lipid nanoparticles ( lnp ) 
 are the leading delivery systems for 
 enabling the therapeutic potential of sirna for systemic applications .
lnp sirna systems containing optimized ionizable cationic lipids 
 can exhibit remarkable in vivo potencies at doses as low as 0.02 mg 
 sirna / kg body weight for silencing liver ( hepatocyte ) target genes 
 in rodents following intravenous ( i.v . ) injection .
these systems are relatively nontoxic , leading to therapeutic 
 indices in mice approaching 1000 , indicating potential clinical utility . despite this progress , the structure of these lnp sirna systems 
 is unclear .
some models of lnp sirna suggest a bilayer vesicle structure 
 of the lnp with sirna on the inside in an aqueous interior , however other observations suggest that such 
 models may be incorrect . for example , recent cryo - transmission electron 
 microscopy ( cryo - tem ) studies of lnp sirna systems formed by mixing 
 an ethanol stream containing lipid with an aqueous stream containing 
 sirna using a t - tube mixing system results in lnp that have electron - dense 
 cores rather than the less dense aqueous 
 cores observed for vesicular systems . in 
 addition , formulation of lnp sirna systems using the t - tube mixer can result in sirna encapsulation efficiencies 
 above 70% , an observation that is difficult to reconcile with bilayer 
 vesicular structure .
this is because encapsulation depends on the 
 presence of cationic lipid and it would therefore be expected that 
 the maximum encapsulation efficiency should be approximately 50% for 
 bilayer systems , assuming that the cationic lipid is equally distributed 
 on both sides of the bilayer . in this work , we characterize 
 the structure of lnp sirna systems 
 produced using a rapid microfluidic mixing technology by employing a variety of biophysical assays as well as 
 in silico simulations .
cryo - tem studies show that these lnp sirna 
 systems exhibit an electron - dense core ( in contrast to bilayer vesicle 
 systems ) , and that  limit size
 systems can be generated 
 at high peg - lipid contents that are consistent with the ability of 
 sirna to stimulate formation of inverted micelles in association with 
 cationic lipid .
fluorescent energy resonance transfer ( fret ) , p nmr , and rnase digestion studies show that encapsulated 
 sirna is associated with internalized cationic lipid , is effectively 
 immobilized on the nmr time scale and is fully protected from external 
 rnase .
density gradient studies show that the density of lnp sirna 
 systems can vary from being significantly less dense than bilayer 
 vesicle systems to exhibiting increased densities at higher sirna 
 contents .
taken together , these experimental results suggest that 
 sirna resides in inverted micelles within the overall lnp sirna structure . 

these results are consistent with molecular modeling studies that 
 indicate these lnp sirna systems have a nanostructured core consisting 
 of a periodic arrangement of aqueous compartments , some of which contain 
 sirna duplexes .
the lipids 1-palmitoyl-2-oleoyl - sn - glycero-3-phosphocholine ( popc ) , 1,2-distearoyl - sn - glycero-3-phosphocholine ( dspc ) , 1,2-dioleoyl - sn - glycero-3-phosphoserine ( dops ) , 1,2-dioleoyl - sn - glycero-3-phosphoethanolamine - n-(7-nitro-2 - 1,3-benzoxadiazol-4-yl ) 
 ( nbd - pe ) , and 1,2-dioleoyl - sn - glycero-3-phosphoethanolamine - n-(lissamine rhodamine b sulfonyl ) ( rh - pe ) were obtained 
 from avanti polar lipids ( alabaster , al ) .
4-(2-hydroxyethyl ) piperazine-1-ethanesulfonic 
 acid ( hepes ) , cholesterol , sephadex g-50 , tetrasodium edta , trizma 
 base , and xylene cyanol were obtained from sigma - aldrich ( st . louis , 
 mo ) .
triton x-100 and 2-(n - morpholino)ethanesulfonic 
 acid ( mes ) were obtained from bdh ( westchester , pa ) .
ammonium acetate , 
 boric acid , sodium acetate , and sodium chloride were obtained from 
 fisher scientific ( fair lawn , nj ) .
bovine pancreatic rnase a was purchased 
 from applied biosystems / ambion ( austin , tx ) .
the phosphodiester sirna used in this 
 study was obtained from invitrogen ( carlsbad , ca ) , and phosphorothioate 
 sirna were 25-mer blunt - end duplexes ( sense -5 -gccuuaacuuuggugaucaaggaua-3 ) 
 and were obtained from dharmacon ( lafayette , co ) .
the ionizable cationic 
 lipid 2,2-dilinoleyl-4-(2-dimethylaminoethyl)--dioxolane ( dlinkc2-dma ) 
 and n-[(methoxy polyethylene glycol 2000 carbamyl]-1,2-dimyristyloxlpropyl-3-amine 
 ( peg - c - dma ) were obtained from alcana technologies inc . ,
cholesterol e total cholesterol assay kit was obtained from wako 
 diagnostics ( richmond , va ) .
quant - it ribogreen rna assay kit was obtained 
 from molecular probes ( eugene , or ) .
bilayer popc / cholesterol ( 1:1 ; mol / mol ) vesicles were prepared 
 by hydration of a dried lipid film with pbs , and the dispersion was 
 then freeze  thawed five times using liquid nitrogen .
unilamellar 
 vesicles were then prepared by extruding the frozen and thawed lipid 
 suspension 10 times through two stacked 80 nm pore size polycarbonate 
 filters .
lnp were prepared by mixing appropriate volumes of lipid stock solutions 
 in ethanol buffer with an aqueous phase containing sirna duplexes 
 employing a microfluidic micromixer as described elsewhere . for the encapsulation of sirna , the desired amount 
 of sirna was dissolved in 25 mm sodium acetate , ph 4.0 .
equal volumes 
 of the lipid in ethanol and the sirna in buffer were combined in the 
 microfluidic micromixer using a dual - syringe pump ( model s200 , kd 
 scientific , holliston , ma ) to drive the solutions through the micromixer 
 at a combined flow rate of 2 ml / min ( 1 ml / minute for each syringe ) . 

the mixed material was diluted into an equal volume of 25 mm sodium 
 acetate buffer , ph 4.0 , upon leaving the micromixer outlet , thus reducing 
 the ethanol content to 25% .
the lipid mixture was then dialyzed for 
 4 h against 1000 volumes of 50 mm mes/50 mm sodium citrate buffer 
 ( ph 6.7 ) followed by an overnight dialysis against 1000 volumes of 
 1 phosphate buffered saline , ph 7.4 ( gibco , carlsbad , ca ) using 
 spectro / por dialysis membranes ( molecular weight cutoff 12  00014  000 
 da , spectrum laboratories , rancho dominguez , ca ) .
the mean diameter 
 of the lnp after dialysis was 41.3  14.9 nm as determined by 
 dynamic light scattering ( number mode ; nicomp 370 submicrometer particle 
 sizer , santa barbara , ca ) .
lipid concentrations were determined by 
 measuring total cholesterol using the cholesterol e enzymatic assay 
 from wako chemicals usa ( richmond , va ) .
encapsulation efficiency was 
 calculated by determining unencapsulated sirna content by measuring 
 the fluorescence upon the addition of ribogreen ( molecular probes , 
 eugene , or ) to the sirna - lnp ( fi ) and 
 comparing this value to the total sirna content that is obtained upon 
 lysis of the lnp by 1% triton x-100 ( ft ) : % encapsulation = ( ft  fi)/ft  100 .
limit size particles were prepared by mixing 20 mm dlinkc2-dma / peg - c - dma 
 ( 90/10 , mol % ) dissolved in ethanol with five volumes of 25 mm sodium 
 acetate , ph 4.0 containing sirna .
mixing of the two streams are accomplished 
 using the above - mentioned herring micromixer device with a flow - rate 
 of 0.5 ml / min for the lipid / ethanol stream and 2.5 ml / min for the 
 aqueous stream .
ethanol was removed by first diluting the lipid mixture 
 with sodium acetate buffer , ph 4.0 and then removing the ethanol - containing 
 buffer with an amicon ultra , 10  000 mwco , regenerated cellulose 
 concentrator ( millipore , billerica , ma ) .
cyro - tem samples were prepared 
 by applying 3 l lnp at 1020 mg / ml total lipid to a 
 standard electron microscopy grid with a perforated carbon film .
excess 
 liquid was removed from the grid by blotting and then the grid was 
 plunge - frozen in liquid ethane to rapidly freeze the sample using 
 a vitrobot system ( fei , hillsboro , oregon ) .
images were taken under 
 cryogenic conditions ( 88 k ) at a magnification of 50  000 
 with an amt hr ccd side mount camera .
samples were loaded with a gatan 
 70 degree cryo - transfer holder in an fei g20 lab6 200 kv tem ( fei , 
 hillsboro , or ) under low dose conditions with an under - focus of 46 
 m to enhance image contrast .
experiments were performed at 
 the university of british columbia bioimaging centre ( vancouver , bc ) . 

particle diameters were measured from the micrographs with the aid 
 of imagej ( national institute of health , bethesda , md ) .
factor vii 
 sirna was encapsulated with lnp formulations containing 40% dlinkc2-dma , 
 11.5% dspc , 47.5% cholesterol , and 1% peg - c - dma ( mol % ) .
an amount 
 of 1.0 g of sirna ( entrapped in lnp ) was incubated with 0.05 
 g of bovine pancreatic rnase a ( ambion , austin , tx ) in 50 l 
 of 20 mm hepes ( ph 7.0 ) at 37 c for 1 h. at the end of the incubation , 
 a 10 l aliquot of the reaction mix was added to 30 l 
 of fa dye ( deionized formamide , tbe , pbs , xylene cyanol , bromophenol 
 blue , triton x-100 ) to halt the rnase reaction .
gel electrophoresis 
 was performed using 20% native polyacrylamide gel , and nucleic acids 
 were visualized by staining with sybr - safe ( invitrogen , carlsbad , 
 ca ) .
proton - decoupled p nmr spectra were obtained using a bruker avii 400 spectrometer 
 operating at 162 mhz .
free induction decays ( fid ) corresponding to 
 10 scans were obtained with a 15 s , 55 
 degree pulse with a 1 s interpulse delay and a spectral width of 64 
 khz .
an exponential multiplication corresponding to 50 hz line broadening 
 was applied to the fid prior to fourier transformation .
experiments were performed 
 at the centre for the drug research and development , vancouver , bc .
fusion 
 between lnp sirna nanoparticles and anionic dops bilayer vesicles 
 was assayed by employing a fluorescence resonance energy transfer 
 lipid mixing assay . labeled dops vesicles containing 
 nbd - pe and rh - pe ( 1 mol % each )
were prepared by hydration of the 
 lipid in a thin film with 20 mm hepes buffer at ph 7.0 followed by 
 10 extrusions through two stacked 100 nm pore size polycarbonate filters 
 ( nuclepore ) using the extruder ( northern lipids , vancouver , bc ) .
lnp 
 composed of 40% dlinkc2-dma , 11.5% dspc , 47.5% cholesterol , and 1% 
 peg - c - dma were prepared with an sirna - to - total lipid ratio ( d / l ratio , 
 wt / wt ) of 0 , 0.06 , and 0.24 .
a d / l ratio of 0.24 represents a charge 
 ratio of negative ( sirna  phosphate ) to positive ( cationic lipid  amine ) 
 of one .
unlabeled lnp were added to a stirred cuvette 
 containing nbd - pe / rh - pe labeled dops vesicles at a 2:1 lipid molar 
 ratio ( 200 m lnp/100 m dops vesicles ) in 2 ml of 10 
 mm ammonium acetate , 10 mm mes , 10 mm hepes , and 130 mm nacl equilibrated 
 to ph 5.5 .
fluorescence of nbd - pe was monitored using 465 nm excitation 
 and 535 nm emission using a perkin - elmer ls-55 fluorimeter with a 
 1  1 cm cuvette under continuous low speed stirring .
lipid mixing 
 was monitored for approximately 10 min , after which 20 l of 
 10% triton x-100 was added to disrupt all lipid vesicles , representing 
 infinite probe dilution ( 0.1% triton x-100 vol / vol final ) .
lipid mixing 
 was expressed as a percentage of infinite probe dilution determined 
 using the equation : % lipid mixing = ( f  f0)/(fmax 
f0 )  100 , where f is the 
 fluorescence intensity measured by the assay , f0 is the initial fluorescence intensity of nbd - pe / rh - pe / dops 
 vesicles , and fmax is the maximum fluorescence 
 intensity at infinite probe dilution after the addition of triton 
 x-100 ( 0.1% v / v final ) .
solutions of 1% , 2.5% , 5% , 10% and 15% sucrose ( wt / vol ) were prepared 
 in distilled water and used to make a 10 ml step gradient .
gradients 
 were prepared successively overlaying 2 ml of less concentrated sucrose 
 on top of the more concentrated ones .
five hundred l of sample 
 was applied to the gradient and was centrifuged at 39000 rpm in a 
 beckman sw41 swing bucket rotor using a beckman coulter optima le-80k 
 ultracentrifuge ( brea , ca ) for 18 h. these conditions results in an 
 average centrifugal force of approximately 190  000 g in the middle of the tube .
lipid content from each fraction 
 was determined using the cholesterol e enzymatic assay from wako chemicals 
 usa ( richmond , va ) described above .
the lnp containing nucleic acids was formed by first simulating 
 the self - assembly of a smaller building block , then creating a large 
 particle by spatial translations of the building block , coating the 
 large particle with a polymer - grafted lipid , and resolvating the system . 

the building block was self - assembled starting from random configurations 
 in a small system ( figure 7a ) .
the small system 
 contained all components excluding the polymer lipid and was prepared 
 at low , intermediate , and high hydration levels ( 12 , 20 , and 40 water 
 molecules per lipid , respectively ) .
self - assembly resulted in a periodic 
 nanostructure with enclosed water compartments ( figure 7b ) .
we also simulated self - assembly of a medium - sized system 
 with all components including the polymer - grafted lipid as a test 
 case .
it resulted in the structure similar to the small system at 
 low hydration , and the low - hydration building block was selected for 
 simulations of the large system .
the solvent was removed , and the 
 building block was multiplied 3  3  3 times to obtain 
 a large nanoparticle , retaining a small spacing ( < 1 nm ) between 
 the translated unit cells .
the coating layer contained the polymer lipid in random conformations 
 distributed within a thin slab ( 3 nm ) , and was placed in close 
 proximity ( 1 nm ) at all facets of the nanoparticle .
this system 
 was then resolvated ( water placed inside and outside of lnp ) in a 
 large simulation box .
the small systems contained 8 duplex 12 
 bp dna complexes , 576 dlinkc2-dmalipids , 144 dspc lipids , and 576 
 cholesterol molecules .
the medium - sized system contained 64 dnas , 
 4608 dlinkc2-dmalipids , 1152 dspc , 4608 cholesterols , and 1152 peg - lipids . 

the large system contained 216 dnas , 15552 dlinkc2-dmalipids , 3888 
 dspc , 15  552 cholesterols , and 3888 peg - lipids .
the total ratio 
 in the medium and large systems was dlinkc2-dma / dspc / cholesterol / peg - lipid 
 4:1:4:1 ( molar ) , and dna to lipid ratio 0.05 wt / wt , similar 
 to experimental conditions .
the small 
 system at low , intermediate , and high hydration levels contained 4030 , 
 6530 , and 12  560 water particles , respectively ; the medium 
 and large systems contained 400  000 and 900  000 
 water particles , respectively ; cl ions were added 
 to all systems to neutralize the charge .
the large system included 
 1  400  000 particles in total , had a box size 
 of 54  54  54 nm , and was simulated for 10 
 s ( actual simulation time is indicated ) .
two independent runs 
 of 5 s each were performed for the medium - sized system , and 
 six copies of the small system ( two for each level of hydration ) were 
 simulated for 1 s . the martini coarse - grained ( cg ) force 
 field was employed . in this force field , 
 typically one martini particle 
 represents 4 heavy atoms .
sirna duplexes were represented by 12 base 
 pair double stranded dnas ; the rna molecules would have minor differences 
 in the lennard  jones interactions , and lower chain flexibility 
 which would be important for longer strands .
models for dna and dspc lipid were downloaded from the martini 
 web site ( http://md.chem.rug.nl/cgmartini/index.php/downloads ) .
the headgroup of dlinkc2-dmalipid comprised charged ( qd type ) 
 and apolar ( c4 ) particles , and the linker was constituted by a ring 
 of sc4 and 2 sp1 particles .
available models for sphingomyelin and 
 peg polymer were combined to produce 
 the peg - grafted ceramide lipid ( containing a chain of 7 beads ) .
while 
 longer polymer chains are used in experimental studies to control 
 the nanoparticle size , here a short chain length was incorporated 
 to avoid unnecessary and computationally expensive increase of the 
 simulation box volume ( to accommodate longer chains ) .
short polymers 
 combined with a moderate water volume surrounding the nanoparticle 
 were sufficient to keep it disconnected from its periodic image in 
 our simulations .
however , a somewhat larger molar concentration of 
 the short peg - lipid ( as compared to longer polymers in the experimental 
 setups ) was required to cover the whole nanoparticle surface .
jones potential 
 was shifted to zero between 0.9 and 1.2 nm , the coulomb potential 
 was shifted to zero between 0 and 1.2 nm , with a relative dielectric 
 constant of 15 .
the time step was 10 fs with the neighbor list updates 
 every 10 steps . the system was coupled to an isotropic pressure of 
 1 bar using the berendsen barostat with 
 a time constant of 4 ps .
lipids , water , and nucleic acids were coupled 
 separately to a temperature of 310 k using the velocity rescaling 
 thermostat with a time constant of 1 
 ps .
as noted above , high sirna encapsulation levels 
 for sirna in lnp systems are inconsistent with bilayer structure , 
 where a maximum encapsulation efficiency of 50% would be expected . 
 here
we characterized the encapsulation efficiencies of lnp sirna 
 systems over a range of sirna / cationic lipid charge ratios using the 
 microfluidic mixing process as described in the experimental 
 section and employing the lipid mixture dlinkc2-dma / dspc / chol / peg - lipid 
 ( 40/11.5/47.5/1 ; mol / mol ) .
the resulting lnp sirna systems exhibited 
 diameters of approximately 50 nm ( number mode ) as measured by dynamic 
 light scattering .
essentially complete (  95% as indicated 
 using the ribo - green assay ) encapsulation was achieved over a wide 
 range of sirna / cationic charge ratios , including charge ratios as 
 high as 1.25 ( see figure 1 , supporting information ) .
these observations suggest that the microfluidic mixing technique 
 for formulating sirna allows somewhat more efficient encapsulation 
 than the t - tube mixing technique where trapping efficiencies of 70% 
 have been noted . in the next set of experiments , 

the cryo - tem characteristics of the lnp sirna systems produced by 
 microfluidic mixing were investigated for the dlinkc2-dma / dspc / chol / peg - lipid 
 ( 40/11.5/47.5/1 ; mol / mol ) lipid composition . as shown in figure 1a and b , lnp sirna systems prepared at sirna / total 
 lipid ratios of 0.06 and 0.24 , corresponding to sirna - to - cationic 
 charge ratios of 0.25 and 1 , exhibited an electron dense core similar 
 to
the electron dense lnp sirna 
 structure contrasts with the less dense interior of a vesicle system 
 with an aqueous interior generated from popc / cholesterol ( 1:1 ; mol / mol ) 
 ( figure 1c ) and is visually similar to the 
 electron dense interior exhibited by cryo - tem of a colloidal fat emulsion 
 as reported by kuntsche et al .
lnp containing 
 dlinkc2-dma exhibit electron dense cores both in 
 the presence and absence of encapsulated sirna as indicated by cryo - tem . 

lnp were prepared by microfluidic mixing employing a herringbone mixer 
 as indicated in the experimental section . 

popc / cholesterol ( 50/50 ; mol / mol ) bilayer vesicles were prepared by 
 extrusion through polycarbonate filters with 80 nm pore size .
( a ) 
 cryo - tem micrograph obtained from lnp sirna with lipid composition 
 dlinkc2-dma / dspc / chol / peg - lipid ( 40/11.5/47.5/1 ; mol / mol ) and sirna 
 at a sirna / lipid ratio of 0.06 , wt / wt , corresponding to an sirna / cationic 
 lipid charge ratio of 0.25 .
( b ) lnp with the same lipid composition 
 as for ( a ) but prepared at an sirna / lipid ratio of 0.24 wt / wt , corresponding 
 to an sirna / cationic lipid charge ratio of 1 .
( c ) cryo - tem micrograph 
 of popc / cholesterol ( 1:1 ) vesicles .
( d ) lnp with the same lipid composition 
 as for ( a ) and ( b ) but prepared in the absence of sirna .
the lnp sirna formulation employed in figure 1a contains sirna at a 0.06 sirna / lipid ( w / w ) ratio 
 which corresponds 
 to an sirna - to - cationic lipid charge ratio of 0.25 . as a result , when 
 the lnp sirna is formulated at ph 4.0 , approximately 75% of the cationic 
 lipid is not complexed to sirna in the lnp .
it is therefore of interest 
 that the lnp sirna particles observed in figure 1a exhibit an electron dense interior with no evidence of an internal 
 aqueous core .
this suggests that the cationic lipid may contribute 
 to the electron dense interior even when not complexed to sirna . in 
 order to determine whether this is the case , lnp systems with the 
 same lipid composition but no sirna
were formulated employing the 
 microfluidic process and characterized by cryo - tem . as shown in figure 1d ,
an electron dense core was observed in the absence 
 of sirna , indicating that cationic lipids such as dlinkc2-dma , possibly 
 in combination with dspc and cholesterol , contribute to electron dense 
 structures in the lnp interior .
the structures that could give rise to the electron dense cores detected 
 by cryo - tem are of interest . in the absence of sirna
it may be proposed 
 that the cationic lipid , in association with a counterion , adopts 
 an inverted structures such as inverted micelles , consistent with 
 the propensity of these lipids for highly curved  inverted  

these structures are inverted 
 in the sense that the polar headgroups are oriented toward interior 
 aqueous cores with diameters as small as 3 nm
. the actual equilibrium radius of an inverted micelle could 
 be larger as dictated by the intrinsic or spontaneous radius of curvature 
 of the constituent lipids . in turn , assuming 
 a bilayer thickness of 4 nm this would suggest that lnp systems composed 
 of pure cationic lipid should exhibit limit sizes with diameters in 
 the range of 11 nm or larger , which is essentially a bilayer surrounding 
 an aqueous interior with diameter as small as 3 nm .
alternatively , 
 in the presence of sirna , it is logical to suppose that the limit 
 size particle consists of a distorted inverted micelle of cationic 
 lipid surrounding the sirna oligonucleotide . in turn
, this would suggest 
 a limit size system as small as 14 nm diameter , assuming that the 
 sirna contained in this inverted micelle is surrounded by an inner 
 monolayer of cationic lipid within an outer monolayer of surface lipid 
 and that the dimensions of the sirna are 2.6 nm in diameter and 5.8 
 nm in length .
here we explored 
 whether limit size particles compatible with such structures could 
 be generated using peg - lipid as the surface lipid . in this regard , 
 a vesicle containing an internal aqueous core of 3 nm diameter has 
 an outside - to - inside surface area ratio of 6.8 ( assuming a bilayer 
 thickness of 4 nm ) , indicating that the outer monolayer requires the 
 presence of lipids that provide an interfacial area approximately 
 7 times larger than the inner monolayer area . assuming that the interfacial 
 area for a lipid such as dlinkc2-dma is similar to that of dioleoylphosphatidylcholine 
 ( 0.7 nm ) , it is straightforward 
 to show that approximately 10 mol % peg2000-lipid ( surface 
 area per molecule 36 nm ) would 
 be required to coat inverted micelles composed of dlinkc2dma with 
 an aqueous core 3 nm in diameter .
we therefore examined the 
 limit size lnp that could be achieved 
 for a dlinkc2-dma / peg - c - dma system ( 90:10 , mol : mol ) produced by the 
 microfluidic mixing process .
the size determined from cryo - tem micrographs 
 ( figure 2a ) was 14.7  6.9 nm , consistent 
 with an ability of the cationic lipid to form inverted micellar structures 
 with interior aqueous diameters in the range of 8 nm .
on the other 
 hand , the limit size of lnp sirna systems formulated at an sirna - to - cationic 
 lipid charge ratio of one resulted in limit size systems of 22.7  
 6.1 nm in diameter ( figure 2b ) , consistent 
 with the presence of inverted micelles with interior diameters of 
 approximately 15 nm consisting of cationic lipid complexed to internalized 
 sirna .
lnp exhibit limit sizes consistent with inverted micelle structure 
 in presence and absence of sirna .
limit size lnp were prepared by 
 microfluidic mixing as indicated in the experimental 
 section .
( a ) cryo - tem micrograph obtained from lnp with lipid 
 composition dlinkc2dma / peg - lipid ( 90/10 ; mol / mol ) in the absence of 
 sirna .
( b ) lnp with the same composition as for ( a ) but prepared with 
 sirna at an sirna - to - cationic lipid charge ratio of 1 .
( c ) size distribution 
 of lnps in figure 2a and ( d ) size distribution of lnps in ( b ) .
particle 
 diameters are determined with the aid of image j ( nih , bethesda , md ) , 
 and average diameters are calculated from over 150 particles .
if the sirna is complexed to cationic lipid 
 and localized in an inverted micelle inside the lnp , it would be expected 
 to be less mobile than if freely tumbling in the aqueous interior 
 of a bilayer vesicle system .
the mobility of the sirna can be probed 
 using p nmr techniques . in particular
, it would be expected 
 that limited motional averaging would be possible for complexed sirna , 
 leading to very broad  solid state 
p nmr 
 resonances due to the large chemical shift anisotropy of the phosphate 
 phosphorus .
if , on the other hand , the 
 sirna is able to freely tumble in an aqueous environment , rapid motional 
 averaging would be expected to lead to narrow , readily detectable p nmr spectra . in order to avoid conflicting p nmr signals arising from phosphorus in phospholipids and sirna , 
 phosphorothoiate sirna , which gives a p nmr signal that 
 is shifted downfield from the normal phosphate resonance , was used 
 to ascertain the motional environment of the sirna . as shown 
 in figure 3a ,
the p nmr signal 
 from free phosphorothioate sirna is a doublet peak , shifted approximately 
 56 ppm downfield from the phosphate resonance .
the doublet structure may be attributed to the presence of rp and 
 sp isomers of the sirna strands . for 
 lnp sirna systems with the lipid composition
dlinkc2-dma / dspc / chol / peg - c - dma 
 ( 40/11.5/47.5/1 mol % ) and containing sirna ( 0.06 sirna / lipid ; wt / wt ) 
 no p nmr signal was observable for the encapsulated sirna 
 ( figure 3b ) , consistent with immobilization 
 within the lnp core .
detergent solubilization of the lnp particle 
 using sodium dodecyl sulfate ( 1% vol / vol ) resulted in recovery of 
 the sirna signal ( figure 3c ) .
note that phosphothioate 
 sirna , which gives rise to a p nmr resonance 56 
 ppm downfield of the phosphodiester peak , was used to avoid overlap 
 with the p nmr signal arising from the dspc phosphorus . 

( b ) p nmr spectrum of phosphothioate sirna encapsulated 
 at a sirna / lipid ratio of 0.06 ( w / w ) in lnp containing dlinkc2-dma / dspc / chol / peg - lipid 
 ( 40/11.5/47.5/1 ; mol / mol ) .
( c ) p nmr signal arising from 
 the same sample as ( b ) after the addition of 1% sds to solubilize 
 the particle .
the spectra depicted were obtained from 15  000 
 transients as described in the experimental section .
sirna sequestered in the 
 lnp core should be fully protected from degradation by externally 
 added rnase .
lnp sirna systems with the lipid composition dlinkc2-dma / dspc / chol / peg - c - dma 
 ( 40/11.5/47.5/1 , mol % ) were incubated with bovine pancreatic rnase 
 a to determine the protection of encapsulated sirna .
as shown in figure 4 , gel electrophoresis indicates that free sirna 
 is degraded , while the sirna encapsulated in the lnp particles formulated 
 by the microfluidic method is completely protected .
addition of the 
 detergent triton x-100 dissolves the lnp , releases the sirna , and 
 results in sirna degradation in the presence of rnase .
sirna was either employed in the free form or encapsulated in lnp 
 containing dlinkc2-dma / dspc / chol / peg - lipid ( 40/11.5/47.5/1 ; mol / mol ) 
 at an sirna / lipid ratio of 0.06 ( w / w ) .
the integrity of the sirna was challenged 
 with 1 g / ml bovine pancreatic rnase a. 5% triton x-100 was 
 added to solubilize the lnp .
gel electrophoresis was performed on 
 20% native polyacrylamide gel and sirna visualized by staining with 
 sybr - safe . as indicated above
, the electron dense core 
 of the lnp sirna systems may be suggested to consist of encapsulated 
 sirna complexed to cationic lipid and the remaining lipid ( cationic 
 lipid , phospholipid , cholesterol and peg - lipid ) is either present 
 in the core in inverted micellar or related nanostructures , or is 
 resident on the lnp exterior .
it would then be expected that at high 
 sirna contents corresponding to sirna - to - cationic lipid charge ratios 
 of 1 , where all the cationic lipid is complexed with internalized 
 sirna , little or no cationic lipid would be present on the external 
 monolayer . in order to test whether this is the case , a fluorescence 
 resonance energy transfer ( fret ) assay for exterior cationic lipid 
 was developed .
this assay , which is essentially a membrane fusion 
 assay , utilized negatively charged bilayer lipid vesicles composed 
 of dioleoylphosphatidylserine ( dops ) that contained the fret pair , 
 nbd - pe and rh - pe at 1 mol % each .
lnp sirna systems consisting of 
 dlinkc2-dma / dspc / chol / peg - lipid ( 40/11.5/47.5/1 mol % ) were added 
 to the dops vesicles and incubated at ph 5.5 .
the pka of dlinkc2-dma is 6.7 and 
 thus more than 90% of the dlinkc2-dma on the outside of the lnp will 
 be charged at ph 5.5 , potentially promoting fusion with negatively 
 charged dops lnp .
as indicated elsewhere fusion is 
 observed as an increase in the nbd - pe fluorescence at 535 nm as the 
 nbd - pe and rh - pe probes become diluted following lipid mixing . as shown in figure 5 , when the lnp systems 
 contained no sirna , substantial fusion was observed , consistent with 
 a considerable proportion of the dlinkc2-dmaresiding on the outer 
 monolayer of the lnp system .
when the lnp systems contained sirna 
 at a sirna - to - total lipid ratio of 0.06 ( w / w ) , which corresponds to 
 an sirna - to - cationic charge ratio of 0.25 , however , the extent of 
 fusion was reduced ( figure 5 ) , and for lnp 
 sirna systems prepared with an sirna - to - cationic lipid charge ratio 
 of one little or no fusion was observed , indicating that little or 
 no dlinkc2-dmawas present on the lnp sirna exterior .
this supports 
 the hypothesis that in lnp with high sirna content all the cationic 
 lipid is complexed with sirna and sequestered in the lnp interior .
the amount of external cationic lipid in lnp sirna systems 
 was assayed as a function of the sirna phosphate - to - cationic lipid 
 charge ratio using the fret lipid mixing assay described in the experimental section .
three lnp systems dlinkc2-dma / dspc / chol / peg - lipid 
 ( 40/11.5/47.5/1 ; mol / mol ) were prepared at charge ratios of 0 ( solid 
 line ) , 0.25 ( dotted line ) , and 1 ( dash line ) .
the lipid mixing assay 
 was performed at ph 5.5 to ensure that essentially all external dlinkc2-dma 
 was positively charged .
the reaction was initiated by injecting the 
 lnp ( at t = 30 s ) into a stirred cuvette containing 
 the anionic dops / nbd - pe / rh - pe ( 98:1:1 molar ratio ) vesicles .
a remaining question concerns why the maximal dequenching 
 for the 
 unloaded sample plateaus at approximately 25% .
it is logical to suppose 
 that the dops vesicles fuse with positively charged lnp to the point 
 that all available cationic lipid is complexed with dops , after which 
 there is no electrostatic attraction driving lnp - dops vesicle fusion 
 and therefore no further dilution of the fret pairs .
the above experiments 
 were performed using 200 mol lnp ( 80 mol cationic lipid ) 
 and 100 mol dops vesicles , indicating that when all possible 
 fusion has occurred , approximately 20% of the dops vesicles would 
 remain intact and the fret pairs in the remaining 80% of the dops 

this 
 may be insufficient to produce maximum dequenching . in order to test 
 this hypothesis ,
the concentration of fret pairs in the dops vesicles 
 was lowered to maximize dequenching effects . as shown in supporting information figure 2 ,
the maximal 
 dequenching then increased to approximately 80% on fusion with unloaded 
 lnp , consistent with hypothesis .
if the lnp sirna systems 
 exhibit a lipid core , they would be expected to exhibit a different 
 density as compared to vesicles with an aqueous core . in the absence 
 of sirna where the interior consists of inverted micelles of cationic 
 lipid the density should be less than vesicular systems , and the density 
 should increase as more sirna is encapsulated . as shown in figure 6
, when empty lnps without sirna and lnps containing 
 sirna at 0.06 sirna - to - total lipid ratio are centrifuged on a 115% 
 sucrose step gradient as described under in the experimental 
 section , the lnps remained on the top of the gradient ( figure 6 ) . in a parallel experiment ,
popc / cholesterol ( 1:1 ; 
 mol / mol ) bilayer vesicles were centrifuged on an identical gradient 
 and the vesicles distributed as a broad peak centered at around fraction 
 7 of the gradient .
increasing sirna content in the lnp results in 
 an increase in density , as lnps containing sirna at a 0.24 sirna - to - total 
 lipid ratio ( corresponding to a 1:1 sirna / cationic lipid charge ratio ) 
 were much denser , exhibiting a peak centered at fraction 15 of the 
 column . in an additional experiment
( data not shown ) empty lnps and 
 lnp containing sirna at 0.06 sirna - to - lipid ratio were introduced 
 first , at the bottom of the centrifuge tube , and a 110% sucrose 
 step gradient layered on top .
after centrifugation , the lnp were found 
 to have redistributed to the top of the gradient .
these results provide 
 strong evidence that lnp sirna systems contain a lipid core with a 
 density dependent on the amount of sirna encapsulated .
the density of lnp sirna 
 systems is consistent with a hydrophobic 
 lipid core as indicated by density gradient ultracentrifugation .
. fractions ( 500 l ) were 
 successively removed from the top of gradient following centrifugation 
 at 190  000 g for 18 h and were assayed for 
 cholesterol in popc / cholesterol bilayer vesicles ( open circles ) , empty 
 lnp system ( filled squares ) , lnp sirna systems at a sirna / cationic 
 lipid charge ratios of 0.25 and 1 ( filled triangles and filled diamonds , 
 respectively ) . the first step in computer 
 modeling was to simulate the self - assembly of a putative unit cell 
 for a lipid nanoparticle . to this end , a mixture of dlinkc2-dma , distearoylphosphatidylcholine 
 ( dspc ) , cholesterol , and nucleic acids was placed in a small box in 
 a random configuration ( figure 7a ) .
the self - assembly was performed in several independent 
 simulations at different hydration levels to explore possible structures 
 formed in the mixture .
we also simulated self - assembly of a medium - sized 
 system containing a polymer - grafted lipid in addition to other components . 
 comparing resulting structures
, we found that a small system at low 
 hydration ( figure 7b ) was similar to the core 
 of the self - assembled medium sized system and selected it as a building 
 block for a larger lnp .
the large lnp was constructed by multiplying 
 the building block , coating the resulting structure with a peg - lipid 
 layer , and resolvating the system ; see the experimental 
 section .
small spacing between the unit cells and between the 
 polymer layer and the nanoparticle was allowed to adjust the water 
 contents upon equilibration of the lnp .
self - assembly from a 
 random configuration ( a ) into a building block 
 ( b ) for a lipid nanoparticle ( lnp ) .
a mixture of dlinkc2-dma , dspc and cholesterol ( 576 dlinkc2-dmalipids , 144 dspc lipids and 
 576 cholesterol molecules ; 44/11/44 ; mol / mol ) is placed in a small 
 simulation box at a low hydration level ; see text .
dlinkc2-dmais shown 
 in yellow , cholesterol in pink , dspc in gray , lipid polar moiety in 
 cyan , and nucleic acids ( 12 bp duplex dna ) in red ; water not shown 
 for clarity . during the equilibration ( 1 s ) , 
 the water compartments 
 closed and the peg - grafted lipids adsorbed on its surface with polymer 
 chains oriented toward the solution . on the simulation time , the lnp 
 gradually transformed into a smooth rounded capsule with an averaged 
 diameter of ca .
the 
 outer layer of the lnp is constituted by a roughly homogeneous coating 
 of peg - lipids . inside the lnp , irregular water filled compartments 
 of diameters ranging from ca .
3 to 9 nm are separated by bilayer membranes 
 with dnas bound to the membrane surface ( see figure 8b  d ) .
the structure of the lnp core resembles an inverted 
 hexagonal phase ( hii ) distorted at a high hydration level .
6 waters / lipid which corresponds to 0.10 l/mol lipid , 
 in good agreement with experimental data ( data not shown ) .
given the 
 significant fraction of lipids on the lnp surface ( 1/4 ) , we 
 expect this number to be slightly higher for a larger size lnp .
a lipid nanoparticle 
 ( lnp ) contains irregular water - filled cavities 
 separated by bilayer membranes , with nucleic acids bound to membrane 
 surfaces : side ( a ) , cross - section ( b , c ) , and zoom - in ( d ) views .
cationic 
 lipid dlinkc2-dmais shown in yellow , cholesterol in pink , dspc in 
 gray , lipid polar moiety in cyan , peg - lipid in violet , and nucleic 
 acids ( duplex dna ) in red ; water not shown for clarity .
the lipid 
 composition was dlinkc2-dma / dspc / cholesterol / peg - lipid ( 4:1:4:1 ; mol / mol ) 
 and dna to lipid ratio 0.05 wt / wt . in the lnp core ,
the numbers of nearest neighbors 
 in the first 
 coordination shell are given in table 1 .
there 
 are strong preferential interactions between the dna phosphates and 
 positively charged groups of dlinkc2-dmalipids .
increased numbers 
 of dlinkc2-dma- dspc neighbors originate from favorable interactions 
 of the positively charged group of the cationic lipid with the negatively 
 charged phosphate groups of dspc .
these interactions lead to formation 
 of molecular pairs which manifest in the radial distribution function 
 ( rdf ) as a pronounced second peak at a distance of 1 nm ( data 
 not shown ) . estimated numbers in the absence 
 of preferential interactions
the numbers are calculated for 
 dna phosphates , positively charged moiety of dlinkc2-dma , the cholesterol 
 polar group , and the dspc phosphate group ( or choline group in the 
 case of dna neighbors ) by integrating the rdfs over the first maximum .
the densities of headgroups are higher 
 in the lnp core ( figure 9a ) than on the lnp 
 surface ( figure 9b ) .
this is expected from 
 a high negative curvature of the water - filled compartments inside 
 the lnp , and the presence of polymer - grafted lipids on the surface . 

the distribution of dlinkc2-dmaaround dspc lipids is shifted towards 
 the dna phosphates , and cholesterol is distributed somewhat away from 
 dlinkc2-dma(figure 9a ) . due to a high cationic 
 lipid to dna ratio
these free ( figure 9c ) and bound ( figure 9d ) cationic lipid fractions have a noticeably different 
 3d neighborhood .
a somewhat heterogeneous surrounding of dspc and 
 cholesterol aligned along the linker of the free lipid becomes for 
 the bound lipid displaced away and to the side by dna phosphates .
spatial 
 density distributions for selected molecular groups around 
 dspc in the lnp core ( a ) and on the lnp surface ( b ) , and dlinkc2-dmain 
 the lnp core free ( c ) and bound to dna ( d ) .
the surfaces of constant 
 number densities are plotted at the values corresponding approximately 
 to the average density in the first coordination shell . in the molecular 
 representations ,
dspc headgroup is shown as dark gray , glycerol - ester 
 region as light gray , and hydrocarbon chains as transparent gray beads ; 
 dlinkc2-dmaheadgroup is shown as yellow , linker as light yellow , and 
 chains as transparent yellow beads . in the density distributions , 

dna phosphates are colored in semitransparent red , dlinkc2-dmaheadgroup 
 in cyan and linker in yellow , cholesterol polar group in pink , dspc 
 headgroup in dark gray , and glycerol - ester in light gray .
the model for 
 lnp sirna structure developed by molecular simulation ( figure 8) is fully consistent with the experimental results 
 presented here .
the model shows that encapsulated sirna is located 
 in internalized distorted inverted micelles complexed to cationic 
 lipid , with remaining lipids organized around small internal aqueous 
 compartments .
such organization could account for the increased electron 
 density as compared to bilayer vesicle systems as evidenced by cryo - tem 
 studies .
this organization is also consistent with the ability of 
 particles composed of dlinkc2-dma and peg - lipid to adopt structures 
 as small as 14 nm diameter in the absence of sirna and 23 nm diameter 
 in the presence of sirna , as well as the lack of external cationic 
 lipid in lnp sirna systems prepared at high sirna - to - cationic lipid 
 charge ratios and the essentially complete protection of encapsulated 
 sirna from external rnase .
previously dspc has been 
 included to provide increased stability to the preformed vesicles 
 used in the preformed vesicle formulation method and has been thought to play a stabilizing role in the 
 lnp structure formed . the results presented here suggest that dspc 
 could also be forming ion pairs between the dspc phosphate group and 
 cationic lipid headgroup , leaving the dspc choline function to associate 
 with sirna phosphates .
a final point is that , 
 as suggested by the molecular modeling results , it is likely that 
 cholesterol is distributed roughly homogeneously in the lnp core and 
 surface , given its ability to partition into both lamellar and inverted 
 lipid structures such as the hexagonal hii phase .
a major advantage of the model presented 
 in figure 8 is that it can explain how sirna 
 encapsulation efficiencies 
 approaching 100% can be achieved during the formulation process . specifically , 
 during the microfluidic synthesis process , the following steps are 
 achieved : first , rapid mixing between the aqueous phase containing 
 sirna and the ethanol phase containing cationic lipid , second the 
 association of cationic lipid with sirna to form hydrophobic nucleating 
 structures , and third , as the polarity of the medium increases further , 
 coating of the nucleating structures by remaining lipids as they reach 
 their solubility limits in the ethanol / water system , forming the final 
 lnp sirna structure . in this picture , it is clear how essentially 
 all of the sirna could be incorporated into lnp systems at nonsaturating 
 sirna loading , leading to complete encapsulation .
there are , 
 however , other structures that could be compatible with 
 high encapsulation efficiencies . in previous work , we have encapsulated antisense oligonucleotides into lnp systems 
 containing ionizable cationic lipids with encapsulation efficiencies 
 of up to 70% using the pfv method .
the lnp formed are small multilamellar 
 vesicles where the oligonucleotide is apparently located at the lamellar 
 interfaces .
these systems contained higher levels of the bilayer forming 
 lipid dspc and lower levels of cationic lipid than employed here . 
 in order to show that the systems we observe here are part of a continuum 
 of structures that are sensitive to the proportions ( and types ) of 
 lipid components
, we examined the cryo - tem morphology of an lnp sirna 
 system containing a larger proportion of dspc and a lower proportion 
 of cationic lipid ( dlinkc2-dma / dspc / chol / peg - lipid in the proportions 
 20/31.5/47.5/1 ; mol / mol , as opposed to 40/11.5/47.5/1 ; mol / mol ) .
this 
 system also exhibited high encapsulation efficiencies above 90% but 
 also showed evidence of multilamellar structure , as shown in supporting information figure 3 .
it would therefore 
 appear that the internal structure of lnp sirna systems changes from 
 multilamellar to inverted micellar as the proportion of bilayer - forming 
 species is reduced , as may be expected based on polymorphic phase 
 preferences of component lipids .
it is likely that the results 
 presented here for lnp sirna systems 
 produced by microfluidic mixing will also extend to lnp sirna systems 
 composed of the same or similar lipids but prepared by other in - line 
 mixing protocols such as the t - tube mixer , as long as the mixing rates 
 are sufficiently fast . in this regard
, it may be noted that lnp sirna 
 systems containing ionizable cationic lipids prepared by t - tube mixing 
 show the distinctive electron - dense core by cryo - tem .
in summary , the results 
 presented in this work demonstrate a new 
 type of lipid nanoparticle with a structured core formed by the rapid 
 mixing of cationic lipid - containing ethanol solutions with aqueous 
 solutions of sirna oligonucleotides .
such structures are consistent 
 with the electron dense core observed by cryo - tem and the efficient 
 loading characteristics associated with these particles .
this work 
 also provides , for the first time , an understanding of the mechanism 
 of formation of lnp sirna formulations formed by mixing cationic lipids 
 in ethanol with sirna in aqueous solution .
it is anticipated that 
 this understanding will lead to the design of more sophisticated lnp 
 structures .
 lipid nanoparticles ( lnp ) containing ionizable cationic 
 lipids 
 are the leading systems for enabling therapeutic applications of sirna ; 
 however , the structure of these systems has not been defined . here 
 
 we examine the structure of lnp sirna systems containing dlinkc2-dma(an 
 ionizable cationic lipid ) , phospholipid , cholesterol and a polyethylene 
 glycol ( peg ) lipid formed using a rapid microfluidic mixing process . 
 
 techniques employed include cryo - transmission electron microscopy , 31p nmr , membrane fusion assays , density measurements , and 
 molecular modeling . 
 the experimental results indicate that these lnp 
 sirna systems have an interior lipid core containing sirna duplexes 
 complexed to cationic lipid and that the interior core also contains 
 phospholipid and cholesterol . 
 consistent with experimental observations , 
 molecular modeling calculations indicate that the interior of lnp 
 sirna systems exhibits a periodic structure of aqueous compartments , 
 where some compartments contain sirna . 
 it is concluded that lnp sirna 
 systems formulated by rapid mixing of an ethanol solution of lipid 
 with an aqueous medium containing sirna exhibit a nanostructured core . 
 
 the results give insight into the mechanism whereby lnp sirna systems 
 are formed , providing an understanding of the high encapsulation efficiencies 
 that can be achieved and information on methods of constructing more 
 sophisticated lnp systems . 
Introduction
Experimental Section
Results and Discussion
Conclusions