10218 DOI: 10.1021/la1003808 Langmuir 2010, 26(12), 10218–10222Published on Web 03/19/2010

pubs.acs.org/Langmuir

© 2010 American Chemical Society

Investigating Lyophilization of Lipid Nanocapsules with Fluorescence

Correlation Spectroscopy

Polina B. Smith, Kimberly A. Dendramis, and Daniel T. Chiu*

Department of Chemistry, University of Washington, Seattle, Washington 98195-1700

Received January 26, 2010. Revised Manuscript Received March 6, 2010

This paper describes characterization of lyophilized lipid nanocapsules loaded with Alexa 488 by fluorescencecorrelation spectroscopy (FCS). Fluorimetry analysis of nanocapsules containing self-quenching concentrations of5- and 6-carboxyfluorescein was performed to establish a point of reference for FCS. FCS results complemented theresults obtained by fluorimetry for a bulk nanocapsule solution and provided additional information about the size anddye retention by individual nanocapsules. Using this method, we determined that nanocapsules composed of the thiol-functionalized lipids showed the best dye retention and the most consistent results. Dye retention, size, and photolysisefficiency of these thiol-functionalized nanocapsules doped with a far-red photosensitizer did not change substantiallyupon lyophilization and storage at-20 �C for up to 2months,making lyophilization a suitablemethod for the long-termstorage of nanocapsules with the appropriate lipid composition.

Introduction

Lipid nanocapsules have received much attention as deliveryvessels for drug and other biological stimuli to cells.1-3 Our groupis interested in perturbing single cells with high spatiotemporalresolution and thus has focused on the development of light-addressable nanocapsules as an alternative for use in the photo-lysis of caged compounds.4-8 Chemically caged molecules sufferfrom several drawbacks, including tediousness of design andsynthesis as well as the difficulty in chemically caging largebiological molecules, such as peptides and proteins. Nanocap-sules, in contrast, offer a versatile platform for “caging” a widerange of biologically relevant molecules. Recently, we have alsodemonstrated the light-triggered release of biological stimuli fromphotosensitized nanocapsules using a single nanosecond laserpulse in the far-red wavelength region,9,10 a feat that has yet to beachieved with chemically caged molecules.

Despite the versatility and potential usefulness of light-addres-sable lipid nanocapsules, there is one important practical limita-tion. Upon their storage in solution, lipid nanocapsules tend todegrade fairly fast (over a few days or less) and thus usually haveto be freshly prepared for each experiment. To facilitate anddisseminate the use of light-addressable nanocapsules, it is of highpractical value to have nanocapsules that could be stored overlong periods of time (weeks to months) without a substantial lossof encapsulated material and photolysis efficiency.

One of the most widely used techniques for storage of bio-logically relevant materials is lyophilization (or freeze-drying).Indeed, liposomes have been successfully lyophilized previouslyand were shown to preserve their encapsulated content best whenfreeze-dried in solutions containing saccharides.11-16 The short-and long-term storage of lyophilized liposomes were also in-vestigated.17-19 For these studies, only the bulk behavior of theliposomes was monitored. Leakage from the liposomes wascharacterized using a fluorimetry method based on the analysisof liposomes loaded with self-quenching concentrations of 5- and6-carboxyfluorescein (CF).11,12,17,18 Although the fluorimetrymethod is easy to use and is a well-established technique, itmeasures parameters of the liposome solution in bulk, relies onself-quenching concentrations of encapsulated dyes, and does notoffer any insight on what is happening at a single-nanocapsulelevel. For example, it would be impossible to tell from the resultsobtained by fluorimetrywhether the decrease in retention ofCF iscaused by rupture of a few nanocapsules or whether all of thenanocapsules had leaked to some degree. It is important for ourapplications, however, to know the change in dye concentrationand size of individual nanocapsules upon lyophilization andrehydration. To overcome the drawbacks of the standard fluori-metry method, this paper describes the use of fluorescencecorrelation spectroscopy (FCS) for monitoring the retention ofdye molecules in nanocapsules as well as the aggregation ofnanocapsules, information that is inaccessible to the fluorimetrymethod.

*Corresponding author. E-mail: chiu@chem.washington.edu.(1) Chrai, S. S.; Murari, R.; Ahmad, I. BioPharm (Duluth, MN, U. S.) 2002, 15,

40-43, 49.(2) Immordino, M. L.; Dosio, F.; Cattel, L. Int. J. Nanomed. 2006, 1, 297–315.(3) Ruysschaert, T.; Germain, M.; Gomes, J.; Fournier, D.; Sukhorukov, G. B.;

Meier, W.; Winterhalter, M. IEEE Trans. Nanobiosci. 2004, 3, 49–55.(4) Sun, B.; Chiu, D. T. J. Am. Chem. Soc. 2003, 125, 3702-3703.(5) Sun, B.; Mutch, S. A.; Lorenz, R.M.; Chiu, D. T.Langmuir 2005, 21, 10763–

10769.(6) Sun, B. Y.; Chiu, D. T. Langmuir 2004, 20, 4614–4620.(7) Sun, B. Y.; Chiu, D. T. Anal. Chem. 2005, 77, 2770–2776.(8) Sun, B. Y.; Lim, D. S. W.; Kuo, J. S.; Kuyper, C. L.; Chiu, D. T. Langmuir

2004, 20, 9437–9440.(9) Dendramis, K. A.; Allen, P. B.; Reid, P. J.; Chiu, D. T. Chem. Commun.

2008, 4795–4797.(10) Dendramis, K. A.; Chiu, D. T. J. Am. Chem. Soc. 2009, 131, 16771–16778.

(11) Ausborn, M.; Schreier, H.; Brezesinski, G.; Fabian, H.; Meyer, H. W.;Nuhn, P. J. Controlled Release 1994, 30, 105–116.

(12) Crowe, J. H.; Crowe, L. M. Biochim. Biophy. Acta 1988, 939, 327–334.(13) Hauser, H.; Strauss, G. Adv. Exp. Med. Biol. 1988, 238, 71–80.(14) Hua, Z. Z.; Li, B. G.; Liu, Z. J.; Sun, D.W.Drying Technol. 2003, 21, 1491–

1505.(15) Miyajima, K. Adv. Drug Delivery Rev. 1997, 24, 151–159.(16) Vemuri, S.; Yu, C.D.; Degroot, J. S.;Wangsatornthnakun, V.; Venkataram,

S. Drug Dev. Ind. Pharm. 1991, 17, 327–348.(17) Sun, W. Q.; Leopold, A. C.; Crowe, L. M.; Crowe, J. H. Biophys. J. 1996,

70, 1769–1776.(18) van Winden, E. C. A.; Crommelin, D. J. A. J. Controlled Release 1999, 58,

69–86.(19) vanWinden, E. C. A.; Crommelin, D. J. A. Eur. J. Pharm. Biopharm. 1997,

43, 295–307.

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Smith et al. Article

FCS has been previously used by our group and others to studythe parameters associatedwith single particles.20-24 In addition tothe information about brightness, and therefore leakage, ofindividual nanocapsules, FCS can simultaneously collect datathat can be used to determine nanocapsule size/aggregation. Thispaper provides the first report of the characterization of lyophi-lized lipid nanocapsules by FCS. Dye retention and change insize/aggregation of individual nanocapsules upon lyophilizationand rehydration are reported. Using this method, we found thiol-functionalized lipid nanocapsules offer the best solution for long-term storage using lyophilization.

Materials and Methods

Materials. All lipids and polycarbonate membranes werepurchased fromAvanti Polar Lipids, Inc. (Alabaster, AL). Alexa488 carboxylic acid succinimidyl ester mixed isomers (Alexa 488),10-dioctadecyl-3,3,30,30-tetramethylindodicarbocyanine perchlo-rate (DiD), and 5- and 6-carboxyfluorescein mixed isomers(CF) were purchased from Invitrogen (Carlsbad, CA). Alexa488 was hydrolyzed by addition of water to get a free dye. Allother reagents were used as received. The miniextruder waspurchased from Avestin (Ottawa, Ontario). Spin columns werepurchased from Epoch Biolabs (Missouri City, TX). All otherreagents and materials were purchased from Sigma-Aldrich(St. Louis, MO).

Nanocapsule Preparation. Nanocapsules were prepared byrehydration of dry lipid cakes in the buffer containing dye for theencapsulation. In a typical procedure, 0.2 mg of lipid or lipidmixture was dried from its solution in chloroform for 1 h undernitrogen and then for aminimumof 2 h under vacuum.To the drylipid cake, we added 200 μL of 100 mM solution of CF in ourlyophilization buffer (which was composed of 10 wt % sucroseand 20 mMHEPES of pH 7.4) or 200 μL of lyophilization bufferand 5 μL of 10 mg/mLAlexa 488 in water. The liposome mixturewas then heated to 70 �C in a water bath, vortexed, and freeze-thawed three times by submersion in liquid nitrogen for 45 sand then thawing in room-temperature water bath. Liposomesolutions were then heated to 70 �C and passed 21 times through100 nm polycarbonate membrane using a miniextruder. Unen-capsulated dye was then removed by running nanocapsule solu-tions through size-exclusion microspin columns containingSephacryl-100HR equilibrated with the lyophilization bufferusing a centrifuge with RCF 2700g. Eluent was diluted 1:10 withlyophilization buffer to get the final lipid nanocapsule solution.

Lyophilization. 200 μL of each of the lipid nanocapsulesolutions was placed in 1.5 drum glass vials, frozen in liquidnitrogen for 2 min, and immediately placed under vacuum on aLabconco Freezone 6 freeze-dryer. The rest of the nanocapsulesolutions were stored at 4 �C. After 18-20 h, lyophilized sampleswere removed from the freeze-dryer, and 180 μL of deionizedwater (Milli-Q) was added to each.

Fluorimetry. Solutions for fluorimetry experiments weretypically prepared by adding 50 μL of nanocapsule solution to3 mL of lyophilization buffer. A nanocapsule solution wasanalyzed right after its preparation, and after the analysis, partof the solution was lyophilized and the rest was stored at 4 �C.Rehydrated lyophilized solution and nonlyophilized solutionstored at 4 �C were analyzed again on the following day.Fluorimetry spectra for each sample were obtained before and

after the addition of 20μLof 5%aqueous solution ofTritonX100detergent. Fluorimetry spectra were collected on Fluorolog Tau 3fluorimetry with excitation at 488 nm by a xenon lamp. Fluores-cence intensitywas determinedby integrating the emission spectrain the 500-700 nm area. Dye retention was determined asdescribed in the literature:12

% retention ¼ ½ðFa0 -Fb

0Þ=Fa0�=½ðFa -FbÞ=Fa��100%

where Fb andFa are the fluorescence intensity before and after theaddition of detergent for nanocapsule solution right after pre-paration and Fb

0 and Fa0 are the fluorescence intensity before and

after addition of detergent to lyophilized or nonlyophilizedsample (part of the solution that was stored at 4 �C while thelyophilized part was drying).

Fluorescence Correlation Spectroscopy (FCS). Solutionsfor FCS experiments were used as prepared. In the same mannerwith fluorimetry experiments, a nanocapsule solution was ana-lyzed byFCS right after its preparation.After the analysis, part ofthe solution was lyophilized and the rest was stored at 4 �C. Bothlyophilized and nonlyophilized solutions were analyzed again onthe following day. The setup and experimental conditions werepreviously described.20-23 In short, 40-50 μL droplets of solu-tions were placed on a glass coverslip above the objective andexcited by 488 nm solid-state diode pumped laser (Coherent,Santa Clara, CA). The laser probe volume was focused 25 μmabove the surface of the coverslip. Fluorescence signal wascollected via an avalanche photodiode (APD SPCM-AQR-16;Perkin-Elmer, Fremont, CA) and autocorrelated using a Flex-02digital correlator (Correlator.com, Bridgewater, NJ). At the startand end of each experiment, calibration data were collected using190 nm polystyrene beads doped with Suncoast Yellow dye(Bangs Laboratories, Fishers, IN). Radius of the nanocapsuleswas determined from the diffusion coefficient obtained fromfitting the autocorrelation data. To produce an autocorrelationcurve, sample fluorescence was collected for 2 min, with threeautocorrelation curves collected for each droplet. Three dropletsfor each sample were analyzed.

Intensity data were collected concurrently with the autocorre-lation data in 5 ms bins. 60 000 bins were collected for eachdroplet. Intensity data were then exported into MS Excel andanalyzed by RobStat.xla Version 1.0 Robust Statistics Tool Kit,based on the Royal Chemical Society Analytical MethodsCommittee (RSC AMC) Technical Brief No. 6 to get medianand median absolute difference (MAD) values. The baseline(background) was set at a value equal to 3 � (median þ 3 �

Figure 1. Lipids used for nanocapsule preparation. All lipids havethe same hydrophobic tail and differ only in the nature of theirhydrophilic heads.

(20) Budzinski, K. L.; Allen, R. W.; Fujimoto, B. S.; Kensel-Hammes, P.;Belnap, D. M.; Bajjalieh, S. M.; Chiu, D. T. Biophys. J. 2009, 97, 2577–2584.(21) Gadd, J. C.; Kuyper, C. L.; Fujimoto, B. S.; Allen, R.W.; Chiu, D. T.Anal.

Chem. 2008, 80, 3450–3457.(22) Kuyper, C. L.; Budzinski, K. L.; Lorenz, R. M.; Chiu, D. T. J. Am. Chem.

Soc. 2006, 128, 730–731.(23) Kuyper, C. L.; Fujimoto, B. S.; Zhao, Y.; Schiro, P. G.; Chiu, D. T. J. Phys.

Chem. B 2006, 110, 24433–24441.(24) Rigler, P.; Meier, W. J. Am. Chem. Soc. 2006, 128, 367–373.

10220 DOI: 10.1021/la1003808 Langmuir 2010, 26(12), 10218–10222

Article Smith et al.

MAD) for a particular sample. The average intensity for eachsample was then calculated as the sum of intensities in bins abovethe background divided by the number of bins above the back-ground and minus the background. Average intensity of everysample was normalized to the average bead intensities measuredbefore and after sample data collection.

Photolysis Efficiency. Lipid nanocapsules were photolyzedand photolysis efficiencies were calculated as previously de-scribed.10 Briefly, the photolysis efficiency was calculated as thepercent of 50 nanocapsules that photolyzed upon a single laserpulse delivered to eachnanocapsule. Three sampleswere analyzedfor each data point.

Results and Discussion

Lipids for Nanocapsules. To select a lipid that produced themost robust nanocapsules, the following set of lipid nanocapsuleswas prepared: (1) DPPC (1,2-dipalmitoyl-sn-glycero-3-phos-phocholine), (2) 9:1 DPPC:DPPG (1,2-dipalmitoyl-sn-glycero-3-phospho-(10-rac-glycerol) [sodium salt]), (3) DPPtdEG (1,2-di-palmitoyl-sn-glycero-3-phospho(ethylene glycol) [sodium salt]),and (4) DPPTE (1,2-dipalmitoyl-sn-glycero-3-phosphothioetha-nol [sodium salt]) (Figure 1). All of the lipids have the samehydrophobic tail but vary in the hydrophilic headgroup.AneutralDPPC lipid is often used for liposome preparation. It is, however,frequently combined with negatively charged DPPG to improvethe robustness of the liposomes. Nanocapsules composed solelyof DPPG, however, do not form well. We also attempted to formnanocapsules composed of the positively charged lipid DPTAP

(1,2-dipalmitoyl-3-trimethylammonium propane [chloride salt])but failed to produce quality nanocapsules.

Besides increased robustness, negatively charged nanocapsuleswould also minimize nonspecific cell adsorption. Thus, we se-lected two other negatively charged lipids not commonly used forliposome preparation, DPPtdEG and DPPTE, and tested themfor liposome formation.We found both to formhigh-quality lipidnanocapsules. DPPtdEGandDPPTE structures differ only in theend group of the hydrophilic head. DPPtdEG has a hydroxylfunctionality, and DPPTE contains a thiol functional group thathas a potential to be oxidized to form disulfide bonds and renderlipid nanocapsules even more robust.Fluorimetry. To establish a reference for FCS, fluorimetry

experiments were carried out in a similar manner as described inthe literature.12 Buffer containing 100 mM of CF was used as aloading solution, which produced a self-quenching CF concen-tration inside the liposomes. Figure 2 shows the amount of dyeretained by the nanocapsules that were lyophilized then rehy-drated aswell as the nonlyophilizednanocapsules thatwere storedat 4 �C for 19 h. It can be seen that lyophilized nanocapsulescomposed of purely negatively charged lipids, DPPTE andDPPtdEG, retained about 80% of the encapsulated CF, whileliposomes made of DPPC and DPPC/DPPG retained only about50%. Since all of the nonlyophilized nanocapsules stored at 4 �Cretained close to 100% of the encapsulated dye, the leakage ofencapsulated dye from lyophilized samples was triggered byfreeze-drying and rehydration.

Although the fluorimetry technique is easy to use, is wellestablished, and provides a good estimate for the dye retentionby the nanocapsules, it suffers from several drawbacks. First,fluorimetry technique relies on self-quenching dye concentrationsinside the liposomes. Upon dye leakage, there is a possibility thatthe concentration inside the nanocapsuleswill dropbelow the self-quenching level. In this case, the increase in fluorescence intensitywill no longer be caused solely by the leaked dyes; dye retainedwithin the liposomes may also contribute to an increase influorescence intensity, which would overestimate the amount ofdye that has leaked from the liposomes. Second, it is impossible totell whether the increase in leaked dye was caused by rupture of asmall number of nanocapsules or was due to homogeneousleakage from all nanocapsules. To overcome these drawbacks,we performed FCS analysis of lipid nanocapsules.Fluorescence Correlation Spectroscopy (FCS). For FCS

experiments, lipid nanocapsules were loaded with Alexa 488 dye,chosen for its better photostability than CF. It can be seen fromFigure 3 that the average dye retention for the lyophilized lipidnanocapsules was at or above the levels obtained by the fluori-metry method. The average brightness of nonlyophilized nano-capsules measured after storage for 19 h at 4 �Cwas the same as itwas right after the nanocapsules were prepared. In general, it wasfound that lipid nanocapsules containing the encapsulated dyeretained their dye content at most for a few days upon storage at4 �C. There is wide variability, however, in the duration thatnanocapsules can be stored at 4 �C, mostly dependent on thetype of lipids used to form the nanocapsules. For example,nanocapsules made from neutral DPPC only last for hours (lessthan 1 day) while those made with negatively charged lipidscan last several days. It is notable that nonlyophilized DPPCnanocapsules displayed an increase in the average brightness,which can be attributed to aggregate formation, evident fromthe pronounced average radius increase of DPPC liposomes(Figures 4 and 5).

The averagedye retentionof lyophilizednanocapsules appearedto be greater than thedye retentionmeasured by fluorimetry for all

Figure 2. Fluorimetry analysis of the retention of encapsulated 5-and 6-carboxyfluorescein (CF) by nonlyophilized (NL; part of thesolution that was stored at 4 �C for 19 h, while the lyophilized partwas drying) and lyophilized (L; rehydrated right after lyophilization)lipidnanocapsules.RetentionofCFwas calculatedas%retention=[(Fa

0 - Fb0)/Fa0]/[(Fa - Fb)/Fa] � 100%, where Fb and Fa are the

fluorescence intensitybeforeandafter the additionofdetergent intoananocapsule solution rightafternanocapsulesweremadeandFb

0 andFa

0 are the fluorescence intensity before and after the addition ofdetergent to a lyophilized (L) or a nonlyophilized (NL) sample.

Figure 3. FCSanalysis of brightness of nonlyophilized (NL;part ofthe solution that was stored at 4 �C for 19 h, while the lyophilizedpart was drying) and lyophilized (L; rehydrated right afterlyophilization) lipid nanocapsules containing Alexa 488. Data nor-malized to intensity of nanocapsules right after their preparation.

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Smith et al. Article

nanocapsules. There are two possible reasons for such behavior:(1) dye retentionmeasuredby fluorimetry does not account for therelease of the dyedue to thenanocapsule rupture and (2) nonlinearincrease in fluorescence due to dequenching of CF. For mostapplications, the presence of nonencapsulated material in theexternal matrix due to rupture of nanocapsules will not be anissue because a quick filtration through the size exclusion chro-matography (SEC) column should remove the nonencapsulatedmaterial. A significant difference in size between the lipid nano-capsules and the encapsulated molecules that makes purificationvia SEC possible is another advantage of using lipid nanocapsulesinstead of chemically caged compounds, which often are hard toseparate from their dark hydrolysis products.

Figure 4 shows the radii of the lipid nanocapsules determinedby FCS. DPPTE nanocapsules showed the smallest increase insize after lyophilization, with the normalized average radius afterthe lyophilization lying within the standard deviation of theaverage radius measured right after the preparation. The mostpronounced increase in radius was observed for DPPC nanocap-sules. The increase in size forDPPCnanocapsules can also be seenin Figure 5. Images of DPPC nanocapsules after storage at 4 �Cfor 19 h (Figure 5B) and after lyophilization and rehydration(Figure 5C) contain spots that are much larger than spots inimages taken right after nanocapsule preparation (Figure 5A),indicating the formation of aggregates and/or fused nanocap-sules. Spots in images of DPPTE nanocapsules (Figure 5D-F)are of the same size before and after storage or lyophilization.Wenote that the number of observed spots in each image does notserve as an accurate indicator of the concentration of thenanocapsules because the density of nanocapsules on the surfacedepends on many variables, including the settling time and the“stickiness” of the surface.

The slight increase in the radii of the DPPC/DPPG andDPPtdEG nanocapsules seen in Figure 4 was most probablycaused by their slight aggregation and/or fusion. The images ofthese nanocapsules (data not provided) did not reflect anyqualitative change in size, meaning that the size of these aggre-gated and/or fused nanocapsules would have to be smaller thanthe microscope resolution.

The increase in the viscosity of the nanocapsule solution afterthe lyophilized solutions were rehydrated could also have resultedin the increased measured radius, although we believe this tobe unlikely. Lyophilized samples were rehydrated by addition of180 μL of deionized water to the dry cake produced by freeze-drying of 200 μL of nanocapsule solution in 10 wt % sucrosebuffer. Fluorimetry measurements of the samples after the

addition of Triton X100 detergent showed that the total concen-tration ofCF in the rehydrated samples on average was 102( 2%of theCF concentration in the nonlyophilized samples. Therefore,it can be assumed that the concentration of all the other solutes,including sucrose, in the lyophilized samples stayed within 4% oftheir original concentrations as well. A 4% increase in the sucroseconcentration would have resulted in less than 2% increase in thecalculated radius of the nanocapsules.Effect of Lipid Headgroup on Dye Leakage. Data in

Figures 2 and 3 show that there was virtually no leakage of theencapsulated dye from the nonlyophilized nanocapsules aftertheir storage at 4 �C for 19 h. Thus, the nature of the lipidheadgroup did not affect the leakage of the encapsulated dyesfrom the nanocapsules stored in a solution. Upon lyophilization,however, nanocapsules composed of the negatively charged lipidsretained more dye than the neutral DPPC nanocapsules. Becauselyophilized DPPC nanocapsules also formed the most aggregatedand/or fused species, this aggregation and/or fusion could be theleading cause of their increase in the dye leakage.

This hypothesis is also supported by the fact that the electro-static repulsion between the negatively charged lipid nanocapsuleswould decrease their aggregation and/or fusion and result in abetter retention of the encapsulated dye. Nanocapsules composed

Figure 4. Radius of nonlyophilized (NL; part of the solution thatwas stored at 4 �C for 19 h, while the lyophilized part was drying)and lyophilized (L; rehydrated right after lyophilization) lipidnanocapsules containing Alexa 488 determined by FCS. Radiuswas calculated from the diffusion coefficient determined by auto-correlation curves. Data normalized to radius of nanocapsulesright after their preparation.

Figure 5. Images of lipid nanocapsules loaded with Alexa 488obtained with total internal reflection fluorescence (TIRF) micro-scopy: (A) DPPC right after preparation; (B) DPPC after storageat 4 �C for 19 h; (C) DPPC after lyophilization and rehydration;(D) DPPTE right after preparation; (E) DPPTE after storage at4 �C for 19 h; (F) DPPTE after lyophilization and rehydration.Scale bar is 5 μm.

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of purely negatively charged lipids, DPPTE and DPPtdEG,showed a better dye retention than DPPC/DPPG nanocapsulesthat contained only 10 mol % of negatively charged lipid. Inaddition, Figure 4 shows that DPPTE nanocapsules exhibited thesmallest increase in their radius after the lyophilization. Since thiolgroups in DPPTE are more acidic than their alcohol counterpartsin DPPtdEG, the potential presence of an additional negativecharge on the DPPTE headgroup could contribute to an evengreater repulsion between the nanocapsules and even lower dyeleakage due to the aggregation and/or fusion of nanocapsules.Incorporation of Photosensitizer Dye. On the basis of the

results from Figures 2-5, DPPTE was chosen as the best-perform-ing lipid for the nanocapsule formation due to the lower dye leakageand a better reproducibility. Tomake nanocapsules photolyzable, afar-red absorbing lipophilic dye, DiD (1,10-dioctadecyl-3,3,30,30-tetramethylindodicarbocyanineperchlorate),was incorporated intotheDPPTEnanocapsules at 6mol%, an amount shown to have anoptimal photolysis efficiency previously.10 The effect of DiDincorporation on the dye retention, size, and photolysis efficiencyof lyophilized nanocapsules was investigated (Figure 6). A slightincrease in the dye leakage inDPPTE/DiDnanocapsules comparedto pure DPPTE nanocapsules was observed by fluorimetry, butFCS results showed 99%retention ofAlexa 488 after lyophilizationand rehydration. The average radius of DPPTE/DiD nanocapsuleswas measured to be 42 ( 2 nm and had increased slightly upon

lyophilization (Figure 6C). The photolysis efficiency of lyophilizedDPPTE/DiDnanocapsuleswas the sameaswith thenonlyophilizednanocapsules (Figure 6D).Long-Term Storage. The effects of the long-term storage on

the dye retention, size, and photolysis efficiency of lyophilizedDPPTE/DiD nanocapsules stored at -20 �C can be seen inFigure 7. No significant change in photolysis efficiency or sizewas observed even after 2 months of storage. A slight decrease inthe brightness after 2 weeks of storage was, in all probability,induced by freezing of the lyophilized samples at-20 �C, since nofurther decrease in brightness was observed after 1 and 2 monthsof storage. The lyophilized nanocapsuleswere stored at-20 �C tosimulate the storage conditions of nanocapsules containing bio-logically active molecules. Overall, DPPTE/DiD nanocapsulesappeared to be suitable for long-term storage with the encapsu-lated material.

Conclusions

This paper describes the firstFCS characterizationof lyophilizedlipid nanocapsules. Therewere threemajor sets of findings: (1) FCSwas more appropriate for the analysis of the dye retention byindividual lyophilized lipid nanocapsules, since fluorimetry did notaccount for the dye leaked due to the rupture of a small populationof the liposomes; (2) out of all the lipids tested, DPPTE gavethe most robust nanocapsules and the most consistent results;and (3) lyophilization could be used for long-term storage ofthe photolyzable nanocapsules without substantial loss of theencapsulated material or of the photolysis efficiency. In addition,the conditions we used to lyophilize and store nanocapsules aresimilar to the conditions used to store sensitive biological materi-als, such as antibodies. The ability to store photolyzable lipidnanocapsules has an important practical advantage and opensnew avenue for their use in a growing number of applications.

Acknowledgment. K. A. Dendramis gratefully acknowledgessupport by the NIH Training Program No. 1T32CA138312-01.This work is supported by the NSF (CHE-0924320) and the NIH(AG 029574).

Figure 6. Analysis of nonlyophilized (partof the solution thatwas storedat 4 �Cfor 19h,while the lyophilizedpartwasdrying) and lyophilized(rehydrated right after lyophilization) DPPTE/DiD lipid nanocapsules: (A) retention of encapsulated 5- and 6-carboxyfluorescein determinedby fluorimetry; (B) retention of Alexa 488 determined by FCS; (C) radius of nanocapsules loaded with Alexa 488, determined by FCS; (D)photolysis efficiency of nanocapsules containing Alexa 488. Data normalized to measurements of nanocapsules right after their preparation.

Figure 7. Fluorescence intensity (O), radius (]), and photolysisefficiency (0) of lyophilizedDPPTE/DiDnanocapsules containingAlexa 488 and stored at -20 �C over time. Data normalized tonanocapsule parameters measured right after their preparation.