Remotely Triggered Liposome Release by Near-Infrared Light Absorption via Hollow Gold Nanoshells

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Abstract

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Supporting Info

An elusive goal for systemic drug delivery is to provide both spatial and temporal control of

drug release. Liposomes have been evaluated as drug delivery vehicles for decades, but their

clinical significance has been limited by slow release or poor availability of the encapsulated

drug. Here we show that near-complete liposome release can be initiated within seconds by

irradiating hollow gold nanoshells (HGNs) with a near-infrared (NIR) pulsed laser. Our findings

reveal that different coupling methods such as having the HGNs tethered to, encapsulated

within, or suspended freely outside the liposomes, all triggered liposome release but withdifferent levels of efficiency. For the underlying content release mechanism, our experiments

suggest that the microbubble formation and collapse due to the rapid temperature increase of

the HGN is responsible for liposome disruption, as evidenced by the formation of solid gold

particles after the NIR irradiation and the coincidence of a laser power threshold for both

triggered release and pressure fluctuations in the solution associated with cavitation. These

effects are similar to those induced by ultrasound and our approach is conceptually analogous

to the use of optically triggered nano-sonicators deep inside the body for drug delivery. We

expect HGNs can be coupled with any nanocarriers to promote spatially and temporally

controlled drug release. In addition, the capability of external HGNs to permeabilize lipid

membranes can facilitate the cellular uptake of macromolecules including proteins and DNA

and allow for promising applications in gene therapy.

Abstract

Remotely Triggered Liposome Release by Near-Infrared Light

Absorption via Hollow Gold Nanoshells

Guohui Wu, Alexander Mikhailovsky, Htet A. Khant,

Caroline Fu, Wah Chiu and Joseph A. Zasadzinski

Department of Chemical Engineering and Department of

Chemistry, University of California, Santa Barbara,

California 93106, and Department of Biochemistry and

Molecular Biology, Baylor College of Medicine, Houston,Texas 77030

J. Am. Chem. Soc., 2008, 130 (26), pp 81758177

DOI: 10.1021/ja802656d

Publication Date (Web): June 11, 2008

Copyright 2008 A merican Chemical Society

[email protected], Department of Chemical Engineering, University of California, Santa Barbara.,

Department of Chemistry, Univers ity of California, Santa Barbara., Baylor College of Medicine.

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motely Triggered Liposome Release by Near-Infrared Light Absorption... http://pubs.acs.org/doi/abs/10.1021/ja802656d?prevSearch=guohui%2...

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Remotely Triggered Liposome Release by Near-Infrared Light Absorption viaHollow Gold Nanoshells

Guohui Wu, Alexander Mikhailovsky, Htet A. Khant, Caroline Fu, Wah Chiu, andJoseph A. Zasadzinski*,

Department of Chemical Engineering and Department of Chemistry, UniVersity of California,Santa Barbara, California 93106, and Department of Biochemistry and Molecular Biology,

Baylor College of Medicine, Houston, Texas 77030

Received April 11, 2008; E-mail: [email protected]

A major challenge for drug delivery is to control drug release

both spatially and temporally. Liposomes have been evaluated as

drug nanocarriers for decades,15 but their clinical applications are

often limited by slow release or poor availability of the encapsulated

drug.6 Here we show that near-complete liposome release can be

initiated within seconds (burst kinetics) by irradiating hollow gold

nanoshells (HGNs) with a near-infrared (NIR) pulsed laser. NIR

light penetrates into the tissue up to 10 cm,

7

allowing these HGN/liposome complexes to be addressed noninvasively within a

significant fraction of the human body. Our findings on the

underlying release mechanism reveal that this approach is conceptu-

ally analogous to using optically triggered nano-sonicators deep

inside the body for drug delivery.

It has proven difficult to create liposomes that are simultaneously

resistant to drug leakage in the circulation8,9 and able to rapidly

release their contents at the site of interest. Many of the current

strategies to enhance temporal or spatial control of drug release

focus on incorporating components into the liposome membranes

to achieve thermal, pH, photochemical, or enzymatically triggered

release.35,10 Unfortunately, destabilizing agents often promote

release in the circulation as well as the site of interest. Active

targeting requires specific ligands with high affinities to receptors

overexpressed on diseased cells, which can lead to binding-site

barriers where the tightly bound nanocarriers prevent drug

penetration into the tissue.4 In addition, targeting a different site

requires the synthesis and characterization of a new ligand.

A new strategy is to delegate the task of controlled drug release

to an externally triggered agent, while optimizing liposome

composition and structure to enhance circulation time and drug

retention. Recently, 2-3 nm gold particles incorporated into

thermally sensitized liposome membranes were shown to enhance

contents release over 10-20 min during continuous irradiation by

UV-light (which limits application to the body surface).11 A NIR

light-based approach has been shown to work on polymer

carriers,1215

which unfortunately are still in the research stage fordrug delivery.16 Besides, it would be difficult to continuously

irradiate a given nanoparticle for 10-20 min before it convects or

diffuses out of the irradiation zone. Liposomes were the first type

of nanoparticles in clinical use; however, little work on controlled

release using NIR light has been reported. Several challenges are:

(1) to develop easily synthesized, biocompatible triggering agents

with a strong NIR absorption small enough (


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HGNs, similar to the cavitation effects induced by ultrasound. That

means release is not due to simple heating as reported in previous

work.15

As proof of concept, a fluorescent dye, 6-carboxyfluorescein

(CF), was encapsulated inside liposomes and used as a soluble

model drug. HGNs with a maximum absorption at 820 nm (Figure

1d) were synthesized via galvanic replacement chemistry19,22

(Supporting Information). HGNs were then coated with 750-Da

polyethylene glycol-thiol (PEG) to enhance particle stability and

were concentrated by ultracentrifugation. The diameter of the HGNs

was 33 ( 13 nm with shell thickness of 3.4 ( 0.9 nm. HGNs were

encapsulated within dipalmitoylphosphatidylcholine (DPPC) lipo-

somes together with CF at a sufficient concentration that CFs

fluorescence was self-quenched (Figure 1a and Supporting Informa-

tion). The unencapsulated HGNs and CF were removed by size-

exclusion chromatography and centrifugation. On release from the

liposomes, CF is diluted to micromolar concentrations so that the

CF fluorescence intensity is proportional to its concentration. The

release of CF from the liposomes was quantified by the increase in

fluorescence intensity above the background relative to the fluo-rescence intensity after all liposomes were lysed.8

Disruption of liposomes was triggered by irradiation with NIR

pulses from a Ti:Sapphire laser (0 ) 800 nm, 130-fs duration, 1

kHz frequency, energy up to 670 J/pulse, corresponding to a mean

power density of 16.1 W/cm2). We monitored the in situ CF release

by recording the evolution of two-photon luminescence over time

to determine the release kinetics (Figure 2a). Irradiation with the

pulsed-NIR laser at a power exceeding 2.2 W/cm2 triggered a near

instantaneous increase of fluorescence intensity in the solution of

liposomes encapsulating HGNs and CF. NIR laser pulses had no

effect on the CF fluorescence intensity in control solutions of DPPC

liposomes with CF but no HGNs, unencapsulated CF, or a mixture

of HGNs and CF (Supporting Information).

To reveal the mechanism of contents release, we varied the laser

power density and compared the fractional CF release. Figure 2b

shows a threshold power density is needed to trigger release: no

fluorescence increase was detected for a power density less than

1.5 W/cm2; while for power densities greater than 4.3 W/cm2,

the maximum fractional release remained constant at about 71%

and 27%, for liposomes encapsulating HGNs (Figure 1a) or mixedwith free HGNs (Figure 1c), respectively. The growth rate and

magnitude of fluorescence intensity during NIR irradiation increased

with the laser power density above the threshold (Figure 2a). With

laser power density at 1.3 W/cm2 (below the threshold) the

fluorescence intensity was constant. At the maximum power level,

release is complete within seconds. A similar power threshold (1.5

W/cm2) was reported necessary to damage cancer cells treated with

NIR irradiation of gold nanocages.21

We investigated the changes of HGN/liposome complexes

induced by pulsed laser irradiation. Cryo-EM shows that only minor

changes in liposomal morphology are visible after irradiation (Figure

3); the membranes are less circular and appear to be under less

tension than before irradiation (Figure 1), which is consistent with

the decrease in the osmotic pressure caused by CF release. This

lack of change in liposome morphology suggests that irradiation

of the HGNs leads to transient defects in the lipid membrane that

enable fast contents release, after which the membrane integrity is

restored. Meanwhile, there was no observable change in the total

CF fluorescence induced by the laser-heated nanoshell indicating

that there was little, if any, chemical degradation of the dye. Cryo-

EM also shows the change in morphology of the HGNs; the hollow

core collapses on itself to form solid gold nanoparticles (red arrow

in Figure 3). The HGN changes were confirmed by UV-vis

spectroscopy (Supporting Information); the 820 nm absorption peak

of HGNs gradually disappears with irradiation,22 along with the

growth of a peak at 530 nm, which is typical for solid gold

nanoparticles. The collapse of HGNs indicates they reach suf-ficiently high temperatures after absorbing NIR pulses to melt and

anneal into more stable shapes. Even though the gold nanoshells

are heated above their melting point, the temperature increase of

the bulk solution was less than 1 C above ambient. Hence, the

rapid CF release was not due to the increased permeability of DPPC

membranes known to occur at the phase transition temperature of

41 C.3

The laser power threshold and the lack of permanent damage to

the liposomes suggest that the triggered release occurs through

perforation of lipid bilayers by microbubble formation and collapse,

referred to as transient cavitation.20,27,28 When an HGN is irradiated,

its temperature rises substantially; heat dissipation to the surrounding

water is slower than the electron dynamics in plasmon-mediated

Figure 2. Effect of pulsed-laser power: (a) Kinetics of in situ fluorescenceintensity shows the rate of liposome release induced by encapsulated HGNsat various laser powers. The solid lines are single exponential fits, F) Fo+ Ae-x/ to the data. (b) Liposome release as a function of laser powerinduced by HGNs encapsulated inside and suspended freely outside after 9min of irradiation. The solid curves are sigmoidal fits to the data: y ) (ymax

- ymin)/(1 + e(E-E

0)/E

) + ymin. The maximum release is different for thetwo coupling methods, but the threshold power density for release is thesame (2.2 W/cm2). (c) Typical photoacoustic signal of pressure fluctuationsassociated with cavitation recorded by a hydrophone from a 0.142 mMHGN solution after a single laser pulse (16.1 W/cm2). The inset is anenlarged view of the first 100 s. (d) Acoustic signal amplitude as a functionof pulsed-laser power. 2.3 W/cm2 is necessary to induce the cavitation signal,which is similar to the threshold needed to trigger liposome contents release(Figure 2b).

Figure 3. Morphology of HGN/liposome complex after laser irradiation.Cryo-EM images showing that HGNs become solid-core nanoparticle (redarrows) after NIR pulsed-laser irradiation (16.1 W/cm2) both inside (left)and outside (right) of the liposomes (blue arrows).

8176 J. AM. CHEM. SOC. 9 VOL. 130, NO. 26, 2008

C O M M U N I C A T I O N S


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heating.22,29 Substantial temperature gradients around the HGNs

can then cause the formation of unstable vapor microbubbles, which

may grow rapidly and then collapse violently producing the

mechanical and thermal effects associated with transient cavitation

similar to those induced by ultrasound.28,30 Continuous-wave (cw)

laser (0 ) 820 nm) irradiation for 4 h produced no release of CF

from HGN/liposome complexes even at an increased power density

(89 W/cm2). Under cw irradiation, HGNs are always near thermal

equilibrium with their surroundings; the lack of temperature

gradients prevents microbubble formation and liposome dis-ruption.22,29

The characteristic acoustic signals of pressure fluctuations in

HGN solutions associated with cavitation were detected using a

hydrophone (Figure 2c). These acoustic signals were absent in CF

or buffer solutions which contained no HGNs under the same

irradiation. Figure 2d shows the acoustic signal amplitude in the

HGN solution as a function of laser power density. The acoustic

signal amplitudes were at background up to the laser power density

of 2.3 W/cm2 which coincides with the power threshold for

liposome contents release (Figures 2b). Above the threshold, there

was a sharp increase in the acoustic signal amplitudes (Figure 2d).

The increased laser power leads to higher HGN temperatures,22

which are then translated into larger pressure fluctuations in solution

while this energy is dissipated. These results are consistent with

reports on laser-induced cavitation.31

Membrane permeabilization by microbubble cavitation is ex-

pected to be induced by NIR-absorbing HGNs as long as HGNs

are within an optimal distance from the lipid membrane. To test

this hypothesis, we mixed DPPC liposomes containing CF with

free HGNs (Figure 1c) at various concentrations. Upon pulsed laser

irradiation, CF was released and the fractional release increased

linearly with external gold concentration up to 0.0315 mM resulting

in a maximum release of 35%. To minimize and control the distance

between HGNs and the lipid membrane, HGNs were tethered to

the liposomes via a thiol-PEG-lipid linker32,33 (Figure 1b, Sup-

porting Information). Tethering HGNs directly to the outer surface

of the liposomes increased the release fraction to 93%. Therefore,the efficacy of phototriggered contents release is strongly affected

by the proximity of the HGN to the lipid membrane which is

consistent with the hypothesis that mechanical disruption by

microbubbles is responsible for the transient membrane rupture.20,23,34

In conclusion, pulsed NIR light absorbed by HGNs triggers the

near instantaneous release of liposome contents providing precise

spatial and temporal control. The laser-heated HGNs act as optically

triggered nano-sonicators to temporally disrupt the lipid mem-

brane. HGNs tethered to, encapsulated within, or suspended freely

outside liposomes all induce liposome disruption; however tethering

achieves the highest release efficacy due to the HGNs proximity

to the lipid membrane. With this new NIR-activated release, disease

cells can be synergistically targeted by combining drug carryingparticles (liposomes) and energy absorbing particles (HGNs);

continued irradiation of the HGNs can induce localized hyperther-

mia or permeabilize cell membranes, both of which can facilitate

the cellular uptake of large macromolecules including proteins and

DNA. This general approach will allow for better control of drug

delivery to selected disease sites while minimizing systemic toxicity;

no targeting ligands are needed to address different receptors and

no binding-site barriers would limit drug penetration.4 In future

work, in vivo testing and the long-term HGN safety need to be

examined thoroughly, although preliminary studies suggest that gold

nanoparticles are nontoxic.35,36

Acknowledgment. We thank Dr. Samir Mitragotri, Dr. Sumit

Paliwal, and Dr. Makoto Ogura for helpful discussions on cavitation

and for generously lending the hydrophone. This work was

supported by NIH Program of Excellence in Nanotechnology Grant

HL080718, NSF Grant CTS-0436124, and the UCSB ICB supported

by U.S. Army Grant No. DAAD19-030D-0004. W.C. was supported

by NIH Grant P41RR02250.

Supporting Information Available: TEM image of HGNs, data

showing pulsed NIR laser has no effect on the control solutions,

absorption spectra of liposome/HGNs/CF complex exposed to pulsed

laser, cryo-EM micrographs of tomographic imaging to demonstrate

encapsulation and tethering, comparison of different methods of

coupling HGNs to liposomes, as well as Materials and Methods. This

material is available free of charge via the Internet at http://pubs.acs.org.

References

(1) Sengupta, S.; Eavarone, D.; Capila, I.; Zhao, G.; Watson, N.; Kiziltepe,T.; Sasisekharan, R. Nature 2005, 436, 568572.

(2) Allen, T. M.; Cullis, P. R. Science 2004, 303, 18181822.(3) Ponce, A. M.; Vujaskovic, Z.; Yuan, F.; Needham, D.; Dewhirst, M. W.

Int. J. Hyperthermia 2006, 22, 205213.(4) Peer, D.; Karp, J. M.; Hong, S.; Farokhzad, O. C.; Margalit, R.; Langer,

R. Nat. Nanotechnol. 2007, 2, 751760.(5) Davidsen, J.; Jorgensen, K.; Andresen, T. L.; Mouritsen, O. G. Biochim.

Biophys. Acta, Biomembr. 2003, 1609, 95101.(6) Abraham, S. A.; Waterhouse, D. N.; Mayer, L. D.; Cullis, P. R.; Madden,

T. D.; Bally, M. B. Methods Enzymol. 2005, 391, 7197.(7) Weissleder, R. Nat. Biotechnol. 2001, 19, 316317.(8) Boyer, C.; Zasadzinski, J. A. ACS Nano 2007, 1, 176182.(9) Lee, S.-M.; Chen, H.; Dettmer, C. M.; OHalloran, T. V.; Nguyen, S. T.

J. Am. Chem. Soc. 2007, 129, 1509615097.(10) Shum, P.; Kim, J.-M.; Thompson, D. H. AdV. Drug DeliVery ReV. 2001,

53, 273284.(11) Paasonen, L.; Laaksonen, T.; Johans, C.; Yliperttula, M.; Kontturi, K.; Urtti,

A. J. Controlled Release 2007, 122, 8693.(12) Goodwin, A. P.; Mynar, J. L.; Ma, Y.; Fleming, G. R.; Frechet, J. M. J.

J. Am. Chem. Soc. 2005, 127, 99529953.(13) Das, M.; Sanson, N.; Fava, D.; Kumacheva, E. Langmuir2007, 23, 196

201.(14) Radt, B.; Smith, T. A.; Caruso, F. AdV. Mater. 2004, 16, 21842189.(15) Skirtach, A. G.; Dejugnat, C.; Braun, D.; Susha, A. S.; Rogach, A. L.;

Parak, W. J.; Moehwald, H.; Sukhorukov, G. B. Nano Lett. 2005, 5, 13711377.

(16) Portney, N. G.; Ozkan, M. Anal. Bioanal. Chem. 2006, 384, 620630.(17) Chen, J.; Wang, D.; Xi, J.; Au, L.; Siekkinen, A.; Warsen, A.; Li, Z.-Y.;Zhang, H.; Xia, Y.; Li, X. Nano Lett. 2007, 7, 13181322.

(18) Hirsch, L. R.; Stafford, R. J.; Bankson, J. A.; Sershen, S. R.; Rivera, B.;Price, R. E.; Hazle, J. D.; Halas, N. J.; West, J. L. Proc. Natl. Acad. Sci.U.S.A. 2003, 100, 1354913554.

(19) Sun, Y.; Mayers, B.; Xia, Y. AdV. Mater. 2003, 15, 641646.(20) Tong, L.; Zhao, Y.; Huff, T. B.; Hansen, M. N.; Wei, A.; Cheng, J.-X.

AdV. Mater. 2007, 19, 31363141.(21) Chen, J. Y.; Wang, D.; Xi, J.; Au, L.; Siekkinen, A.; Warsen, A.; Zhi-

Yuan, L.; Zhang, H.; Xia, Y. N.; Li, X. Nano Lett. 2007, 7, 13181322.(22) Prevo, B. G.; Esakoff, S. A.; Mikhailovsky, A.; Zasadzinski, J. A. Small

2008, in press.(23) Huang, X.; El-Sayed, I. H.; Qian, W.; El-Sayed, M. A. J. Am. Chem. Soc.

2006, 128, 21152120.(24) Norman, R. S.; Stone, J. W.; Gole, A.; Murphy, C. J.; Sabo-Attwood, T. L.

Nano Lett. 2008, 8, 302306.(25) Chiruvolu, S.; Warriner, H. E.; Naranjo, E.; Idziak, S.; Radler, J. O.; Plano,

R. J.; Zasadzinski, J. A.; Safinya, C. R. Science 1994, 266, 12221225.(26) Kisak, E. T.; Coldren, B.; Zasadzinski, J. A. Langmuir2002, 18, 284288.

(27) Paliwal, S.; Mitragotri, S. Expert Opin. Drug DeliVery 2006, 3, 713726.(28) Pecha, R.; Gompf, B. Phys. ReV. Lett. 2000, 84, 13281330.(29) Link, S.; Burda, C.; Nikoobakht, B.; El-Sayed, M. A. Chem. Phys. Lett.

1999, 315, 1218.(30) Popinet, S.; Zaleski, S. J. Fluid Mech. 2002, 464, 137163.(31) Lin, C. P.; Kelly, M. W. Appl. Phys. Lett. 1998, 72, 28002802.(32) Karyakin, A. A.; Presnova, G. V.; Rubtsova, M. Y.; Egorov, A. M. Anal.

Chem. 2000, 72, 380511.(33) Yang, M.; Yau, H. C. M.; Chan, H. L. Langmuir 1998, 14, 61216129.(34) Pitsillides, C. M.; Joe, E. K.; Wei, X.; Anderson, R. R.; Lin, C. P. Biophys.

J. 2003, 84, 40234032.(35) Connor, E. E.; Mwamuka, J.; Gole, A.; Murphy, C. J.; Wyatt, M. D. Small

2005, 1, 325327.(36) Stern Joshua, M.; Stanfield, J.; Lotan, Y.; Park, S.; Hsieh, J.-T.; Cadeddu

Jeffrey, A. J. Endourol. 2007, 21, 93943.

JA802656D

J. AM. CHEM. SOC. 9 VOL. 130, NO. 26, 2008 8177

C O M M U N I C A T I O N S


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Wu ET Al., PAGE S1

Supporting Information for

Remotely triggered liposome release by near-infrared light

absorption via hollow gold nanoshells

Guohui Wu, Alexander Mikhailovsky, Htet A. Khant, Caroline Fu, Wah Chiu,

and Joseph A. Zasadzinski *

*To whom correspondence should be addressed. E-mail: [email protected]

This PDF file includes:

Figs. S1 to S6

Materials and Methods

References


6/16

Wu ET Al., PAGE S2

SUPPORTING FIGURES AND LEGENDS

Figure S1 | TEM image of HGNs. All of the nanoshells have a hollow core, i.e., the light grey

area near the center of each nanoshell, surrounded by the gold shell, which is black in the images.

The measured diameter of HGNs is 33 13 nm (mean s.d., n = 3280) and the shell thickness is

3.4 0.9 nm (mean s.d., n = 269).


7/16

Wu ET Al., PAGE S3

Figure S2 | Pulsed NIR laser has no effect on the control solutions. (a) Fractional increase in

fluorescence intensity. The red bar shows that 71% of the CF was released from 0.2 mg/ml

DPPC liposomes encapsulating HGNs and CF. DPPC liposomes (0.2 mg/ml) encapsulating CF

dye but no HGNs showed no release of CF (green bar). The CF dye alone (9.7 M, blue) or a

mixture of HGNs and CF dye (0.08 mM and 9 M, respectively, cyan) showed no fluorescence

increase during irradiation. (b) Kinetics ofin situ fluorescence shows the fluorescence intensity

of control solutions remains constant during their exposure to the pulsed NIR laser at 16.0

mJ/cm2.

a b


8/16

Wu ET Al., PAGE S4

Figure S3 | UV-Vis spectrophotometry of a suspension of 0.1 mg/ml DPPC liposomes

encapsulating HGNs and CF inside, exposed to 16.0 W/cm2 fs-pulsed laser. The surface plasmon

peak of the HGN at ~ 820 nm gradually disappears with irradiation along with the growth of a

peak at ~ 530 nm, consistent with the melting and annealing of the nanoshells into solid

nanoparticles (See Fig. 3).

400 500 600 700 800 900 1000 1100

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

Absorbance

Wavelength (nm)

10 pulses

100 pulses

5000 pulses

485000 pulses

CF

HGN

Solid Au

particle


9/16

Wu ET Al., PAGE S5

Figure S4 | Cryo-EM images of the same set of liposomes with encapsulated HGN inside takenat different goniometer tilt angles: -45; 0; +45. The purpose of tilting the goniometer is tocomplement the otherwise missing information along the direction of e-beam in the 2-dimensional projected images. The tilt series clearly show which HGNs are inside theliposomes. The red arrows indicate the same HGNs during the 90 o range tilt to confirm thatHGNs are inside the liposomes.


10/16

Wu ET Al., PAGE S6

Figure S5 | Cryo-EM images of the same set of liposomes with HGN tethered to the outer

surface of the bilayer taken at different goniometer tilt angles: -45; 0; +45. The red arrows are

examples to mark the HGNs followed during the 90 tilt to confirm that HGNs are tethered to the

liposome surface.


11/16

Wu ET Al., PAGE S7

Figure S6 | Comparison of different methods for coupling HGNs to liposomes. The proximity of

the HGN to the lipid membrane strongly influences the fractional release of CF. HGNs directly

tethered to the bilayer induce 93% CF release; encapsulated HGNs induce 71% release and

HGNs in solution (not encapsulated) induce 28% release on irradiation with pulsed NIR laser.

0

20

40

60

80

100

HGNout

side

HGNinsi

de

HGNteth

ered

Trigger

edRelease(%)

liposome

toliposom

e

liposome


12/16

Wu ET Al., PAGE S8

Supplementary Materials

MATERIALS AND METHODS

Materials. Silver nitrate (AgNO3) and sodium borohydride (NaBH4) were purchased from Fisher

Scientific (Atlanta, GA). Sodium citrate was purchased from J.T.Baker Chemical Co.,

Phillipsburg, NJ. Hydroxylamine hydrochloride (NH2OH HCl), gold tetrachloroaurate (HAuCl4),

reduced Triton X100, Trauts regent (2-iminothiolaneHCl), and methoxypolyethylene glycol

amine (750PEG-NH2, molecular weight 750 Da) were purchased from Sigma Aldrich (St. Louis,

MO). Distearoylphosphoethanolamine-amino (polyethylene glycol) 2000 (DSPE-2000PEG-NH2)

and Dipalmitoylphosphatidylcholine (DPPC) were purchased from Avanti Polar Lipids

(Alabaster, AL). 6-CarboxyFluorescein (CF) was purchased from Invitrogen (Eugene, Oregon).

Synthesis of hollow gold nanoshells. Hollow gold nanoshells (HGN) were prepared as

described previously 1-3. Silver seed nanoparticles were prepared by reducing a well-stirred

solution of 600 mL 0.2 mM AgNO3 with 0.6mL 1.0 M NaBH4 in the presence of 0.5mM sodium

citrate. The solution was stirred for at least half an hour to allow NaBH4 to fully hydrolyze.Larger silver nanoparticles to be used as sacrificial templates for the gold nanoshells were grown

from the silver seed solution by adding 0.6 ml of 2.0 M NH2OHHCl and 1.5 ml 0.1 M AgNO3

and stirring overnight. The HGN were formed via sacrificial galvanic replacement of silver with

gold by quickly mixing 3.8 ml of 25 mM HAuCl4 solution with the silver nanoparticles at 60 C.

The silver template is oxidized to Ag+ ions as the gold is reduced to metal, coating the original

silver nanoparticle template, resulting in a hollow gold nanoshell 1-3.

The HGNs were sterically stabilized against aggregation by reacting the HGN with

polyethylene glycol via a Au-SH linkage. The 750PEG-SH was converted from 750PEG-NH2 using

Trauts regent with 1-fold molar excess of 2-iminothiolaneHCI in buffer (2.28 mM Na2HPO4,

pH 8.8). 0.5 ml of the as-prepared 0.0379 M 750PEG-SH was added to 600 ml HGN to achieve a

1000:1 ratio of thiol: gold. The PEG stabilized HGN solution was centrifuged at 21000 g for 30

minutes and dispersed in milli-Q water for two cycles to remove unattached soluble chemicals.

The pellet readily re-disperses in buffer. The UV-VIS absorption spectra of HGN were recorded

on a Jasco V-530 spectrometer (JASCO Corp., Tokyo) and showed a surface plasmon resonance


13/16

Wu ET Al., PAGE S9

peak at 820 nm. Sizing and morphology were analyzed by transmission electron microscopy on a

FEI Tecnai T20 microscope (Fig. S1).

Encapsulation of HGN and CF inside liposomes. CF was dissolved in water together with 6

equivalents of concentrated NaOH, which converts the CF from its acid form to the water-

soluble salt form. The CF solution described was used to disperse the HGN pellet described

above. Liposomes were prepared via the interdigitated phase transition. Firstly, the dry lipid was

hydrated by Milli-Q water and vortexed at 55 oC. DPPC unilamellar vesicles (50 nm in diameter)

were prepared by sonication at room temperature using a 60 Sonic Dismembrator (Fisher

Scientific, Atlanta, GA, USA) for 4 minutes at a power of 4 W. Secondly, the transition from thenormal (L) bilayer phase to the interdigitated (LI) bilayer phase was induced by the addition of

0.106 mL of ethanol (3 M net ethanol concentration) to 0.5 mL of a 50 mg/mL DPPC vesicle

suspension. The initially bluish vesicle suspension turned milky white, and its viscosity increased

significantly. After sitting at 4 oC for overnight, the interdigitated sheets were centrifuged and

dispersed in milli-Q water 3 times to remove ethanol. Thirdly, the pellet of interdigitated DPPC

sheets was mixed with the solution of CF and HGN. The mixture was then heated at 50 C for 2

hours under vortex mixing, driving the sheets to close around the HGN in suspension to form the

interdigitation-fusion vesicles 4, 5. Mixing of the CF and HGN with the pellet of DPPC

interdigitated sheets gives final concentrations of 32 mM CF (110 mOsM), 12 mM HGN and 22

mg/ml DPPC. Based on earlier work, 5, about 50 60% of the HGN is expected to be

encapsulated in liposomes. Fig. S4 shows a few cryo-EM micrographs during tomography tilt

imaging that demonstrate the HGN are indeed encapsulated within the liposomes after this

procedure.

After the encapsulation step, phosphate buffer saline (PBS: 20 mM Na2HPO4/NaH2PO4,

34.5 mM NaCl, pH = 7.4) was used to disperse the liposomes to minimize osmotic stress across

the membrane. The unencapsulated CF was removed by size-exclusion chromatography using a

Sephadex G-75 column (Amersham Biosciences Corp., Piscataway, NJ) eluted with PBS buffer.

The eluted suspension was centrifuged at 100 g for 20 minutes and re-dispersed in PBS buffer

twice to remove any unencapsulated HGN.


14/16

Wu ET Al., PAGE S10

Tethering HGN to liposomes containing CF. The HGNs were tethered through Au-SH-PEG-

lipid linkage to liposomes with 32 mM CF encapsulated inside. First, the pellet of interdigitated

DPPC sheets was prepared as described above. The interdigitated sheets were modified by

mixing with 2 mole% DSPE-2000PEG-NH2 powder along with CF solution. The mixture was then

heated at 50 C for 1 hour under vortex mixing, driving DSPE-2000PEG-NH2 to be incorporated

into DPPC sheets as the sheets closed and formed the interdigitation fusion vesicles 4, 5. Next,

the amine groups at the liposome surfaces were converted to thiol by mixing with 100% excess

2-iminothiolane solution. The thiolated liposomes encapsulating CF were incubated with a

solution of HGN and CF for 48 hours to allow HGN to tether to the outer surfaces of liposomes

(See Fig. S5). The final concentrations in the solution were 18 mg/ml phospholipid (98 mole %DPPC and 2 mole% DSPE-2000PEG-SH), 18 mM HGN and 32 mM CF. The liposomes with

tethered HGNs were eluted through a Sephadex G-75 size-exclusion column and centrifuged to

remove unencapsulated CF and free HGNs.

Mixing liposomes containing CF with external HGNs. DPPC liposomes containing 32 mM

CF were prepared by the same interdigitation-fusion method described above except no HGNs

were added. The preformed vesicles were eluted through a Sephadex G-75 column to remove

external CF, and then dispersed in different concentrations of HGN solutions.

Pulsed laser irradiation. The samples were irradiated by the output of the femtosecond (fs) Ti:

Sapphire regenerative amplifier (Spectraphysics Spitfire) running with 1 kHz repetition rate. The

laser beam was collimated by a Galilean telescope to achieve a Gaussian diameter of 2.3 mm.

Pulse duration was monitored by a home-built single-shot optical autocorrelator and was kept at

about 130 fs. The spectral FWHM of the laser radiation was ~ 12 nm centered around 800 nm.

The laser beam was directed onto the sample by a system of mirrors, no focusing optics were

used. The energy of the optical pulse was controlled by Schott neutral density glass filters. A

thermopile power meter (Newport Inc., Irvine, CA) was used to measure the incident optical

power. The maximum power available was 670 mW, which corresponds to 670 J/pulse and

energy density of 16.1 mJ/cm2 (or mean power density of 16.1 W/cm2). The temperature of the

HGN suspensions was measured using an Omegaette HH306 digital thermometer (Omega) with

a K-type thermal couple probe (Omega Engineering, Inc., Stamford, CN), which was immersed


15/16

Wu ET Al., PAGE S11

into the solution ~ 5 mm above the laser beam. The solution was stirred to ensure a good mixing

of the sample during irradiation.

Luminescence was excited in the sample via two-photon absorption process. The

emission was collected at a 90 angle by a system of lenses and focused on the entrance slit of a

monochromator (Acton Research SpectraPro 300). The laser radiation was blocked by a Schott

colored glass filter (BG38). The light dispersed by the monochromator was detected by a

spectroscopic CCD camera (PI Acton PIXIS-400) and transferred into a PC. The evolution of the

photoluminescence was recorded by collecting consecutive spectra over a 600 nm bandwidth

with a constant interval.

To quantify the fractional release of the dye, fluorescence was measured using a PTIQuantaMaster spectrofluorimeter (Photon Technology International, Lawrenceville, NJ). Any

release from the liposomes was detected by an increase in fluorescence intensity from the

background as the external concentration of CF increased. The fractional release can be

quantified as % release =omax

olaser

I-I

I-I, where Ilaser is the fluorescence intensity of the solution after

laser treatment, Imax is the maximum fluorescence intensity after solubilization or lysing of the

liposomes by reduced Triton X100 5, and Io is the background fluorescence intensity before

either treatment.

Continuous-wave Laser Irradiation. Irradiation of samples in continuous wave (CW) regime

was performed using Spectraphysics 3900S Ti:Sapphire CW laser. The laser wavelength was

tuned to 820 nm and the output power was controlled by changing the power of the pump laser

source (Spectraphysics Beamlok-2060) to be 0.7 W. The Gaussian beam diameter of the CW

laser is 1.0 mm.

Electron Cryo-Microscopy (Cryo-EM). Particles were suspended across a thin layer of

vitreous ice, which was prepared under controlled temperature and humidity conditions within a

VitRobot (FEI Company, Oregon) then vitrified by rapid plunging into liquid ethane 6-8. Cryo-

EM imaging was performed on a FEI Tecnai T20 microscope operating at 200 kV with a Gatan

liquid nitrogen specimen cryo-holder. Single-axis tomographic imaging was performed with a

JEOL 2010A microscope using 0o to 60o tilt angles in 2o increments with a total dose of less


16/16

Wu ET Al., PAGE S12

than 100 electrons per 2. To verify the location of HGNs relative to liposomes, 3-dimensional

reconstruction was done by IMOD (Boulder Laboratory for 3-D Electron Microscopy of Cells)

using HGNs as fiducial markers (3D movie not shown, instead 3 representative images at

goniometer tilt angles of -45, 0 and 45 o are shown in Fig. S4 and S5.).

Hydrophone measurements of pressure fluctuations. The pressure fluctuations in samples

undergoing laser irradiation were measured using a hydrophone (model TC4013, Reson, Goleta,

CA). The bandwidth of the hydrophone is 1 Hz-170 kHz. The hydrophone diameter is 0.5 cm

and the length is ~2 cm. The hydrophone was immersed into the solution ~ 5 mm above the laser

beam in a quartz cuvette with 10 mm lightpath and 3.0 ml capacity. The solutions volume was2.5 ml. The sample was irradiated by the pulsed fs Ti:Sapphire laser and the solution inside

cuvette was not stirred during the laser irradiation to minimize the acoustic noise. The output of

the hydrophone was collected by a digital oscilloscope (Tektronix TDS5032B). The data

collection was synchronized with the laser cavity dumping event by using triggering signal from

the laser control electronics. The acoustic transients were averaged over several hundred laser

pulses to improve the signal/noise ratio. A typical resultant waveform is shown in Fig. 2c of the

manuscript. Each measurement was summarized by the standard deviation of the amplitude of

the pressure fluctuation signal collected by the hydrophone as in Fig. 2d of the manuscript.

REFERENCES

(1) Prevo, B. G.; Esakoff, S. A.; Mikhailovsky, A.; Zasadzinski, J. A. Small2008.(2) Hao, E.; Li, S. Y.; Bailey, R. C.; Zou, S. L.; Schatz, G. C.; Hupp, J. T. J. Phys. Chem. B

2004,108, 1224-1229.(3) Sun, Y.; Mayers, B.; Xia, Y. Advanced Materials2003,15, 641-646.(4) Kisak, E. T.; Coldren, B.; Zasadzinski, J. A. Langmuir2002,18, 284-288.(5) Boyer, C.; Zasadzinski, J. A. ACS Nano2007,1, 176-182.

(6) Chiruvolu, S., Warriner, H. E., Naranjo, E., Idziak, S., Radler, J. O., Plano, R. J.,Zasadzinski, J. A. and Safinya, C. R. Science1994,266, 1222-1225.

(7) Chiruvolu, S.; Walker, S.; Leckband, D.; Israelachvili, J.; Zasadzinski, J. Science1994,264, 1753-1756.

(8) Jung, H. T.; Lee, S. Y.; Coldren, B.; Zasadzinski, J. A.; Kaler, E. W. Proceedings of theNational Academy of Sciences2002,99, 15318-15322.

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