over the past several 
 decades , the entrapment of proteins in transparent , 
 mesoporous silica has been of significant interest to scientists and 
 engineers spanning a broad spectrum of disciplines . in more recent 
 history ,
integral membrane proteins ( imps ) have been of particular 
 interest for entrapment in sol  gel - derived silica due to their 
 differing functionalities that can be exploited to tailor these systems 
 for accommodating various applications , such as biosensing , affinity 
 chromatography , high - throughput drug screening , and bioreaction engineering .
imps contain both hydrophobic and hydrophilic amino acid residues ; 
 thus , they are either partially or completely embedded within amphiphilic 
 lipid bilayers of cell membranes .
the necessity of lipid bilayers 
 for proper imp functionality requires an entrapment system that minimally 
 modulates the physical and structural properties of the lipid bilayers ; 
 direct modification of the lipid bilayer structures would adversely 
 affect protein conformation within it .
therefore , the investigation 
 of the stability of lipid - bilayer - derived structures ( i.e. nanolipoprotein 
 particles and liposomes ) entrapped within silica gel is essential 
 to the development of viable , efficient imp - derived bioinorganic hybrid 
 materials . during the 1990s , research groups of bright , friedman , 
 kostic , 
 and brennan examined the properties of various water - soluble proteins 
 entrapped in silica gels derived from alkoxysilane precursors .
their work spurred the development of optimized , biocompatible techniques 
 for a variety of water - soluble proteins .
one of the main techniques 
 included the addition of glycerol and osmolytes , such as sugar , to 
 alter protein hydration .
however , this approach 
 did not address the problematic presence of high concentrations of 
 alcohol that resulted from the hydrolysis reactions of alkoxysilane 
 precursors .
the presence of alcohols is especially detrimental to 
 lipid bilayers , as sufficiently high concentrations will lead to alcohol 
 significantly partitioning into the bilayer , causing it to interdigitate . to address this , brennan s group further 
 pioneered the development of biocompatible sol 
gel chemistries 
 that consisted of modified alkoxysilane precursors bearing covalently 
 attached sugar moieties and/or glycerol .
depending on the specific precursor , the quantity of alcohol liberated 
 during hydrolysis reactions was either greatly reduced or completely 
 removed , and the additives were unable to leach from the gel . in 2002 , besanger et al . examined the stability of 1,2-dipalmitoyl - sn - glycero-3-phosphatidylcholine ( dppc ) liposomes within 
 silica gels derived from three different precursors : an unmodified 
 alkoxysilane ( tetraethyl orthosilicate or teos ) , an alkoxysilane with 
 covalently attached glycerol ( diglycerylsilane or dgs ) , and sodium 
 silicate ( ss ) .
their work demonstrated 
 that the use of dgs- and ss - derived gels permitted the dppc liposomes 
 to exhibit phase transitions as they would in solution , while the 
 use of teos - derived gels did not . also , they depicted , via confocal 
 fluorescence imaging , that the liposomes are capable of undergoing 
 a variety of conformational changes once trapped inside of the gel , 
 forming aggregates or bicelles .
though the dgs- and ss - derived silica 
 gels seemed to work favorably at first , the dppc liposomes eventually 
 lost the ability to undergo phase transitions several days later . 

halder et al . would corroborate this theory in 2004 by examining the 
 solvation dynamics of coumarin 480 inside of liposomes entrapped in 
 silica gel .
silica 
 interactions have elucidated that silica has a propensity for deforming 
 liposomes , causing them to rupture and fuse to the surface .
moreover , silica is actually a very common substrate 
 for performing liposomal fusion to make supported lipid bilayers .
an alternate approach for circumventing the problematic alcohol 
 presence was presented in 2002 by ferrer et al .
it consisted of a 
 simple technique where rotary evaporation was used prior to incorporation 
 of biological species .
this approach 
 was later utilized by luo et al . in 2005 to entrap liposomes bearing 
 the imps bacteriorhodopsin and atp - synthase in silica gels derived 
 from the precursor tetramethyl orthosilicate ( tmos ) .
though protein activity was observed after entrapment , 
 the condition of the liposomal hosts was not examined . on the basis 
 of their analysis
, it is unknown whether or not the protein was functioning 
 near its optimal activity , if the liposomes retained their structure 
 over time , or if this approach would work well for other biologically 
 significant imps . from these previous works
, it can be seen 
 that liposomes undergo 
 structural changes and altered lipid dynamics upon entrapment ; thus , 
 they are not optimal biological membrane hosts for imps inside of 
 silica gel .
in addition , the size mismatch of liposomes ( 100200 
 nm in solution ) with mesoporous silica ( 550 nm pores ) is a 
 limiting factor in their successful implementation as biological membrane 
 host for imps .
here we look to improve 
 upon the use of liposomes in silica gel by instead utilizing nanolipoprotein 
 particles ( nlps ) as biological membrane hosts .
nlps are discoidal 
 patches of lipid bilayer associated with amphiphilic scaffold proteins 
 that interact with the hydrophobic domain of the bilayer by wrapping 
 around the particle periphery , making the entire structure water - soluble . 

nlps have an average thickness of 5 nm , with a diameter ranging from 
 10 to 25 nm depending on the stoichiometric ratios and types of lipids 
 and scaffold proteins being used .
this 
 allows nlps to be more compatible with the pore size ( 550 
 nm ) of mesoporous silica and bear more resemblance to water - soluble 
 proteins  the molecules for which this architecture was optimized  than 
 liposomes .
therefore , here we perform entrapment of nlps using a quick , 
 simple sol 
gel processing technique for tmos that includes 
 evaporation of the majority of the methanol after the hydrolysis reactions . 

the lipid phase behavior of entrapped nlps in comparison to entrapped 
 liposomes was observed using fluorescence anisotropy measurements , 
 while the secondary structure of the scaffold protein was examined 
 via circular dichroism spectroscopy .
we found that liposomes exhibited 
 more significant modulations in their phase behavior upon entrapment 
 in silica gel than nlps and that modulations caused by residual methanol 
 for both liposomes and nlps are relatively small .
lipid 
 interactions , thus strongly suggesting that there were minimal alterations 
 in structure for nlps .
our results demonstrate that nlps are more 
 favorable for silica gel entrapment than liposomes .
msp is a his - tagged membrane 
 scaffold protein 
 ( msp1e3d1 , sigma - aldrich , inc ) , which is comprised of residues 56243 
 of human apoa - i and a 22 amino acid n - terminal fusion containing the 
 his tag , a spacer sequence , and the tev protease site .
imidazole ( 99% ) , 
 tmos ( 99% ) , 1,6-diphenyl-1,3,5-hexatriene ( dph ) ( 98% ) , 
 ethanol ( 200 proof ) , sodium chloride ( 99% ) , methanol ( 99% ) , 
 and sodium cholate ( 99% ) were also purchased from sigma - aldrich , 
 inc .
1,2-dipentadecanoyl - sn - glycero-3-phosphocholine 
 ( di15:0pc ) was purchased in chloroform ( 10 mg / ml concentration ) from 
 avanti polar lipids , inc .
the tris(hydroxymethyl)aminomethane ( mb grade ) and hydrochloric 
 acid ( 12.1 n ) used to prepare a tris - hcl buffer stock solution ( 500 
 mm , ph 7.5 ) were purchased from usb corp . and fisher scientific international , 
 inc .
all water used in these experiments was purified 
 in a barnstead nanopure system ( barnstead thermolyne , dubuque , ia ) 
 with a resistivity 17.9 mcm . for a single preparation
, 
 an appropriate aliquot of di15:0pc was removed from the 10 mg / ml chloroform 
 stock solution , placed into a glass conical vial , dried with nitrogen , 
 and then placed under mild vacuum for at least 4 h to fully evaporate 
 all of the chloroform .
the lipid film was then rehydrated with a reconstitution 
 buffer ( 20 mm tris , 100 mm nacl , ph 7.4 ) to a final lipid concentration 
 of 2 mg / ml and heated to 80 c for at least 5 min . after hydration 
 and heating
, the lipids were extruded through 100 nm pore membranes 
 in an extruder ( avestin , inc . ,
finally , a small fraction 
 of the resulting liposome solution was used for size determination 
 via a particle size analyzer ( brookhaven instruments corp .
nlp batches were synthesized 
 by first placing a stoichiometric excess ( 6 mg ) of di15:0pc in chloroform 
 solution inside of a glass conical vial .
the contents were first dried 
 using nitrogen and then placed under mild vacuum for at least 4 h. 
 afterward , the dried lipid film was rehydrated and solubilized with 
 a sodium cholate reconstitution buffer ( 40 mm sodium cholate , 20 mm 
 tris , 100 mm nacl ) and transferred to a plastic centrifuge tube , where 
 it was allowed to further mix at room temperature ( 22  1 c ) 
 on a vortex mixer ( fischer scientific , hampton , nh ) for 30 min .
next , 
 0.93 mg of his - tagged msp was added to the centrifuge tube and allowed 
 to incubate at room temperature and 300 rpm for 1 h. after incubation , 
 the nlp reaction mixture was transferred to a 10  000 mwco ( molecular 
 weight cutoff ) dialysis filter ( thermo scientific , rockford , il ) and 
 dialyzed against a reconstitution buffer ( 20 mm tris , 100 mm nacl ) 
 at 250 volume excess to remove cholate .
dialysis was performed 
 for 4 h at room temperature and 20 additional hours at 4 c . 
 over the course of the 24 h dialysis
, the buffer was exchanged three 
 times ( 250 overall cholate dilution factor ) .
nta agarose at 4 c 
 for at least 2 h. the ratio of nlps and ni  nta agarose was 
 prepared such that the concentration of nlps was far below the maximum 
 binding capacity of the ni 
after incubation , the 
 agarose was separated via gentle centrifugation ( 500 rpm ) and the 
 supernatant aqueous phase was removed .
centrifugation was also used 
 during the subsequent wash and elution steps to separate the agarose 
 from the aqueous phase .
the agarose was washed four times with a wash 
 buffer ( 20 mm imidazole , 20 mm tris , 100 mm nacl ) .
the agarose was 
 then eluted four times with an elution buffer ( 400 mm imidazole , 20 
 mm tris , 100 mm nacl ) .
the supernatant liquid removed after each elution 
 was concentrated using 100  000 mwco centrifugal concentrators 
 ( vivaproducts , inc .
littleton , ma ) and combined for a total volume 
 of roughly 1 ml .
nlps were then dialyzed again under the same conditions 
 previously mentioned for the purpose of imidazole removal .
the concentration 
 of msp was determined using a uv  vis spectrophotometer ( shimadzu 
 scientific instruments , columbia , md ) to measure the concentration 
 of msp via absorbance at 280 nm .
the lipid concentration was determined 
 by synthesizing a separate batch of nlps using di15:0pc that was laced 
 with a trace amount of fluorescent oregon green 488 dhpe ( life technologies , 
 carlsbad , ca ) and using an oregon green standard curve to measure 
 fluorescence intensities of the resulting nlp batch with a fluorescence 
 spectrophotometer ( perkinelmer , inc . ,
the lipid to protein 
 ratio ( 4 mg:1 mg ) was consistent with the expected ratio of msps to 
 lipids ( 2:375 ) .
the size of the nlps 
 was measured using a particle size analyzer ( brookhaven instruments 
 corp . ,
the acquired stokes diameter was converted 
 to a discoidal diameter using known thicknesses of phosphatidylcholine 
 bilayers ( see the supporting information ) . for 
 a typical preparation ,
5.6 ml of 0.01 m hcl in nanopure water was 
 combined with 7.6 ml of tmos in a round - bottom flask and swirled until 
 a uniform , cloudy phase was observed .
the solution was then subjected 
 to sonication in a bath sonicator for 10 min , followed by rotary evaporation 
 ( 340 mbar reduced pressure , 50 c ) for 20 min to promote rapid 
 removal of methanol liberated during the hydrolysis reactions .
the 
 solution was then passed through a 0.45m filter , resulting 
 in roughly 4 ml of a clear silica sol .
of the 4 ml of silica sol , 
 1 ml was placed into a methacrylate cuvette , followed by 1 ml of a 
 stronger reconstitution buffer ( 34 mm tris , 100 mm nacl ) in order 
 to neutralize the ph .
afterward , 1 ml of either a 20 diluted 
 liposome solution or 20 diluted nlp solution in regular reconstitution 
 buffer ( 20 mm tris , 100 mm nacl ) was added to the methacrylate cuvette . 

the liposome stock solution was typically 2.8 mm 15:0 pc ( final concentration 
 56 m inside gel ) , while the nlp stock solution was typically 
 20 m msp ( final concentration 400 nm inside gel ) .
gelation 
 typically occurred within 2 min after addition of the liposomes or 
 nlps .
ten microliters 
 of a 100 m dph stock solution in ethanol was added to either 
 liposome or nlp solutions in 1 ml volumes of reconstitution buffer 
 ( 20 mm tris , 100 mm nacl ) .
afterward , these samples were either diluted 
 further ( 3 ) and used for solution anisotropy readings or added 
 as the final 1 ml aliquot during the aforementioned entrapment / gelation 
 process and used for gel anisotropy readings .
anisotropy values ( r ) are determined by the difference in polarized light intensity 
 emitted parallel and perpendicular to the excitation source normalized 
 by the total light intensity emitted , as shown in eq 1.1 the measurements were carried out on 
 a perkinelmer ls 55 fluorescence spectrometer equipped with a ptp-1 
 fluorescence peltier system ( perkinelmer , inc . ,
a wavelength 
 of 360 nm was used for excitation , while emission intensities at 440 
 nm were used for determining anisotropy values .
band passes of 3 and 
 5 nm were used on the excitation and emission monochromators , respectively . 

the anisotropy values were recorded at varying temperature intervals 
 ( 4 c ) as temperature was raised at an average rate of 
 0.4 c / min .
anisotropy values reported are the average of at 
 least four measurements at a given temperature .
there were no fluorescence 
 emissions at 440 nm ( hence no anisotropy signals ) from the silica 
 gel , protein , or lipid ; only dph - containing samples showed fluorescence 
 emission intensities .
the anisotropy vs temperature experimental data was regressed using 
 the method of least - squares for an empirical phase transition model 
 shown in eq 2 , where the parameters rmax , rmin , tm , and n are the maximum anisotropy , 
 minimum anisotropy , melting temperature , and cooperativity index , 
 respectively.2a and b are 
 constant coefficients for the quadratic baseline . ignoring the quadratic 
 baseline , eq 2 depicts a sigmoid function with 
 asymptotic end behavior in the limit as t is significantly 
 far from the phase transition region .
the parameters rmax and rmin represent the 
 vertical displacement of the plot , while tm corresponds to the inflection point ( for significantly small values 
 of b ) and n corresponds to the broadness of the phase 
 transition region .
a quadratic baseline can be used in the vicinity 
 of the phase transition region to more accurately capture the manner 
 in which anisotropy varies with temperature , as well as any asymmetry 
 about the inflection point .
the parameters a and b were chosen to be the average of all of the individually 
 regressed data plots .
this was due to a trade - off between n and the quadratic baseline during the regression process ; a and b can influence the value of n. by fixing a and b , 
 the change in n between different plots is almost 
 entirely attributed to the sample , with minimal effect from other 
 parameters . to implement this ,
each plot was regressed individually , 
 while its values for a and b were 
 recorded .
once all of the values were recorded and averaged , all of 
 the plots were reregressed with fixed values for a and b ( see the supporting information ) .
these values were the overall average from the previous regressions . in order to 
 perform circular dichroism ( cd ) spectroscopy , the 20 mm tris
, 100 
 mm nacl buffer was replaced with a 25 mm phosphate , 100 mm naf buffer 
 due to absorbance of tris and chloride in the uv region of interest . 

solution and gelated measurements were performed on a jasco j-715 
 spectropolarimeter with a 2 cm / min scan speed ( jasco , easton , md ) . 

data points were collected as averages of three scans from 260 to 
 190 nm at room temperature in a demountable close - ended far - uv ( q ) 1 mm path length quartz cuvette cell type 20c ( starna 
 cells ) for the silica gel samples and in a 1 mm path length open - end 
 quartz cuvette for the solution samples .
the final concentrations 
 of msp were 20 m in solution and 60 m in silica gel .
the 
 -helical content was determined by first converting the 
 measured ellipticity at 222 nm to a molar ellipticity using eq 3 , where [ ]molar , mrw , 222 , l , and c are the molar 
 ellipticity ( deg cm dmol ) , msp mean 
 residue weight ( g / mol ) , ellipticity ( deg ) , cuvette path length ( cm ) , 
 and msp concentration ( g / ml ) , respectively.3 once this was obtained , a common correlation shown in eq 4 was used 
 to estimate the percentage of -helical content.4
fluorescence anisotropy 
 of the membrane - inserting probe 1,6-diphenyl-1,3,5-hexatriene 
 ( dph ) was used to investigate the phase behavior of di15:0pc carbonyl 
 tails within liposomes and nlps .
di15:0pc was used instead of other 
 biologically prevalent pc lipids ( such as dmpc or dppc ) due to the 
 compatibility of its phase transition temperature with the heating / cooling 
 speeds of the peltier element in the experimental setup , which allowed 
 for higher throughput of samples in a given period of time ( see the supporting information ) .
liposomes had a stokes 
 diameter of 166.5  1.8 nm , while nlps had a stokes diameter 
 of 11.7  2.2 nm and corresponding discoidal diameter of 14.8 
  4.4 nm by dynamic light scattering .
we obtained fluorescence 
 anisotropy values using eq 1 at a range of temperatures 
 that included the main phase transition and in two environments , aqueous 
 buffer solution and porous silica gel derived from the alkoxysilane 
 precursor , tmos .
as shown in figure 1a , the 
 phase transition from a solid lipid phase to a liquid disordered lipid 
 phase , observed as a decrease in anisotropy with increasing temperature , 
 was broader for nlps than that of the liposomes in buffer .
this 
 corresponds to the disparity in the cooperativity indices ( n ) of the phase transition , obtained by fitting to eq 2 .
when lipid bilayers undergo a phase transition , 
 the transition occurs involving multiple subunits known as cooperative 
 units .
the cooperativity index is a relative scale that is directly 
 proportional to the size of the cooperative unit .
the nlps have an 
 average index of 0.36  0.20 and the liposomes have an average 
 index of 1.79  0.24 , both in solution .
figure 1a also illustrates the shift in the midpoint of the phase 
 transition ( tm ) to higher temperature 
 for nlps in comparison to liposomes . by fitting this data to eq 2
, tm values are found 
 to be 35.1  0.1 and 37.7  0.6 c on average for 
 the liposomes and nlps , respectively . immediately after entrapment 
 in silica gel ( day 1 ) , phase transitions can be observed by the decrease 
 in anisotropy with increasing temperature , as shown in figure 1b .
however , the anisotropy range  the overall 
 ordinate axis difference between the maximum and minimum anisotropy 
 values  was reduced for both nlps and liposomes in comparison 
 to solution anisotropy ranges .
in addition , figure 1 and table 1 show that the tm values of both nlps and liposomes entrapped 
 in silica gel are elevated compared to their solution values . measured anisotropy 
 values of nlps and liposomes in ( a ) 20 mm tris
, 
 100 nm nacl buffer and ( b ) silica gel after initial entrapment , with 
 corresponding regression curves as temperature was increased .
table 1 illustrates that the cooperativity 
 indices for nlps and liposomes entrapped within silica gel were on 
 the same order of magnitude , with the nlp values being slightly higher . 

these values were on the same order of magnitude of nlp cooperativity 
 in solution , but were an order of magnitude lower than that of liposome 
 cooperativity in solution .
this is depicted in figure 2a , where the cooperativity of nlps and liposomes entrapped 
 in silica gel are plotted over a 56 week period , normalized 
 by their respective solution values .
the normalized cooperativity for 
 nlps started near unity and decayed 
 to a constant value of approximately 0.7 over the course of 2 weeks 
 ( figure 2a ) .
the liposomes maintained a constant 
 normalized cooperativity between 0.1 and 0.2 over the entire 5 - 6 week 
 period ( figure 2a ) . in solution
, cooperativity 
 did not exhibit any significant changes over time for both nlps and 
 liposomes as it remained unaffected over the course of 3 weeks ( see 
 tables s1 and s2 , supporting information ) .
in addition to the normalized cooperativity , the phase transition 
 temperature ( tm ) of liposomes entrapped 
 within silica gel remained relatively constant in the 4042 
 c region over the course of the 5 - 6 week period , as shown in 
 figure 2b .
the nlp tm was higher , as it gradually increased and leveled off in the 4650 
 c region , as shown in figure 2b .
as with 
 cooperativity , tm also remained relatively 
 unaffected in solution over the course of 3 weeks for both nlps and 
 liposomes ( see tables s1 and s2 , supporting information ) . for all samples , the observed anisotropy curves were reversible 
 with respect to temperature in solution .
however , they were not reversible 
 in silica gel and appeared somewhat broadened ( data not shown ) . therefore , 
 different samples were independently aged and used to generate single 
 sets of parameters .
the plots in figure 2 are 
 the averaged scatter plots of the regressed values for 20 silica gels 
 entrapping nlps and 9 silica gels entrapping liposomes . regressed values 
 for ( a ) cooperativity and ( b ) phase transition 
 temperature of nlp and liposome samples entrapped in silica gel over 
 the course of 56 weeks . upon synthesis of tmos - derived silica gel
the methanol content in rotary 
 evaporated silica was determined to be roughly 5 v / v% , while untreated 
 silica had a methanol concentration of 24 v / v% ( see the supporting information ) . due to its presence , 
 the effect of methanol on the phase behavior of di15:0pc in nlps and 
 liposomes was examined . as shown in figure 3
, the anisotropy values of nlps in buffer solution decreased in nearly 
 constant intervals as the concentration of methanol was increased 
 at all temperatures .
this was also observed for liposomes ( see figure 
 s3 , supporting information ) .
measured anisotropy 
 values and corresponding regression curves 
 of different nlp samples in 20 mm tris , 100 mm nacl at various concentrations 
 of methanol ( v / v% ) .
moreover , the tm values for nlps and 
 liposomes monotonically decreased and then increased with increasing 
 methanol concentration , as shown in figure 4a , with minimum tm values of 29.1  
 0.1 c at 28  4 v / v% methanol and 28.3  0.1 c 
 at 20  4 v / v% methanol , respectively .
cooperativity indices , 
 obtained by fits to eq 2 , did not show corresponding 
 minima or maxima and instead increased for nlps and decreased for 
 liposomes with increasing methanol concentration , as shown in figure 4b as the methanol concentration was increased 
 in solution , the size 
 of particles in nlp samples also increased . at 0 , 15 , and 30 v / v% , 
 the stokes diameter of particles in nlp solution samples was 11.7 
  2.2 , 17.3  3.2 , and 93.0  3.5 nm , respectively 
 ( see table s3 , supporting information ) . 

the effect of methanol was also examined inside silica gel for nlps , 
 where samples without rotary evaporation during the sol  gel 
 processing were compared to samples utilizing it . in figure 5 and table 1 ,
it is shown 
 that elimination of the rotary evaporation step , which corresponds 
 to higher methanol concentrations , resulted in lowered anisotropy 
 values and a reduced phase transition temperature ( 46.3  0.6 
 c for rotary evaporated samples and 40.0  0.5 c 
 for non - rotary - evaporated samples ) .
in addition , it was observed that 
 the cooperativity change was small ( 0.33  0.06 c for rotary 
 evaporated samples and 0.35  0.05 c for non - rotary - evaporated 
 samples )
. regressed parameters for ( a ) phase transition temperature ( tm ) and ( b ) cooperativity ( n ) of nlps and liposomes in 20 mm tris , 100 mm nacl buffer in methanol 
measured anisotropy values 
 of nlps in silica gel after initial 
 entrapment with and without the use of rotary evaporation during the 
 sol  gel processing .
far - uv circular dichroism 
 spectroscopy was used to examine the secondary structures of the scaffold 
 protein msp in lipid - free and lipid - bound conformations .
figure 6 shows spectra in terms of ellipticity vs wavelength 
 for msp alone ( lipid - free ) and msp assembled in nlps ( lipid - bound ) 
 in both solution and gel - entrapped states .
it can be seen that there 
 were vertical shifts in spectral intensity for msp over the course 
 of 1 week .
these shifts were more prominent for samples where msp 
 was in a lipid - free conformation ( figure 6a , d ) 
 in comparison to samples where it was in a lipid - bound conformation 
 ( figure 6b , e ) in both solution and silica gel , 
 respectively .
circular dichroism spectra of ( a ) lipid - free msp in solution 
 and 
 ( b ) lipid - bound msp ( nlps ) in solution and ( c ) corresponding solution 
 -helix content determined from 222 nm ellipticity .
circular 
 dichroism spectra of ( d ) lipid - free msp in silica gel and ( e ) lipid - bound 
 msp ( nlps ) in silica gel , with ( f ) corresponding gel -helix 
 content . for all cd spectra in figure 6 , two peaks 
 characteristic of substantial -helical content
the actual 
 -helical content was estimated from the 222 nm peak using eqs 3 and 4 , as other secondary 
 structural elements have little contribution to this region of a protein 
 spectrum . in figure 6c , the -helical content in buffer solution was shown 
 to remain at 70% for lipid - bound msp and increase from 35% to 43% 
 for lipid - free msp over the course of 1 week . in figure 6f ,
the -helical content in silica gel was shown to 
 remain constant at 75% for lipid - bound msp and decrease from 73% to 
 65% for lipid - free msp over the course of 1 week .
liposomes can contain 
 tens to hundreds of thousands of lipids per structure and have cooperativity 
 units that have been estimated to include up to 1700 lipid molecules .
having fewer lipids per structure , nlps are 
 not capable of forming cooperative units as large as those present 
 in liposomes .
in addition , a fraction of the lipids , located in a two - lipid - thick 
 belt along the scaffold protein boundary , undergo a concomitant loss 
 of cooperativity after nlp formation .
therefore , 
 in solution , nlps inherently have a lower cooperativity index than 
 liposomes , as we have observed here .
the presence of scaffold proteins 
 has also been shown to elevate the phase transition temperature in 
 solution , as observed here , for a given lipid incorporated into nlps 
 compared to when incorporated into liposomes due to an increase in 
 lateral pressure from the protein  lipid interactions . upon entrapment in the silica gel ,
cooperativity 
 of the nlp phase transition decreased slightly ( 93%  10% of 
 solution value ) while cooperativity of the liposomes decreased significantly 
 ( 12%  3% of solution value ) .
this leads us to believe that the 
 liposomes are undergoing significant structural changes upon entrapment , 
 while the nlps are not .
a significant reduction in size of liposomes 
 has been shown to result in lowered cooperativity .
the rupturing of liposomes and formation of smaller lipid 
 aggregates or bicellar structures could reasonably explain this observed 
 behavior and has previously been observed in other works where liposomes 
 were entrapped in silica gel .
moreover , only a slight 
 reduction in cooperativity and elevation in phase transition temperature 
 ( compared to liposomes ) for nlps observed over the course of 56 
 weeks could indicate that they are not undergoing significant alterations 
 in their size and structure after entrapment . upon silica gel 
 entrapment
, there was an observed elevation in 
 phase transition temperature for nlps and liposomes , which is consistent 
 with previous work where elevated phase transitions for entrapped 
 liposomes were observed due to speculated excluded volume effects .
this is analogous to effects observed in work 
 with thermal unfolding of proteins in silica gel , where a higher free 
 energy ( thus higher unfolding temperature ) was imposed upon entrapped , 
 folded proteins due to decreased volume available for the unfolded 
 form .
since the lipid tails become less dense 
 when undergoing a gel phase to liquid crystalline transition , it is reasonable to believe that the excluded 
 volume effect can be involved .
the observed decreased in anisotropy 
 range is due either to a change in the packing and motion of the lipid 
 tails in each phase or release of a portion of the dph into the pores 
 of the silica gel ( see the supporting information ) .
nonetheless , a decrease in the anisotropy range does not have 
 significant effect on the regressed values for the cooperativity index 
 or phase transition temperature ( see figure s1 , supporting information ) .
we speculate that the minor 
 ( approximately 20% ) reduction in cooperativity 
 over weeks for entrapped nlps could be related to the known phenomenon 
 of silica gel shrinkage over time .
since phospholipid 
 headgroups are known to interact strongly with silica surfaces , changes in the geometry and size of pores could 
 directly impact adsorbed nlps . a decrease in size of lipid structure 

is correlated to a decrease in cooperativity and could rationalize 
 this observation ; however , the possibility of aggregation is not ruled 
 out .
a decrease in silica gel size corresponds to a decrease in porosity , 
 which would enhance the excluded volume effect , explaining our observed 
 increase in phase transition temperature over weeks for nlps .
further 
 work in analyzing different silane precursors and monolith shrinkage 
 effects on lipid structures would be required to validate this hypothesis . 

overall , these results make it very plausible that the silica sol  gel - entrapped 
 nlps , unlike liposomes , maintain a structure resembling their solution 
 counterparts for weeks at a time .
due to the presence 
 of residual methanol in the silica gels ( 5 v / v% ) , the effect 
 of methanol on anisotropy values and phase behavior for nlps and liposomes 
 in solution was examined . the trend in decreasing anisotropy and minimum tm values for nlps and liposomes
as the methanol 
 concentration was increased is consistent with previous fluorescence 
 anisotropy studies involving short - chain alcohols , as well as similar studies in which lipid absorbance at 
 400 nm was utilized .
short - chained alcohols increase 
 the area per molecule of the lipid bilayer and induce the interdigitated phase ; thus , the dph probe is allowed to more freely rotate , lowering the 
 anisotropy value .
the minimum in the tm value is reported to correspond to the completion of the interdigitation 
 transition .
the elevation in methanol concentration 
 required to fully interdigitate nlps could indicate that they have 
 a slightly higher resistance to bilayer interdigitation .
the presence 
 of the scaffold proteins could perhaps prolong the bilayer structure 
 in elevated methanol concentrations due to direct interactions with 
 the lipid tails , making it more difficult for them to interlace .
the 
 use of tmos instead of the popular precursor teos is favorable due 
 to its liberation of methanol , which requires exponentially higher 
 concentrations than ethanol to significantly modulate lipid bilayer 
 behavior .
we found that in the vicinity 
 of 5 v / v% methanol , anisotropy and tm values 
 for nlps and liposomes were minimally changed .
therefore , the more 
 significant changes in these values for silica sol  gel - entrapped 
 nlps and liposomes were not caused by the presence of methanol . while the liposomes display a decrease in the cooperativity index , 
 cooperativity increases for nlps with increasing methanol concentration . 
 the interdigitated phase of bilayers tends to have a lower phase transition 
 temperature than the gel phase ; thus , the decrease in the cooperativity that is observed is potentially 
 the result of coexistence between the gel and interdigitated phases . 

this coexistence would result in the transition appearing broader , 
 as the phase transition equation used ( eq 2 ) 
 only accounts for one inflection point .
however , at 5 v / v% methanol , 
 the cooperativity of the phase transition is only decreased by approximately 
 10% in comparison to 90% decrease observed for liposomes entrapped 
 in silica gel . in the case of the nlps ,
the measured increase in aggregate 
 size in methanol solutions would account for larger cooperative units , 
 which would directly increase the cooperativity index .
the appearance 
 of the opposite trend , i.e. , slightly decreasing cooperativity , for 
 silica - gel - entrapped nlps illustrates that the methanol concentration 
 is below the threshold necessary for aggregate growth . by removing 
 most of the methanol through rotary evaporation in 
 silica sol  gel - entrapped nlps
, we have avoided significant 
 changes in anisotropy and tm caused by 
 methanol .
if we did not use rotary evaporation , the solution methanol 
 concentration was roughly 24 v / v% with accompanying decreased anisotropy 
 and tm values , which were consistent in 
 magnitude with what was observed in solution experiments . however , 
 the increase in cooperativity was minimal compared to that observed 
 in solution .
the size of the pores ( 550 nm ) could perhaps 
 limit the nlps from aggregating or remodeling into larger lipid structures 
 that would have cooperative units comparable to those in solution .
the presence 
 of -helical secondary structure is essential for nlp formation 
 and protein  lipid binding .
we 
 found that the -helical content of the scaffold protein msp 
 is significantly higher in its lipid - bound nlp - associated state ( 70% ) , 
 versus a lipid - free state ( 35%43% ) in solution .
this magnitude 
 of difference is consistent with previous works that examined -helical 
 content in very similar scaffold proteins , such as msp1d1 and apolipoprotein a - i .
for apolipoprotein a - i , this is due to 4 
 of the 10 helical regions forming a bundle in the lipid - free state , 
 while the other 6 helical regions , along with the globular region , 
 fold in a variety of different conformations having relatively higher 
 random coil content .
it is reasonable 
 to believe that similar behavior is involved for msp , as it is derived 
 directly from apolipoprotein a - i . once entrapped in silica gel
interestingly , entrapped msp in its lipid - free state adopted 
 a significantly higher -helical content than in solution , increasing 
 to 73% .
these results are consistent with previous discoveries of 
 the biocompatible environment of silica sol 
gels for water - soluble 
 proteins which are often stabilized against denaturation and aggregation 
 in silica sol  gels .
the stabilization effect has been attributed 
 to the ability of the sol  gel matrix to restrict conformational 
 flexibility and diffusional motion and to promote structural rigidity 
 in the water environment .
the confinement from 
 the pores of the silica gel could rationalize the observed increase 
 in -helical content for msp upon entrapment ; an estimated radius 
 of gyration of 3 nm for randomly structured msp in solution ( see the supporting information ) is on the same order 
 of magnitude as the pore size .
this could potentially cause msp to 
 adopt an alternative conformation that consists of higher -helical 
 content .
the decrease of -helical content over the course of 
 a week ( 73%65% ) for lipid - free msp in silica gel in comparison 
 to the steady -helical content of 75% for lipid - bound msp could 
 indicate that the silica gel promotes lipid association of msp , thereby 
 maintaining the higher helical conformation of this state .
however , the absence of lipids does not allow msp to maintain a 
 constant conformation .
we have demonstrated 
 that nanolipoprotein particles ( nlps ) are 
 more compatible with the nanoscale environment of the silica gel pores 
 in comparison to liposomes .
direct measurement of size of soft matter 
 inside of mesoporous silica is difficult to obtain ; thus , we utilized 
 biophysical characterization in the form of fluorescence anisotropy 
 and circular dichroism spectroscopy to directly investigate lipid 
 phase behavior and scaffold protein secondary structure , as well as 
 indirectly correlate this behavior to aggregate size .
fluorescence 
 anisotropy , which is then used to analyze the cooperativity and temperature 
 of the main phase transition , revealed that nlps entrapped in silica 
 gel exhibit phase behavior with a stronger resemblance to their solution 
 counterparts than liposomes .
in particular , cooperativity indices 
 indicate that entrapment in silica gel causes immediate large - scale 
 changes in lipid aggregation state of liposomes toward smaller cooperative 
 units and only minor changes for nlps over weeks of time .
these large - scale 
 changes in liposomes have been linked in the past to liposome rupture 
 and denaturation of integral membrane proteins . by investigating these 
 same properties for liposomes and nlps in methanol solutions ,
we find 
 that the small amount of methanol remaining after evaporative removal 
 is not sufficient to cause observed changes in these properties upon 
 sol  gel entrapment .
however , if we did not remove the methanol , 
 we found that entrapped nlps displayed shifts in anisotropy and phase 
 transition temperature that were consistent with large fractions of 
 methanol but that the cooperativity was relatively maintained , which 
 we attribute to limitations in the growth of the nlps by confinement 
 in the nanoporous environment of the silica gel . upon further investigation 
 of conformational changes , circular dichroism revealed that the scaffold 
 protein of the entrapped nlps maintained a consistent -helical 
 content necessary for its structural function of belting the phospholipids . 

this is consistent with the known biocompatibility and structure promotion 
 of silica gels for water - soluble proteins .
future work will entail 
 the incorporation of integral membrane proteins inside of nlps and 
 analysis / quantification of their activity retention upon sol  gel - derived 
 entrapment via protein - specific assays .
 the entrapment of nanolipoprotein 
 particles ( nlps ) and liposomes 
 in transparent , nanoporous silica gel derived from the precursor tetramethylorthosilicate 
 was investigated . 
 nlps are discoidal patches of lipid bilayer that 
 are belted by amphiphilic scaffold proteins and have an average thickness 
 of 5 nm . 
 the nlps in this work had a diameter of roughly 15 nm and 
 utilized membrane scaffold protein ( msp ) , a genetically altered variant 
 of apolipoprotein a - i . 
 liposomes have previously been examined inside 
 of silica sol  
 gels and have been shown to exhibit instability . 
 
 this is attributed to their size ( 150 nm ) and altered structure 
 and constrained lipid dynamics upon entrapment within the nanometer - scale 
 pores ( 550 nm ) of the silica gel . by contrast , the dimensional 
 match of nlps with the intrinsic pore sizes of silica gel opens the 
 possibility for their entrapment without disruption . 
 here we demonstrate 
 that nlps are more compatible with the nanometer - scale size of the 
 porous environment by analysis of lipid phase behavior via fluorescence 
 anisotropy and analysis of scaffold protein secondary structure via 
 circular dichroism spectroscopy . 
 our results showed that the lipid 
 phase behavior of nlps entrapped inside of silica gel display closer 
 resemblance to its solution behavior , more so than liposomes , and 
 that the msp in the nlps maintain the high degree of -helix 
 secondary structure associated with functional protein  
 lipid 
 interactions after entrapment . 
 we also examined the effects of residual 
 methanol on lipid phase behavior and the size of nlps and found that 
 it exerts different influences in solution and in silica gel ; unlike 
 in free solution , silica entrapment may be inhibiting nlp size increase 
 and/or aggregation . 
 these findings set precedence for a bioinorganic 
 hybrid nanomaterial that could incorporate functional integral membrane 
 proteins . 
Introduction
Materials and Methods
Results
Discussion
Conclusions