Echogenic liposomes (ELIPs) are an excellent candidate for ultrasound activated therapeutics and imaging. Although multiple experiments have established their echogenicity, the underlying mechanism has remained unknown. However, freeze-drying in the presence of mannitol during ELIP preparation has proved critical to ensuring echogenicity. Here, the role of this key component in the preparation protocol was investigated by measuring scattering from freshly prepared freeze-dried aqueous solution of mannitol—and a number of other excipients commonly used in lyophilization—directly dispersed in water without any lipids in the experiment. Mannitol, meso-erythritol, glycine, and glucose that form a highly porous crystalline phase upon freeze-drying generated bubbles resulting in strong echoes during their dissolution. On the other hand, sucrose, trehalose, and xylitol, which become glassy while freeze-dried, did not. Freeze-dried mannitol and other crystalline substances, if thawed before being introduced into the scattering volume, did not produce echogenicity, as they lost their crystallinity in the thawed state. The echogenicity disappeared in a degassed environment. Higher amounts of sugar in the original aqueous solution before freeze-drying resulted in higher echogenicity because of the stronger supersaturation and crystallinity. The bubbles created by the freeze-dried mannitol in the ELIP formulation play a critical role in making ELIPs echogenic.

1.
Abdelwahed
,
W.
,
Degobert
,
G.
,
Stainmesse
,
S.
, and
Fessi
,
H.
(
2006
). “
Freeze-drying of nanoparticles: Formulation, process and storage considerations
,”
Adv. Drug Delivery Rev.
58
,
1688
1713
.
2.
Akers
,
M. J.
,
Milton
,
N.
,
Byrn
,
S. R.
, and
Nail
,
S. L.
(
1995
). “
Glycine crystallization during freezing: The effects of salt form, pH, and ionic strength
,”
Pharm. Res.
12
,
1457
1461
.
3.
Bader
,
K. B.
,
Haworth
,
K. J.
,
Shekhar
,
H.
,
Maxwell
,
A. D.
,
Peng
,
T.
,
McPherson
,
D. D.
, and
Holland
,
C. K.
(
2016
). “
Efficacy of histotripsy combined with rt-PA in vitro
,”
Phys. Med. Biol.
61
,
5253
5274
.
4.
Buchanan
,
K. D.
,
Huang
,
S.
,
Kim
,
H.
,
Macdonald
,
R. C.
, and
McPherson
,
D. D.
(
2008
). “
Echogenic liposome compositions for increased retention of ultrasound reflectivity at physiologic temperature
,”
J. Pharm. Sci.
97
,
2242
2249
.
5.
Budavari
,
S.
(
1996
). “
XP002191966
,” in
The Merck Index: Encyclopedia of Chemicals, Drugs, and Biologicals
, 13th ed. (
ACS
,
Washington, DC
), pp.
1
2708
.
6.
Cavatur
,
R. K.
,
Vemuri
,
N. M.
,
Pyne
,
A.
,
Chrzan
,
Z.
,
Toledo-Velasquez
,
D.
, and
Suryanarayanan
,
R.
(
2002
). “
Crystallization behavior of mannitol in frozen aqueous solutions
,”
Pharm. Res.
19
,
894
900
.
7.
Chen
,
H.
,
Chen
,
S.
,
Chen
,
H.
,
Wu
,
Y.
, and
Shu
,
G.
(
2015
). “
Effects of sugar alcohol and proteins on the survival of Lactobacillus bulgaricus LB6 during freeze drying
,”
Acta Sci. Pol. Technol. Aliment.
14
,
117
124
.
8.
Chongprasert
,
S.
,
Knopp
,
S. A.
, and
Nail
,
S. L.
(
2001
). “
Characterization of frozen solutions of glycine
,”
J. Pharm. Sci.
90
,
1720
1728
.
9.
Franks
,
F.
(
1998
). “
Freeze-drying of bioproducts: Putting principles into practice
,”
Eur. J. Pharm. Biopharm.
45
,
221
229
.
10.
Fujii
,
K.
,
Izutsu
,
K.-i.
,
Kume
,
M.
,
Yoshino
,
T.
,
Yoshihashi
,
Y.
,
Sugano
,
K.
, and
Terada
,
K.
(
2015
). “
Physical characterization of meso-erythritol as a crystalline bulking agent for freeze-dried formulations
,”
Chem. Pharm. Bull.
63
,
311
317
.
11.
Hartel
,
R. W.
(
2001
).
Crystallization in Foods
(
Springer
,
New York
).
12.
Hartel
,
R. W.
,
Ergun
,
R.
, and
Vogel
,
S.
(
2011
). “
Phase/state transitions of confectionery sweeteners: Thermodynamic and kinetic aspects
,”
Compr. Rev. Food Sci. Food Saf.
10
,
17
32
.
13.
Haworth
,
K. J.
,
Raymond
,
J. L.
,
Radhakrishnan
,
K.
,
Moody
,
M. R.
,
Huang
,
S.-L.
,
Peng
,
T.
,
Shekhar
,
H.
,
Klegerman
,
M. E.
,
Kim
,
H.
, and
McPherson
,
D. D.
(
2016
). “
Trans-stent B-mode ultrasound and passive cavitation imaging
,”
Ultrasound Med. Biol.
42
,
518
527
.
14.
Higashiyama
,
T.
(
2002
). “
Novel functions and applications of trehalose
,”
Pure Appl. Chem.
74
,
1263
1269
.
15.
Huang
,
S. L.
,
Hamilton
,
A. J.
,
Nagaraj
,
A.
,
Tiukinhoy
,
S. D.
,
Klegerman
,
M. E.
,
McPherson
,
D. D.
, and
MacDonald
,
R. C.
(
2001
). “
Improving ultrasound reflectivity and stability of echogenic liposomal dispersions for use as targeted ultrasound contrast agents
,”
J. Pharm. Sci.
90
,
1917
1926
.
16.
Huang
,
S. L.
,
Hamilton
,
A. J.
,
Pozharski
,
E.
,
Nagaraj
,
A.
,
Klegerman
,
M. E.
,
McPherson
,
D. D.
, and
MacDonald
,
R. C.
(
2002
). “
Physical correlates of the ultrasonic reflectivity of lipid dispersions suitable as diagnostic contrast agents
,”
Ultrasound Med. Biol.
28
,
339
348
.
17.
Huang
,
S. L.
, and
MacDonald
,
R. C.
(
2004
). “
Acoustically active liposomes for drug encapsulation and ultrasound-triggered release
,”
Biochim. Biophys. Acta, Biomembr.
1665
,
134
141
.
18.
Izutsu
,
K.-i.
,
Yoshioka
,
S.
, and
Terao
,
T.
(
1994
). “
Effect of mannitol crystallinity on the stabilization of enzymes during freeze-drying
,”
Chem. Pharm. Bull.
42
,
5
8
.
19.
Katiyar
,
A.
, and
Sarkar
,
K.
(
2010
). “
Stability analysis of an encapsulated microbubble against gas diffusion
,”
J. Coll. Interface Sci.
343
,
42
47
.
20.
Katiyar
,
A.
,
Sarkar
,
K.
, and
Jain
,
P.
(
2009
). “
Effects of encapsulation elasticity on the stability of an encapsulated microbubble
,”
J. Colloid Interface Sci.
336
,
519
525
.
21.
Kim
,
A. I.
,
Akers
,
M. J.
, and
Nail
,
S. L.
(
1998
). “
The physical state of mannitol after freeze-drying: Effects of mannitol concentration, freezing rate, and a noncrystallizing cosolute
,”
J. Pharm. Sci.
87
,
931
935
.
22.
Kopechek
,
J. A.
,
Haworth
,
K. J.
,
Raymond
,
J. L.
,
Douglas Mast
,
T.
,
Perrin
,
S. R.
, Jr.
,
Klegerman
,
M. E.
,
Huang
,
S.
,
Porter
,
T. M.
,
McPherson
,
D. D.
, and
Holland
,
C. K.
(
2011
). “
Acoustic characterization of echogenic liposomes: Frequency-dependent attenuation and backscatter
,”
J. Acoust. Soc. Am.
130
,
3472
3481
.
23.
Liao
,
X.
,
Krishnamurthy
,
R.
, and
Suryanarayanan
,
R.
(
2005
). “
Influence of the active pharmaceutical ingredient concentration on the physical state of mannitol—Implications in freeze-drying
,”
Pharm. Res.
22
,
1978
1985
.
24.
Nahire
,
R.
,
Haldar
,
M. K.
,
Paul
,
S.
,
Ambre
,
A. H.
,
Meghnani
,
V.
,
Layek
,
B.
,
Katti
,
K. S.
,
Gange
,
K. N.
,
Singh
,
J.
,
Sarkar
,
K.
, and
Mallik
,
S.
(
2014a
). “
Multifunctional polymersomes for cytosolic delivery of gemcitabine and doxorubicin to cancer cells
,”
Biomaterials
35
,
6482
6497
.
25.
Nahire
,
R.
,
Haldar
,
M. K.
,
Paul
,
S.
,
Mergoum
,
A.
,
Ambre
,
A. H.
,
Katti
,
K. S.
,
Gange
,
K. N.
,
Srivastava
,
D. K.
,
Sarkar
,
K.
, and
Mallik
,
S.
(
2013
). “
Polymer-coated echogenic lipid nanoparticles with dual release triggers
,”
Biomacromolecules
14
,
841
853
.
26.
Nahire
,
R.
,
Hossain
,
R.
,
Patel
,
R.
,
Paul
,
S.
,
Meghnani
,
V.
,
Ambre
,
A. H.
,
Gange
,
K. N.
,
Katti
,
K. S.
,
Leclerc
,
E.
,
Srivastava
,
D. K.
,
Sarkar
,
K.
, and
Mallik
,
S.
(
2014b
). “
pH-Triggered echogenicity and contents release from liposomes
,”
Mol. Pharmaceutics
11
,
4059
4068
.
27.
Nahire
,
R.
,
Paul
,
S.
,
Scott
,
M. D.
,
Singh
,
R. K.
,
Muhonen
,
W. W.
,
Shabb
,
J.
,
Gange
,
K. N.
,
Srivastava
,
D. K.
,
Sarkar
,
K.
, and
Mallik
,
S.
(
2012
). “
Ultrasound enhanced matrix metalloproteinase-9 triggered release of contents from echogenic liposomes
,”
Mol. Pharmaceutics
9
,
2554
2564
.
28.
Ohkuma
,
C.
,
Kawai
,
K.
,
Viriyarattanasak
,
C.
,
Mahawanich
,
T.
,
Tantratian
,
S.
,
Takai
,
R.
, and
Suzuki
,
T.
(
2008
). “
Glass transition properties of frozen and freeze-dried surimi products: Effects of sugar and moisture on the glass transition temperature
,”
Food Hydrocolloids
22
,
255
262
.
29.
Paul
,
S.
,
Russakow
,
D.
,
Nahire
,
R.
,
Nandy
,
T.
,
Ambre
,
A. H.
,
Katti
,
K.
,
Mallik
,
S.
, and
Sarkar
,
K.
(
2012
). “
In vitro measurement of attenuation and nonlinear scattering from echogenic liposomes
,”
Ultrasonics
52
,
962
969
.
30.
Pyne
,
A.
,
Chatterjee
,
K.
, and
Suryanarayanan
,
R.
(
2003
). “
Solute crystallization in mannitol–glycine systems—implications on protein stabilization in freeze-dried formulations
,”
J. Pharm. Sci.
92
,
2272
2283
.
31.
Pyne
,
A.
,
Surana
,
R.
, and
Suryanarayanan
,
R.
(
2002
). “
Crystallization of mannitol below Tg′ during freeze-drying in binary and ternary aqueous systems
,”
Pharm. Res.
19
,
901
908
.
32.
Pyne
,
A.
, and
Suryanarayanan
,
R.
(
2001
). “
Phase transitions of glycine in frozen aqueous solutions and during freeze-drying
,”
Pharm. Res.
18
,
1448
1454
.
33.
Rahman
,
M. S.
, and
Roos
,
Y. H.
(
2017
).
Glass Transition and Phase Transitions in Food and Biological Materials
(
Wiley
,
New York
), p.
824
.
34.
Raymond
,
J. L.
,
Haworth
,
K. J.
,
Bader
,
K. B.
,
Radhakrishnan
,
K.
,
Griffin
,
J. K.
,
Huang
,
S.-L.
,
McPherson
,
D. D.
, and
Holland
,
C. K.
(
2014
). “
Broadband attenuation measurements of phospholipid-shelled ultrasound contrast agents
,”
Ultrasound Med. Biol.
40
,
410
421
.
35.
Rey
,
L.
, and
May
,
J. C.
(
2010
).
Freeze-Drying/Lyophilization of Pharmaceutical and Biological Products
(
CRC Press
,
Boca Raton, FL
), p.
584
.
36.
Roos
,
Y.
(
1997
). “
Frozen state transitions in relation to freeze drying
,”
J. Therm. Anal. Calorim.
48
,
535
544
.
37.
Sarkar
,
K.
,
Katiyar
,
A.
, and
Jain
,
P.
(
2009
). “
Growth and dissolution of an encapsulated contrast microbubble
,”
Ultrasound Med. Biol.
35
,
1385
1396
.
38.
Shekhar
,
H.
,
Bader
,
K. B.
,
Huang
,
S.
,
Peng
,
T.
,
Huang
,
S.
,
McPherson
,
D. D.
, and
Holland
,
C. K.
(
2016
). “
In vitro thrombolytic efficacy of echogenic liposomes loaded with tissue plasminogen activator and octafluoropropane gas
,”
Phys. Med. Biol.
62
,
517
538
.
39.
Sutton
,
J.
,
Haworth
,
K.
,
Shanmukhappa
,
S.
,
Moody
,
M.
,
Klegerman
,
M.
,
Griffin
,
J.
,
Patton
,
D.
,
McPherson
,
D.
, and
Holland
,
C.
(
2016
). “
Delivery of bevacizumab to atheromatous porcine carotid tissue using echogenic liposomes
,”
Drug Delivery
23
,
3594
3605
.
40.
Tang
,
X. C.
, and
Pikal
,
M. J.
(
2004
). “
Design of freeze-drying processes for pharmaceuticals: Practical advice
,”
Pharm. Res.
21
,
191
200
.
41.
Telang
,
C.
,
Yu
,
L.
, and
Suryanarayanan
,
R.
(
2003
). “
Effective inhibition of mannitol crystallization in frozen solutions by sodium chloride
,”
Pharm. Res.
20
,
660
667
.
42.
Thanatuksorn
,
P.
,
Kajiwara
,
K.
,
Murase
,
N.
, and
Franks
,
F.
(
2008
). “
Freeze–thaw behaviour of aqueous glucose solutions—The crystallisation of cubic ice
,”
Phys. Chem. Chem. Phys.
10
,
5452
5458
.
43.
van Winden
,
E. C. A.
(
2003
). “
Freeze-drying of liposomes: Theory and practice
,”
Liposomes, Pt. A
367
,
99
110
.
You do not currently have access to this content.