  program  under  Grant  No.  1359095.    

I   would   like   to   thank   Dr.   Eric   Freeman,   Kengelle  Chukwurah,  Khushboo  Brahmbab,  and  Dr.  Xianqiao  Wang  for  their  mentorship.    

Thank  you  to  Dr.  Leidong  Mao  for  his  dedica1on  to  crea1ng  an  enriching  program  as  the  director  of  the  Nanotechnology  and  Biomedicine  REU  at  UGA.  

A   suitable   polydimethylsiloxane   (PDMS)   substrate   was  developed   to   contain   the   DHB   for   experimenta1on.   Figure   5  and  Figure  6  illustrate  the  mold  in  which  the  PDMS  is  cured  and  the   final   prepared   substrate   aser   altera1ons.   DHB   forma1on  and   characteriza1on   is   executed   with   the   prepared   substrate  taped   onto   a   height-‐adjustable   microscope   stage   centered  above   a   stack   of   permanent   magnets.   The   housing   for   the  overhead  electrode   is  abached  to   the  same  stage   in  order   for  the  electrode  and  the  substrate  to  move  concurrently.  A  front-‐facing  compound  microscope  containing  a  camera   is  placed   in  front   of   the   stage;   a   backlight   sits   behind   the   substrate.   The  ver1cal   channel   of   the   substrate   is   filled  with   an   oil   and   lipid  mixture,   and  a  microfil   is  used   to  manually   inject  a  droplet  of  1.5%   SPION-‐containing   ferrofluid   with   a   polyethylene   glycol  (PEG)  surfactant.  The  hydrogel  and  ferrofluid  are  both  adjusted  to  contain  equal  ionic  concentra1ons  of  0.01  M  MOPS  and  1.0  M   KCl.   Once   the   droplet   falls   and   a   DHB   visibly   forms,   the  headstage  electrode  is  inserted  into  the  ferrofluid  droplet  and  a  triangle   voltage   signal   with   a   frequency   of   10   Hz   and   an  amplitude  of   10  mV   is   applied.   The   output   current   signal   and  visual  deforma1on  of  the  ferrofluid  droplet  are  observed  as  the  stage   is   moved   down   millimeter   by   millimeter,   closer   to   the  magnet,  un1l  the  DHB  fails.    

Figure   5.   Illustra1on   of   the   mold   for   the   PDMS   substrate.   The  trough   shape   provides   an   unmarked   surface   (labelled   as   “slit   in  straw”)   that   serves   as   a   clear   viewing   window   to   the   center  channel  formed  by  the  capillary  tube.    

Figure  6.  Illustra1on  of  the  prepared  substrate.    

A   B  

Figure   7.   Prepared   substrates   as   viewed   through   the   compound  microscope.   (A)   Empty   channel   with   hydrogel-‐electrode   plug  visible.   (B)   Channel   filled   with   an   oil   and   lipid   mixture   with   an  intact  DHB  between  the  hydrogel  plug  and  ferrofluid  droplet.  An  electrode   is   inserted   into   the   droplet   for   electrical  characteriza1on  of  the  DHB.    

A  

B  

Figure   8.   Experimental   setup.   (A)  Overhead  view  and  (B)  side  view  of  adjustable   stage,   microscope,  backlight,   and   headstage   (housing  for  electrode  inserted  from  above)    

Figure   12.   Characterizing   data   of   the   SPIONs   used   in   the   experiments  presented  here.  (A)  TEM  Image  of  par1cle  size  (B)  Par1cle  size  distribu1on,  indica1ng   an   average   core   size   of   about   10   nm.   (C)   A   measure   of   the  SPION’s  magne1c  suscep1bility.    

(b) (c) (a)

Par1cle  Co

unt

40 nm

Magne

1za1

on  (kA/m)

Core  Diameter  (nm) Field  (mT)

Figure  10.  Rela1onship  between  the  distance  from  the  top  of  the  magnet  to  the  bobom  of  the  substrate  and  the   strength   of   the   magne1c   field   as   measured   by   a  Gauss  meter.  Data  points  represent  the  mean  of  three  measurements   of   magne1c   field   strength.   Ver1cal  error  bars  are  taken  to  be  the  standard  error.    

1  mm   3  mm   5  mm   7  mm  

9  mm   10  mm   11  mm   12  mm  

13  mm   14  mm   15  mm   15  mm  Figure   11.   Visual   deforma1on   of   the   ferrofluid   droplet   as   the   stage   is  lowered   towards   the   permanent   magnet.   The   numbers   indicate   the  micrometer   reading   on   the   stage   height   micromanipulator.   A   larger  reading  indicates  a  smaller  distance  from  the  magnet,  with  15  mm  being  the  closest  abainable  posi1on.    

Coarse-‐grained  molecular   dynamics   simula1ons   (Figure   9)   predict   that   nanopar1cles  with   different   ligand   coa1ngs   will   diffuse   across   a   membrane   given   a   sufficient  transloca1on  force5.    The  force  applied  here  is  a  func1on  of  the  magne1c  field  and  the  response  of  the  superparamagne1c  par1cles  suspended  in  the  ferrofluid  (Eq  1,  2  and  Figure  12).  As  the  force  on  the  par1cles  increases,  the  membrane  size  gradually  grows  as  the  ferrofluid  droplet  is  compressed  into  the  hydrogel  (Figure  13).  This  compression  is   reversible;   by   removing   the   magne1c   field,   the   original   membrane   size   may   be  restored.   This   behavior   is   linked   directly   to   the   magne1c   field   and   the   ferrofluid  ac1vity.    

At   the   peak   magne1c   field   strength,   liposome   forma1on   events   are   observed.   The  measured   membrane   area   increases   at   a   higher   rate,   followed   by   a   sudden   sharp  decrease   back   to   the   original   value   (Figure   13).   This   is   accompanied   by   a   visible  increase   in   the   ferrofluid   concentra1on   within   the   hydrogel,   sugges1ng   that   the  magne1c  fluid   is  shearing  droplets  from  the  bulk  membrane  and  pinching  the  bilayer  membrane   into   liposomes   within   the   hydrogel.   This   behavior   is   also   linked   to   the  inevitable   failure   of   the   membrane,   as   the   DHB   membrane   may   fail   during   the  liposome  forma1on.  

A   deeper   understanding   of   the   interac1ons  between   nanopar1cles   and   biological   systems  would  revolu1onize  the  medical  field.  A  number  of  addi1onal   experiments   should   be   conducted   in  order   to   more   comprehensively   assess   the   forces  SPIONs   exert   on   lipid   bilayers.   The   following   tasks  remain:  § Convert  magnet  proximity   to   force  ac.ng  on   the  par.cle.  The  magne1za1on  data  combined  with  the  images   in   Figure   11   provide   a   link   between   the  electrical  measurements   and   the   force   exerted   on  the  par1cles  at  the  DHB.  § Perform   conduc.vity  measurements   during   DHB  membrane   compression .   The   capacitance  measurements   allow   for   insights   into   the  invagina1on   of   the   bilayer   membrane   and   its  gradual   spreading   and   growth   due   to   the   applied  magne1c   field.   However,   the   transloca1on   of  individual   par1cles   may   be   linked   to   temporary  increases   in   the   membrane   conduc1vity   through  the  forma1on  of  pores.  § Assess   the   performance   of   various   ferrofluids.    According   to   the   results   of   computa1onal  simula1ons,  the  nature  of  the  ligand  coa1ng  on  the  nanopar1cles   influences   the   force   require   for  par1cle   transloca1on.  Crea1ng  new   ferrofluids  and  comparing   the   results   will   provide   experimental  data  for  this  predic1on.  

Eq.   1   and   Eq.   2   are   used   to   es1mate   the  force   ac1ng   on   the   SPIONs,   where   Fm   is  the   force   on   a   magne1c   par1cle,   xb,eff   is  the  slope  of  the  linear  region  of  Plot  (c)  in  Figure  107,  Msat   is   the  satura1on,  and  B   is  the   magne1c   field.   Eq.   1   applies   to   only  the  linear  region  of  Plot  (c)  in  Figure  107.    

Figure  13.    Electrical  recordings  of  the  membrane  capacitance  converted  to  area,  assuming  a  specific  capacitance  of  0.8  mF/cm2.    As  the  magnet  is  moved  closer   to   the  DHB,   the  area  gradually   increases  due  to  magneto-‐weqng.     Reduc1on   of   the  magne1c   field   results   in   a   reduc1on   of   the    membrane   area.     At   high   magne1c   fields,   the   membrane   swells   and  pinches,   forming   ferrofluid   liposomes   that  diffuse   into   the  hydrogel  and  rupture.  

1.12

1.14

1.16

1.18

210 255 300 345 390 435 480

Mem

bran

e  Area  (m

m2 )

Time  (seconds)

Stable  Membrane  Areal  Increase  (Magnetowetting)

1.24

1.25

1.26

1.27

760 780 800 820 840 860 880 900

Mem

bran

e  Area  (m

m2 )

Time  (seconds)

Liposome  Formation

liposomeAΔ

1

1.1

1.2

1.3

200 300 400 500 600 700 800 900Mem

bran

e  Area  (m

m2 )

Time  (seconds)

Magnetowetting  ReversibilityStep  Increases Removal  +  Reapplication