U.S. patent application number 13/684809 was filed with the patent office on 2013-05-23 for encapsulation and controlled release of small molecules for intracellular delivery using thermally responsive nanocapsules.
This patent application is currently assigned to UNIVERSITY OF SOUTH CAROLINA. The applicant listed for this patent is University of South Carolina. Invention is credited to Xiaoming He.
Application Number | 20130129829 13/684809 |
Document ID | / |
Family ID | 42540610 |
Filed Date | 2013-05-23 |
United States Patent
Application |
20130129829 |
Kind Code |
A1 |
He; Xiaoming |
May 23, 2013 |
Encapsulation and Controlled Release of Small Molecules for
Intracellular Delivery Using Thermally Responsive Nanocapsules
Abstract
In accordance with certain embodiments of the present
disclosure, a method for intracellular delivery of small molecules
is provided. The method includes encapsulation of small molecules
in a thermally responsive nanocapsule by decreasing the temperature
of the nanocapsule to increase the permeability of the nanocapsule
and allowing the small molecules to be suck into or diffuse into
the nanocapsule. The nanocapsule is delivered into a cell by
increasing the temperature of the nanocapsule. The small molecules
are released from the nanocapsule into the cell in a controllable
manner by cooling and heating treatments.
Inventors: |
He; Xiaoming; (Dublin,
OH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
University of South Carolina; |
Columbia |
SC |
US |
|
|
Assignee: |
UNIVERSITY OF SOUTH
CAROLINA
Columbia
SC
|
Family ID: |
42540610 |
Appl. No.: |
13/684809 |
Filed: |
November 26, 2012 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12705072 |
Feb 12, 2010 |
8318207 |
|
|
13684809 |
|
|
|
|
61207485 |
Feb 12, 2009 |
|
|
|
Current U.S.
Class: |
424/493 ;
424/490; 424/497 |
Current CPC
Class: |
A61K 9/19 20130101; A61K
31/00 20130101; A61K 9/0004 20130101; A61K 9/5161 20130101; A61K
9/51 20130101; A61K 9/5146 20130101 |
Class at
Publication: |
424/493 ;
424/490; 424/497 |
International
Class: |
A61K 9/51 20060101
A61K009/51 |
Claims
1-14. (canceled)
15. A thermally responsive nanocapsule comprising: a polymeric
hydrogel nanocapsule comprising a shell and a core, the shell
having a diameter of greater than 150 nm at a temperature of less
than 25.degree. C. and a diameter of less than 150 nm at a
temperature of greater than 25.degree. C.
16. The nanocapsule of claim 15, wherein the nanocapsule comprises
a polycation comprising polyethylenimine, chitosan, or
poly-l-lysine.
17. The nanocapsule of claim 15, wherein the nanocapsule comprises
a poloxamer, an amphiphilic polymer, or combinations thereof.
18. The nanocapsule of claim 17, wherein the amphiphilic polymer
comprises poly(N-isopropylacrylamide).
19. The nanocapsule of claim 17, wherein the amphiphilic polymer
exhibits a lower critical solution temperature between about
4.degree. C. and about 37.degree. C.
20. The nanocapsule of claim 15, wherein the surface of the core is
modified with polyethylene glycol, stealth materials, folic acid,
or combinations thereof for in vivo drug delivery.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] The present application is based on and claims priority to
U.S. Provisional Application 61/207,485 having a filing date of
Feb. 12, 2009, which is incorporated by reference herein.
BACKGROUND OF THE INVENTION
[0002] Trehalose, a non-reducing disaccharide of glucose, is found
at high concentrations in organisms that are capable of
withstanding extreme drought and/or cold conditions in nature
(i.e., anhydrobiosis or life without water). Moreover, trehalose
has been demonstrated to be a potent, nontoxic bioprotectant for
stabilizing lipids, proteins, viruses, blood cells and even
eukaryotic mammalian cells (e.g., oocytes) at cryogenic and
particularly, ambient temperatures (i.e., cryo and
lyopreservation). Unfortunately, mammalian cells lack a mechanism
to synthesize trehalose and the sugar cannot permeate their plasma
membrane. However, trehalose must present both intra and
extracellularly to protect cells from being damaged by the
dehydration and/or freezing stresses during cryo and
lyopreservation. Therefore, it is crucial to develop an effective
approach that can deliver trehalose into mammalian cells as the
first step toward long-term biostabilization of mammalian cells
using the sugar, particularly at an ambient temperature. Due to the
limited availability of cell sources, long-term cell
biostabilization for future use is critical to the success of the
emerging cell-based medical technologies such as tissue
engineering, regenerative medicine, cell/organ transplantation,
stem cell therapy, and assisted reproduction.
[0003] A number of methods have been explored to introduce
trehalose within mammalian cells over the past two decades. The
most straightforward approach is to deliver exogenous trehalose
into the cytoplasm by direct microinjection. This approach has been
successfully applied to oocytes that have a large size (.about.100
.mu.m in diameter) and are generally in a small quantity (tens or
at most hundreds). However, it has difficulty to be applied to most
mammalian cells that are generally much smaller (<20 .mu.m) and
in large quantities (usually millions). Mammalian cells have been
genetically engineered to synthesize trehalose for
biostabilization. This approach requires the constant production of
adenoviral vectors at high multiplicities of infection that was
found to exhibit significant cytotoxicity. Trehalose has also been
introduced within mammalian cells or their organelles through
engineered or natured transmembrane pores, electroporation,
fluid-phase endocytosis, and lipid phase transition. More recently,
liposomes have being investigated to encapsulate trehalose as a
potential intracellular delivery vehicle of the sugar. However,
consistent report of cryo and lyopreservation using trehalose
delivered intracellularly via the above-mentioned approaches for
small (<20 .mu.m) eukaryotic mammalian cells, is still absent.
This could be due to the inability to deliver a sufficient amount
of intracellular trehalose (i.e., 0.1 M or more) for cellular
protection using some of the approaches (e.g., fluid phase
endocytosis). In addition, cells could be too severely compromised
during the trehalose delivery steps to withstand further
cryo/dehydration stress, considering the highly invasive nature of
some of the approaches (e.g., electroporation).
[0004] Therefore, further investigation to develop a minimally
invasive approach capable of delivering sufficient intracellular
trehalose or similar agents for biostabilization is in need.
Further, there is a more general need for efficient mechanisms for
encapsulation and controlled release of small molecules for
intracellular delivery.
SUMMARY
[0005] In accordance with certain embodiments of the present
disclosure, a method for intracellular delivery of small molecules
is provided. The method includes encapsulation of small molecules
in a thermally responsive nanocapsule by decreasing the temperature
of the nanocapsule to increase the permeability of the nanocapsule
and allowing the small molecules to be sucked into or diffuse into
the nanocapsule. The nanocapsule is delivered into a cell by
increasing the temperature of the nanocapsule. The small molecules
are released from the nanocapsule into the cell.
[0006] In accordance with still other aspects of the present
disclosure, a thermally responsive nanocapsule comprising is
provided. The nanocapsule comprises a polymeric hydrogel
nanocapsule which includes a shell and a core. The shell has a
diameter of greater than 150 nm at a temperature of less than
25.degree. C. and a diameter of less than 150 nm at a temperature
of greater than 25.degree. C.
BRIEF DESCRIPTION OF THE FIGURES
[0007] A full and enabling disclosure of the present subject
matter, including the best mode thereof, to one of ordinary skill
in the art, is set forth more particularly in the remainder of the
specification, including reference to the accompanying figures in
which:
[0008] FIG. 1. Morphology and thermal responsiveness of the
synthesized Pluronic F127-PEI (polyethylenimine) nanocapsules
demonstrated by a typical TEM (transmission electron microscopy)
image of the nanocapsules (A) and the effective diameter and
surface zeta potential measured by DLS (dynamic light scattering,
B), respectively. Error bar represents standard deviation. Scale
bar: 100 nm
[0009] FIG. 2. A schematic representation of the Pluronic F127-PEI
nanocapsules at 22.degree. C. (A) and 37.degree. C. (B): PPO,
polypropylene oxide; PEO, polyethylene oxide; and PEI,
polyethylenimine. The plus symbol represents positive charge.
[0010] FIG. 3. Typical confocal micrographs of 3T3 fibroblasts
after incubating with FITC-labeled nanocapsules in serum free
culture medium containing Hoechst and LysoTracker Red under DIC
contrast (A and E), green channel showing FITC-labeled nanocapsule
(B and F), red channel showing the LysoTracker Red stain for
endosomes/lysosomes (C and G), and the merged view of the green and
red channels (D and H). The upper (A-D) and lower (E-H) panels are
for cells without and with a cold shock treatment, respectively.
Scale Bar: 100 .mu.m
[0011] FIG. 4. Immediate cell viability (A) and 3-day proliferation
(B) of 3T3 cell after being exposed to extracellular nanocapsules
at various concentrations up to 1 mg/ml followed by a 15 min cold
shock at 22.degree. C. The sample with zero nanocapsule
concentration was performed to serve as control. Error bar
represents standard deviation.
[0012] FIG. 5. Release of trehalose to 1 L deionized water at
37.degree. C. from a dialysis bag containing 2 ml solution of 15%
(w/v) dissolved trehalose without nanoencapsulation (trehalose w/o
NE, .DELTA.), mixture of dissolved and nanoencapsulated trehalose
with an overall trehalose concentration of 15% (w/v) and a cold
shock (CS) treatment at 22.degree. C. before transferred to the
dialysis tube (Trehalose w/ NE & CS, O), and mixture of
dissolved and nanoencapsulated trehalose with an overall trehalose
concentration of 15% (w/v) kept at 37.degree. C. all the time
(trehalose w/ NE but w/o CS, .gradient.). Error bar represents
standard deviation.
[0013] FIG. 6. A schematic representation of the process of
trehalose nanoencapsulation and controlled release: Trehalose can
be loaded into the nanocapsule by incubating it with aqueous
trehalose solution at 22.degree. C. (A) followed by freeze-drying
and heating to 37.degree. C. (B). A quick release of the
nanoencapsulated trehalose can be achieved by cooling the trehalose
nanocapsule to 22.degree. C. (C) in aqueous solution followed by
heating back to 37.degree. C. (D).
[0014] FIG. 7. Intracellular concentration of trehalose in 3T3
fibroblasts after incubating the cells in trehalose solution with
both dissolved and nanoencapsulated trehalose (Trehalose w/ NE) at
various concentrations at 37.degree. C. for 40 min. The sample with
the same dissolved extracellular trehalose concentration but no
nanoencapsulation (Trehalose w/o NE) was studied to serve as
control. Error bar represents standard deviation. Asteroids
indicate statistical significance.
[0015] FIG. 8. A schematic representation of the process of
nanocapsule assisted intracellular delivery of trehalose: The
positively charged trehalose-loaded nanocapsule floating in culture
medium at 37.degree. C. (1) is attracted onto the negatively
charged plasma membrane and enwrapped in a clathrin-coated pit on
the plasma membrane (2). The coated pit then buds into the
cytoplasm to form the early endosome (.about.150 nm in size, 3). A
cold shock treatment at 22.degree. C. results in breaking the early
endosome by the swollen nanocapsule to release trehalose into the
cytosol slowly by passive diffusion (4). A quick release of the
nanoencapsulated trehalose can be achieved by heating the cells
back to 37.degree. C. to squeeze the dissolved trehalose out of the
nanocapsule as a result of the more than 15 times of volume
contraction (5).
[0016] FIG. 9. Cell proliferation (A) and collagen production (B)
in 3 days of 3T3 fibroblasts in culture after incubating the cells
for 40 minutes in fresh medium without trehalose (Control), medium
containing 0.22 M dissolved trehalose without nanoencapsulation
(Trehalose w/o NE), and medium containing 0.22 M both dissolved and
nanoencapsulated trehalose using 0.5 mg/ml nanocapsules (i.e., the
condition for delivering .about.0.3 M intracellular trehalose in
FIG. 7, Trehalose w/ NE). Error bar represents standard
deviation.
[0017] FIG. 10. TEM images of nanocapsules in accordance with
certain aspects of the present disclosure.
[0018] FIGS. 11A-B. Thermal responsiveness of a nanocapsule in
accordance with certain aspects of the present disclosure is
illustrated.
[0019] FIG. 12. Activation of the Pluronic 127 and the crosslinking
reaction between the Pluronic F127 and chitosan results in the
formation of the Pluronic F127-chitosan nanocapsule.
[0020] FIGS. 13A-D. Cellular uptake of a nanocapsule in accordance
with the present disclosure does not appear to affect the immediate
and long-term survival of cells.
[0021] FIG. 14. Cellular uptake of a nanocapsule in accordance with
the present disclosure does not appear to significantly affect the
capability of adipogenic differentiation of cells.
[0022] FIG. 15. Confocal images showing cell uptake of the labeled
nanocapsule.
[0023] FIGS. 16A-E. Cellular uptake of the encapsulated EB either
in the presence or absence of free extracellular EB.
[0024] FIG. 17. Shows that a nanocapsule in accordance with the
present disclosure can be used to condense DNA plasmids to
transfect cells and a cold shock treatment can significantly
enhance the transfection efficiency.
DETAILED DESCRIPTION OF REPRESENTATIVE EMBODIMENTS
[0025] Reference will now be made in detail to embodiments of the
disclosed subject matter, one or more examples of which are set
forth below. Each example is provided by way of explanation of the
subject matter, not limitation of the subject matter. In fact, it
will be apparent to those skilled in the art that various
modifications and variations can be made in the present disclosure
without departing from the scope or spirit of the subject matter.
For instance, features illustrated or described as part of one
embodiment, can be used on another embodiment to yield a still
further embodiment.
[0026] In general, the present disclosure is directed to
encapsulation and controlled release of small molecules for
intracellular delivery using thermally responsive
nanoparticles.
[0027] Polymeric nanoparticles are a useful tool to encapsulate
therapeutic drugs, genes, and proteins for their controlled and
sustained delivery. Among them, polymeric hydrogel nanoparticles
with thermal and/or pH responsiveness are particularly attractive
as vehicles for delivery and release of small molecules. Many
polymeric hydrogel nanoparticles exhibit a lower critical solution
temperature (LCST), which can be designed to be between about
20-35.degree. C. The polymeric hydrogel is in a swollen/soluble
state at or below room temperature, while it is in a collapsed/gel
state at the physiological temperature (i.e., about 37.degree. C.).
The sol-gel transition of the hydrogel is accompanied with an
apparent change of its chemical and physical properties, which
could be utilized to achieve controlled release of drug and
therapeutic agents encapsulated in the hydrogel. For example,
hydrophilic and hydrophobic therapeutic agents can be effectively
encapsulated in appropriately designed Pluronic hydrogel
nanocapsules with minimum release (less than about 20%) for up to 2
days at a temperature above the hydrogel LCST. Nanocapsules less
than about 150 nm can be easily internalized by mammalian cells via
endocytosis, a natural pathway of cell self-feeding. Furthermore,
the surface of the nanocapsules can be modified using ligands
and/or other functional moieties such as polyethylenimine (PEI) to
achieve target specific and/or facilitated intracellular delivery
of therapeutic agents.
[0028] In accordance with the present disclosure, a thermally
responsive polymeric hydrogel nanocapsule was synthesized,
characterized, and used as the vehicle for delivering various
therapeutic drugs, genes, and proteins. The nanocapsule was made of
Pluronic F127 and polyethylenimine (PEI), although any suitable
materials could be utilized in accordance with the present
disclosure. For instance, a triblock polymer poly(ethylene
oxide)-polypropylene oxide)-poly(ethylene oxide), which is
commercially available under the PLURONIC.TM. or POLOXAMER.TM.
trade names, can be utilized to form the nanocapsule of the present
disclosure. In addition, the nanocapsule can include
polyethylenimine, chitosan, poly-l-lysine, or other polycations can
be utilized to form the nanocapsule of the present disclosure. In
certain embodiments, the nanocapsule can be formed using
poly(N-isopropylacrylamide) or another amphiphilic polymer that
exhibits a LCST of from about 4.degree. C. to about 37.degree.
C.
[0029] The nanocapsule can be loaded with therapeutic drugs, genes,
and proteins, or the like such as exogenous trehalose. The
nanocapsule can be delivered to any suitable type of cell including
mammalian cells. The temperature dependent properties (i.e.,
thermal responsiveness) of the nanocapsule such as size, surface
charge, and particularly wall permeability were utilized to achieve
nanoencapsulation and controlled release of therapeutic drugs,
genes, and proteins both outside and inside mammalian cells (i.e.,
NIH 3T3 fibroblasts here). In certain embodiments, trehalose can be
utilized in nanocapsules of the present disclosure. In certain
embodiments, the nanoparticles can be loaded with DNA plasmids,
siRNA, microRNA, or combinations thereof.
[0030] For instance, in one particular embodiment, it was found
that a significant amount of trehalose that is sufficient for
biostabilization can be delivered into the cells using the Pluronic
F127-PEI nanocapsule. It was further found that cytotoxicity of the
nanocapsules is negligible for the purpose of trehalose
delivery.
[0031] The nanocapsules of the present disclosure can be
surface-modified using polyethylene glycol or other stealth
materials for in vivo drug delivery. In addition, the nanocapsules
can be surface-modified using folic acid or other targeting
moieties for target specific in vivo drug delivery. However, such
examples are not meant to be limiting and any suitable compounds
can be used to surface-modify the nanocapsules of the present
disclosure.
[0032] Reference now will be made to exemplary embodiments of the
invention set forth below. Each example is provided by way of
explanation of the invention, not as a limitation of the
invention.
EXAMPLE 1
[0033] In the present study, a thermally responsive polymeric
hydrogel nanocapsule made of Pluronic F127 and polyethylenimine
(PEI) was synthesized, characterized, and used as the vehicle for
delivering exogenous trehalose into mammalian cells. The
temperature dependent properties (i.e., thermal responsiveness) of
the nanocapsule such as size, surface charge, and particularly wall
permeability were utilized to achieve nanoencapsulation and
controlled release of trehalose both outside and inside mammalian
cells (i.e., NIH 3T3 fibroblasts here). It was found that a
significant amount of trehalose that is sufficient for
biostabilization can be delivered into the cells using the Pluronic
F127-PEI nanocapsule. It was further found that cytotoxicity of the
nanocapsules is negligible for the purpose of trehalose
delivery.
[0034] 2. Materials and Methods
[0035] 2.1. Materials
[0036] Pluronic F127 (12.6 kDa) manufactured by BASF Corp.
(Wyandotte, Mich.) was used. LysoTracker Red DND-99, and
Viability/Cytotoxicity kit for mammalian cells were purchased from
Invitrogen (Carlsbad, Calif.). The dihydrate of
.alpha.,.alpha.-trehalose (high purity) was purchased from Ferro
Pfanstiehl Laboratories (Waukegan, Ill.). Polyethylenimine (PEI,
MW=2 kDa), 4-nitrophenyl chloroformate, and fluorescein
isothiocyanate (FITC) were purchased from Sigma (St Louis, Mo.) and
used as received.
[0037] 2.2. Synthesis of Pluronic F127-PEI Nanocapsules
[0038] The thermally responsive Pluronic F127-PEI nanocapsule was
prepared using a modified emulsification/solvent evaporation method
with slight modification. Briefly, Pluronic F127 was pre-activated
at both terminals with 4-nitrophenyl chloroformate that contains an
amine-specific reactive group. The activated Pluronic F127 was then
dissolved in dichloromethane (i.e., oil) at a concentration of 20%
(w/v) and added drop-wise into an aqueous solution of 0.75% (w/v)
PEI with a pH of 9. The oil-in-water mixture was emulsified for 4
min using a Branson 450 Sonifier (Danbury, Conn.). Pluronic
F127-PEI nanocapsules were formed as a result of the interfacial
crosslinking reaction between the pre-activated Pluronic F-127 in
oil and PEI in water at the oil-in-water interface. Organic
solvents (i.e., dichloromethane) in the emulsion were then removed
by evaporation using a rotary evaporator until the solution became
clear. The sample was then dialyzed against water at pH 4.0 with a
Spectra/Por (Spectrum Labs, Rancho Dominguez, CA) dialysis tube
(MWCO, 50 kDa) to remove non-crosslinked Pluronic F127 and PEI and
any residual organic solvents. Water in the sample was then removed
by freeze drying and the resultant dry nanocapsules were either
used immediately or kept at -20.degree. C. for future use.
[0039] 2.3. Characterization of Nanocapsule Morphology, Size, and
Surface Charge
[0040] The morphology of the synthesized nanocapsules was studied
using transmission electron microscopy (TEM). For TEM analysis, one
drop (2 .mu.l) of the aqueous nanocapsule solution (2 mg/ml) was
dried on a copper TEM grid for at least 10 min. The dried
nanocapsule specimen was then stained by adding a drop (.about.2
.mu.l) of 2% (w/v) uranyl acetate solution followed by drying for
at least 10 min. The sample was then examined using a Hitachi H-800
transmission electron microscope. All the procedures were performed
at room temperature. The size and surface charge (represented by
the surface .zeta. potential) of the synthesized nanocapsule at
various temperatures from 4-45.degree. C. was further measured
using a Brookhaven (Holtsville, N.Y.) ZetaPlus dynamic light
scattering (DLS) instrument, for which the nanocapsule was
dissolved in 1.times. phosphate-buffered saline (PBS) at a
concentration of 1 mg/ml.
[0041] 2.4. Cell Culture
[0042] NIH 3T3 fibroblasts were cultured in high glucose DMEM
(Invitrogen, Carlsbad, Calif.) supplemented with 10% fetal bovine
serum (Hyclone, Logan, Utah), 100 U/ml penicillin and 100 .mu.g/ml
streptomycin (Hyclone, Logan, Utah) at 37.degree. C. in a
humidified 5% CO.sub.2 incubator.
[0043] 2.5. Cellular Uptake and Intracellular Distribution of the
Nanocapsule
[0044] To study cellular uptake and the subsequent intracellular
distribution of the Pluronic F127-PEI nanocapsules, they were first
labeled with the fluorescent probe FITC. A total of 60 mg of the
freeze-dried nanocapsules was dissolved in 4.6 ml of 0.1 M sodium
carbonate buffer at pH 9, followed by adding dropwise a total of
220 .mu.l of 26 mM FITC solution (in DMSO). FITC labeling of the
nanocapsules was done by allowing the solutions to react for 8 h at
4.degree. C. under gentle and continuous shaking in the dark. A
total of 2.2 mg ammonium chloride was then added into the solution
for 2 h at 4.degree. C. to quench the reaction. The FITC labeled
nanocapsules were further purified by dialysis against deionized
water in the dark for 24 hours with the water being replaced every
3-5 hours.
[0045] To study cellular uptake of the FITC labeled nanocapsules,
NIH 3T3 cells were seeded in 33 mm Petri dishes at a density of
5.times.10.sup.5 cells/dish in 1 ml medium. After 24 h, the culture
medium was replaced with serum-free medium containing FITC-labeled
nanocapsules (100 .mu.g/ml) and LysoTracker Red DND-99 (55 nM). The
latter is a fluorescent probe that can permeate cell plasma
membrane and accumulates in sub-cellular organelles with an acidic
internal environment such as the endosome and lysosome. After
incubation for 40 min at 37.degree. C., cells were washed three
times using warm 1.times. phosphate-buffered saline (PBS). The
cells were then fixed using 4% warm paraformaldehyde for 20 min
either immediately or after a cold shock treatment by incubating
the cells in 1.times. PBS for 15 min at 22.degree. C. After
fixation, the cells were washed using 1.times. PBS and
intracellular distribution of FITC-labeled nanocapsules in the
cells was examined using a confocal microscope (LSM 510, Carl-Zeiss
Inc, Oberkochen, Germany) with fluorescent capability.
[0046] 2.6. Cytotoxicity of the Synthesized Nanocapsules
[0047] Both immediate cell viability and long-term cell
proliferation were studied to test the cytotoxicity of the
nanocapsules synthesized. For immediate cell viability study (i.e.,
short-term toxicity), NIH 3T3 cells were seeded in 33 mm Petri
dishes at a density of 5.times.10.sup.5 cells/dish in 1 ml medium.
After 24 h, the cell culture medium was replaced with warm
(37.degree. C.) serum-free medium containing nanocapsules of
various concentrations. After incubating for 40 min at 37.degree.
C., cells were washed three times using warm (37.degree. C.)
1.times. PBS to remove any extracellular nanocapsules followed by a
cold shock treatment in fresh culture medium for 15 min at
22.degree. C. Cell viability of the cells immediately after cold
shock was determined using the standard live/dead assay kit
purchased from Invitrogen. The cell membrane permeable calcein AM
(5 .mu.M) in the kit could be converted to the intensely green
fluorescent calcein which can be well retained within live cells
with intact plasma membrane. Ethidium homodimer (EthD-1, 5 .mu.M),
the other fluorescent probe in the kit, enters cells with a
compromised plasma membrane (taken as dead cells) and binds to
nucleic acids producing bright red fluorescence while it is
excluded by the intact plasma membrane of viable cells. The cells
were examined using an Olympus BX 51 microscope equipped with
fluorescent cubes and a QICAM CCD digital camera (QImaging, Surrey,
BC, Canada). At least 10 representative images were taken and
processed using the Linksys 32 software (Linkam, UK) to count
viable (green fluorescence) and dead (red fluorescence) cells.
Immediate cell viability was calculated as the ratio of the number
of viable cells to the total number of cells, which were at least
1200 for each sample.
[0048] For long-term cell proliferation studies, cells were seeded
in 33 mm Petri dishes at a relatively low density of
1.times.10.sup.5 cells/dish in 1 ml medium. At 24 h, the cells were
exposed to nanocapsules in the same way as that described above for
immediate cell viability studies. After cold shock, the cells were
further cultured for 3 days to monitor their proliferation
(long-term toxicity). This was done by taking at least 10
representative images of the samples every day including the day
(taken as day 0) when the cells were exposed to nanocapsules. The
total number of cells in each image were counted automatically
using NIH ImageJ.
[0049] 2.7. Nanoencapsulation and Controlled Release of
Trehalose
[0050] Nanoencapsulation of trehalose was done in two steps: 1),
incubating the nanocapsules (10 mg/ml) with trehalose (15% w/v) in
water overnight (.about.12 hr) at room temperature
(.about.22.degree. C.) when the nanocapsules were swollen and their
wall permeability was high and 2) freeze-drying the sample to
remove water both inside and outside the nanocapsules. Trehalose
diffused into the nanocapsule during the incubating step should
remain in the nanocapsule after freeze drying. The resultant
freeze-dried mixture of extra-capsular trehalose and
trehalose-loaded nanocapsules was either used immediately or stored
at -20.degree. C. for future use.
[0051] To determine whether trehalose can be withheld in the
nanocapsule at 37.degree. C. for controlled release, the
freeze-dried mixture of trehalose and trehalose-loaded nanocapsules
was preheated to 37.degree. C. and dissolved in 2 ml warm water at
37.degree. C. in a 15 ml centrifuge tube. The final overall
trehalose (i.e., trehalose both inside and outside the
nanocapsules) concentration in the solution was 15% (w/v) and the
corresponding nanocapsule (excluding the encapsulated trehalose)
concentration was 10 mg/ml. Therefore, the total amount of
trehalose and nanocapsules (excluding the encapsulated trehalose)
in the 2 ml solution was 0.3 and 0.02 g, respectively. The solution
in the centrifuge tube was then transferred either after cooling at
22.degree. C. (i.e., cold shocking) for 15 min or immediately into
a Spectra/Por dialysis bag (MWCO, 50 kDa) and placed in a beaker
containing 1 L of deionized water kept warm at 37.degree. C. with
constant stirring using a hotplate/stirrer for 5 hr. It is expected
that the 15 min cooling at 22.degree. C. followed by heating at
37.degree. C. in the beaker should result in a quick release of
nanoencapsulated trehalose as a result of the more than 15 times of
volume expansion and contraction in response to the temperature
variation. Control experiments were performed similarly except that
2 ml of 15% (w/v) pure trehalose solution (i.e., in the absence of
nanocapsules) was used in the dialysis tube. At various times
(i.e., 0.5, 1, 2, 3, and 5 hr), a total of 0.5 ml of the solution
outside the dialysis tube in the beaker was collected to determine
the trehalose concentration in the 1 L deionized water for each
sample. The total liquid volume outside the dialysis tube in the
beaker was kept at 1 L by adding the same amount of deionized water
at each sampling time. Trehalose concentration in the 0.5 ml
samples was determined using a trehalose assay kit (Megazyme Co.,
Wicklow, Ireland) by following the manufacturer's instructions.
Briefly, trehalose in a sample was hydrolyzed to D-glucose using
trehalase and the D-glucose was phosphorylated using hexokinase
(HK) and adenosine-5'-triphosphae (ATP) to glucose-6-phosphate
(G-6-P). In the presence of the glucose-6-phosphate dehydrogenase
(G6P-DH), the produced G-6-P was oxidized by nicotinamide-adenine
dinucleotide phosphate (NADP+) to gluconate-6-phosphate with the
formation of reduced nicotinamide-adenine dinucleotide phosphate
(NADPH). The absorbance at 340 nm of NADPH was then measured using
a Shimazu (Columbia, Md.) UV-2101PC spectrophotometer to determine
the amount of trehalose in the original sample.
[0052] 2.8. Nanocapsule Assisted Intracellular Delivery of
Trehalose
[0053] For intracellular delivery of trehalose, the freeze-dried
mixture of trehalose and trehalose- loaded nanocapsules were
preheated to 37.degree. C. and dissolved in warm (37.degree. C.)
serum-free culture medium at various trehalose (or correspondingly,
nanocapsule) concentrations to incubate with the 3T3 fibroblasts
for uptake. The procedures performed for cellular uptake of the
trehalose loaded nanocapsules was the same as that described in
section 2.6 for the uptake of empty nanocapsules. After cold shock
at 22.degree. C. for 15 min, the incubating solution was decanted
and the cells were further washed three times using 1.times. PBS.
The cells were then detached/lysed in deionized water with the aid
of a cell scraper. The cells were further lysed using three cycles
of freezing and thawing in liquid nitrogen and 37.degree. C. water
bath, respectively. The lysed cell suspension was homogenized for
10 min by sonication using a Branson ultrasonic cleaner (Danbury,
Conn.) followed by a brief vortex-mixing. After centrifuging at
10,000 g for 10 min, the supernatant of each sample was divided
into two aliquots. One aliquot was used to determine trehalose
concentration in the sample using the trehalose assay kit (Megazyme
Co. Wicklow, Ireland) as described in the previous section.
Interfering reducing sugars mainly from the cellular cytoplasm in
all samples were removed using alkaline borohydride (10 mg/mL
sodium borohydride in 50 mM sodium hydroxide) and excessive
alkaline borohydride was neutralized using 200 mM acetic acid. The
other aliquot was used to determine cell density in the sample
based on its DNA content measured using a method described in Dunn
et. al, Long-term in vitro function of adult hepatocytes in a
collagen sandwich configuration, Biotechnol Prog 1991 May-June;
7(3):237-245. The osmotically active volume of a single NIH 3T3
fibroblast was reported to be 1.45.times.10-15 m.sup.3. With the
trehalose concentration and cell density in the sample being
measured, the total amount of intracellular trehalose and the total
number of cells (and thus the total osmotically active cell volume)
can be calculated. Therefore, the intracellular trehalose
concentration can be determined.
[0054] 2.9. Immediate Viability, Proliferation, and Collagen
Production of Trehalose Loaded Cells
[0055] The immediate cell viability, proliferation, and collagen
production of 3T3 cells exposed to 0.22 M extracellular trehalose
and 0.5 mg/ml nanocapsules (loaded with trehalose) (As will be
shown later in Results and Discussion that the intracellular
trehalose is .about.0.3 M under this loading condition) were
further studied to test the effectiveness of the nanocapsule based
approach for intracellular delivery of trehalose. Immediate cell
viability and proliferation of the 3T3 cells after loading with
trehalose were studied in the same way as that described for 3T3
cells loaded with empty nanocapsules in section 2.6. The collagen
production of cells exposed to 0 M trehalose and 0 mg/ml
nanocapsules (control), 0.22 M extracellular trehalose and 0 mg/ml
nanocapsules (trehalose without NE), and 0.22 M extracellular
trehalose and 0.5 mg/ml extracellular nanocapsules
(nanoencapsulated with trehalose) was quantified using the
Sircol.TM. Assay kit (Biocolor, Belfast, N. Ireland). To do this,
the cells treated under the three conditions were further cultured
for 3 days. The culture medium (1 ml) was collected and the cells
replenished with the same amount of fresh medium every day. To
measure collagen concentration in the collected medium using the
Sircol assay, 20 .mu.l of the collected medium was diluted into 50
.mu.l using deionzed water in a 1 ml centrifuge tube. Blank samples
were prepared using 20 .mu.l fresh medium and 30 .mu.l deionized
water and collagen standard samples were prepared using 20 .mu.l
fresh medium and 30 .mu.l deionized water containing 5, 10, 15 and
20 .mu.g collagen. A total of 0.5 ml Sircol dye reagent was then
added into each sample and mixed with the sample for 30 minutes to
allow binding between the dye and collagen monomers. After
centrifuging for 10 min at 12,000 g, the supernatant with unbound
dye in the tube was removed. The collagen bound dye was then
released by adding 0.5 ml of alkali reagent included in the assay
kit. The absorbance at 540 nm wavelength of the dye was then
measured using a BioTek Synergy 2 microplate reader (Winooski,
Vt.). Collagen concentration in the collected medium was quantified
by comparing its absorbance subtracted with the absorbance of the
blank sample with that of the standards.
[0056] 2.10. Statistical Analysis
[0057] All results were reported as the mean and standard deviation
of data from at least three replicates. Student's two-tailed t-test
assuming equal variance was calculated using Microsoft.RTM. Excel
to determine statistical significance (p<0.05).
[0058] 3. Results and Discussion
[0059] 3.1. Nanocapsule Morphology, Size, and Surface Charge
[0060] A typical TEM image of the synthesized Pluronic F127-PEI
nanocapsules is shown in FIG. 1A. The nanocapsule is round in shape
and its size distribution is quite uniform. The size of the
nanocapsule was further measured by dynamic light scattering (DLS)
at various temperatures from 4 to 45.degree. C. and the results are
shown in FIG. 1B. Clearly, a broad thermal responsiveness of the
Pluronic F127-PEI nanocapsule over the temperature range is
observable: it is less than 100 nm at 37.degree. C. or higher
(e.g., 95.1.+-.15.4 nm at 37.degree. C. and 74.9.+-.14.0 nm at
45.degree. C.) whereas it is more than .about.250 nm at 22.degree.
C. or lower (e.g., 247.2.+-.27.8 at 22.degree. C. and 351.8.+-.38.2
nm at 4.degree. C.). Also shown in FIG. 1B is the temperature
dependent surface charge of the nanocapsule represented by the
surface zeta (.zeta.) potential. On the contrary, the surface
charge of the nanocapsule increases with increasing temperature. It
is much more positively charged at 37 (31.6.+-.7.1 mV) than
22.degree. C. (9.8.+-.4.4 mV) and even become negatively charged at
4.degree. C. (-11.6.+-.4.1 mV).
[0061] Pluronic F127 is an amphiphilic block copolymer with a
formula PEO100-PPO65-PEO100, in which the subscripts 100 and 65
indicate the number of PEO (polyethylene oxide) and PPO
(polypropylene oxide) blocks, respectively. A distinctive feature
of Pluronic copolymers is that it exhibits a sol-gel transition
behavior in aqueous solution when temperature increases from below
to above its lower critical solution temperature (LCST) usually
less than 40.degree. C. The solution to gel transition is
accompanied with a significant volume contraction as a result of
dehydration of the PPO block, which is presumably responsible for
the significant volume change (e.g., more than 15 times contraction
from 22 to 37.degree. C. shown in FIG. 1B) observed for the
Pluronic F127 based nanocapsules synthesized in this study. This
volume change has been reported to be even bigger for other
Pluronic F127 based nanocapsules reported in the literature. For
example, the volume contraction from .about.20 to 37.degree. C. of
a Pluronic F127-PEI nanocapsule synthesized using a higher Pluronic
concentration (30% w/v) and a shorter time (3 vs. 4 minutes in this
study) for cross-linking between the activated Pluronic and PEI
(and presumably the cross-link is weaker) was found to be more than
40 times and it is more than 1000 times for a Pluronic F127-haprin
nanocapsule. The difference in the volume contraction between the
PEI and heparin cross-linked Pluronic nanocapsule was attributed to
the difference between the two cross-links: the latter was reported
to be much more fragile and softer and hence allowing much bigger
volume change. The Pluronic F127-PEI nanocapsules have been shown
to have an empty core-shell like nano-reservoir structure. This
observation may explain why the shrunken Pluronic F127-PEI
nanocapsule (e.g., .about.95 nm in this study and 100-150 nm
reported in the literature is much bigger than a solid monolithic
Pluronic F127 micelle in aqueous solution (.about.30 nm) at
37.degree. C. The empty core of the nanocapsule was proposed to be
a result of the balance between the hydrophobic interactions
(leading to dehydration in the PPO blocks and volume contraction)
and the repulsion between the positive charges in the cross-linked
PEI molecules (resisting collapse). The increase of the nanocapsule
surface charge with temperature probably is a result of the
exposure of more PEI molecules on the surface in response to the
volume collapse with increasing temperature. Putting together the
experimental data and above discussion, a schematic representation
of the nanocapsule at 22 and 37.degree. C. can be given in FIG. 2.
The empty core-shell structured nanocapsule is much smaller and
more positively charged at 37 than 22.degree. C. As will be
discussed later in more detail, the temperature dependent size and
surface charge and other thermally responsive properties of the
nanocapsule play a key role in facilitating their uptake by
mammalian cells, determining their distribution within the cells,
and ultimately encapsulating trehalose for efficient intracellular
delivery.
[0062] 3.2. Cellular Uptake of the Nanocapsule and its
Intracellular Distribution
[0063] Typical confocal micrographs demonstrating cellular uptake
of the FITC-labeled nanocapsule and its intracellular distribution
are shown in FIG. 3 for cells both without (upper panels A-D) and
with (lower panels E-H) a cold shock treatment at 22.degree. C.
Differential interference contrast (DIC) images of the cells (panel
A and E for without and with the cold shock treatment,
respectively) show the morphology of an elongated spindle which is
phenotypical for 3T3 fibroblasts. Cellular uptake of the
FITC-labeled nanocapsules is clearly demonstrated by the bright
green fluorescence in panel B of cells without the cold shock
treatment. However, the green fluorescence becomes fainted in cells
with the cold shock treatment (panel F). Locations of sub-cellular
organelles (mainly the endosome/lysosome) with an acidic internal
environment were made visible with the fluorescent probe
LysoTracker Red (red channels) in panels C and G for cells without
and with the cold shock treatment, respectively. Both panel shows
strong red fluorescence indicating the existence of a significant
amount of endosomes/lysosomes in the cells after incubating the
cells with the medium containing nanocapsules. Merged views of the
green and red channels for cells without and with cold shock are
shown in panels D and H, respectively. A yellowish color of the
merged view in panel D indicates extensive co-staining of the two
fluorescent probes (i.e., FITC and LysoTracker Red) for cells
without the cold shock treatment. The yellowish color is not
readily identifiable in the merged view of the red and green
channels of cells with the cold shock treatment (panel H). These
results indicate that the FITC-labeled nanocapsules were primarily
sequestered in the endosome/lysosome system immediately after being
internalized and before the cold shock treatment, suggesting
endocytosis is the dominant mechanism of cellular uptake of the
nanocapsules. After cold shock, however, the nanocapsules escaped
the endosome/lysosome system into the cytosol (resulting in the
fainted green fluorescence in Panel F), presumably by mechanically
breaking the endosome/lysosome as a result of a significant volume
expansion of the nanocapsule in responsive to cold shock. As shown
in FIG. 1, the diameter (or volume) of the nanocapsule at
22.degree. C. is more than 2.5 (or 15.6) times bigger than that at
37.degree. C. (247.2.+-.27.8 vs. 95.1.+-.15.4 nm) and is more than
1.5 (or 3.4) time bigger than that of the endosome/lysosome
(.about.150 nm).
[0064] The results showing in FIG. 3 clearly demonstrate the
importance of thermal responsiveness of the nanocapsule for its
cellular uptake and intracellular distribution. For example, the
small size (.about.95 nm) of the nanocapsule at 37.degree. C. is
important for its cellular uptake since the nanocapsules must be
small enough to be enwrapped in the clathrin coated endocytotic
endosome (.about.150 nm) before they can be internalized by the
cells via endocytosis. After internalization, the nanocapsule can
mechanically break and escape the endosome/lysosome system as a
result of more than 15 times volume expansion in response to a cold
shock treatment at 22.degree. C. Of note, the capability of the
nanocapsule to break and escape the endosome/lysosome into the
cytosol is of critical importance. This is because degradation or
potentially exocytosis (if not biodegradable) of the endocytosed
material as a result of their sequestration in the
endosome/lysosome system has been reported to be a major bottleneck
for cytosolic drug delivery via endocytosis. FIG. 3 also
demonstrates that a significant amount of nanocapsules could be
internalized in a short incubation period of 40 minutes, indicating
an accelerated endocytosis was involved in the uptake process. The
accelerated endocytosis (also called adsorptive endocytosis) is
presumably attributed to the highly positively charged surface of
the nanocapsule, which has a high affinity with the negatively
charged cell membrane as a result of electrostatic attraction. In
summary, as a result of their thermal responsiveness, the Pluronic
F127-PEI nanocapsules could be easily internalized by mammalian
cells and the thermal responsiveness could be further utilized to
control their intracellular distribution.
[0065] 3.3. Cytotoxicity of the Nanocapsule
[0066] The immediate cell viability and 3-day proliferation of the
3T3 cells after being exposed to the nanocapsules (40 min at
37.degree. C. followed by a cold shock at 22.degree. C. for 15 min)
at different extracellular concentrations from 0 to 1 mg/ml are
shown in panels A and B of FIG. 4, respectively. Studies with an
extracellular nanocapsule concentration of zero were performed to
serve as control. FIG. 4A shows that the immediate cell viability
was more than 95% under all the experimental conditions. Moreover,
the 3-day proliferation of the cells exposed to nanocapsules was
not significantly different from that of control. Since a
significant amount of nanocapsules could be internalized by the
cells during the 40 min incubation period and the nanocapsules
could escape the endosome/lysosome system in response to a cold
shock treatment as demonstrated in FIG. 3, the results shown in
FIG. 4 (i.e., high immediate cell viability and normal
proliferation in 3 days) indicate that the nanocapsules is not
toxic to the cells at least under the concentration tested.
Moreover, the process of breaking the endosome/lysosome by a heat
shock treatment to release the nanocapsule into cytosol did not
result in significant injury to the cells either.
[0067] The low cytotoxicity of Pluronic based nanocapsules has been
reported in the literature and could be partially attributed to the
excellent biocompatibility of its constituent polymers. Pluronic
F127 has been approved by FDA (Food and Drug Administration) for
use as food additives and pharmaceutical ingredients. PEI is a
cationic polymer that has been commonly used for gene delivery.
Although PEI could be toxic to mammalian cells at high
concentrations, its cytotoxicity has been shown to be negligible
when it is cross-linked with Pluronic F-127 in the nanocapsule. The
high immediate cell viability and normal 3-day cell proliferation
of 3T3 fibroblasts shown in FIG. 4 is consistent with this
observation. Nevertheless, the intracellular trafficking of the
nanocapsules released from the early endosome after cold shock may
help to further explain their negligible cytotoxicity.
[0068] 3.4. Nanoencapsulation and Controlled Release of
Trehalose
[0069] Trehalose release patterns from the 2 ml dialysis bag into
the 1 L deionized water at 37.degree. C. under the three different
experimental conditions are shown in FIG. 5. Also shown in the
figure is the maximum possible trehalose concentration (i.e., 0.3
g/L represented by the horizontal dash line) in the 1 L deionized
water under the equilibrium condition (i.e., the trehalose
concentration outside the dialysis bag in the 1 L water is the same
as that inside the dialysis bag). For the control condition with
dissolved trehalose only (i.e., without nanoencapsulation),
trehalose release from the dialysis bag reached equilibrium in
approximately 2 hours according to the figure. For the condition
with nanoencapsulation (NE) but no cold shock (CS) treatment at
22.degree. C., however, trehalose release from the dialysis tube is
apparently much slower than that under the control condition,
particularly after one hour when more than 80% of the
non-encapsulated trehalose was released from the dialysis tube into
the 1 L deionized water in the beaker. The release was far from
complete even after 5 hours for the condition with
nanoencapsulation but no cold shock. Since the release of
non-encapsulated trehalose from the dialysis tube reached
equilibrium at 2 hr, the difference in trehalose concentration
(i.e., 0.03=0.3-0.27 g/L according to the figure) in the 1 L
solution outside the dialysis tube between the control and the
sample with nanoencapsulation (NE) but no cold shock (CS) should be
a result of trehalose nanoencapsulation in the nanocapsules. The
total nanoencapsulated trehalose can then be estimated to be 0.03 g
as the product of the trehalose concentration difference at 2 hr
(i.e., 0.03 g/L) and the total volume of deionized water (.about.1
L) in the beaker. Since the total amount of empty nanocapsules used
is 0.02 g (see section 2.7 in Materials and Methods), a loading
capability defined as the ratio of the weight of nanoencapsulated
trehalose to that of empty nanocapsule can be estimated to be 1.5
(=0.03 g trehalose/0.02 g nanocapsules). In other words, trehalose
takes up approximately 60% of the total weight of the
trehalose-loaded nanocapsules. These results indicate that
trehalose indeed can be encapsulated in the nanocapsule by simply
incubating the nanocapsule in aqueous trehalose solution at
22.degree. C. followed by freeze drying and heating to 37.degree.
C. A schematic representation of the nanoencapsulation process is
given in FIG. 6(A.fwdarw.B). Interestingly, with both
nanoencapsulation (NE) and a cold shock (CS) treatment at 22 for 15
min before dialysis, the trehalose release pattern is much closer
to that of the control condition (i.e., without nanoencapsulation).
This result indicates that the nanoencapsulated trehalose was
quickly released into the 2 ml solution as a result of the cold
shock treatment at 22.degree. C. before it was transferred into the
dialysis bag and the subsequent heating back to 37.degree. C.
during dialysis as schematically demonstrated in FIG.
6(B.fwdarw.C.fwdarw.D). Based on the trehalose concentration data
at 2 hr, it is estimated that approximately 75% of the total
nanoencapsulated trehalose was released from the nanocapsule during
the cold shock and subsequent heating process. Presumably, more
nanoencapsulated trehalose can be released from the nanocapsule by
a second cold shock treatment. In any event, the results given in
FIG. 5 indicate that effective nanoencapsulation of trehalose in
the Pluronic-PEI nanocapsule can be achieved by using a combination
of incubation/freeze-drying/heating procedures. Trehalose can be
withheld within the nanocapsule at 37.degree. C. with minimum
release for at least 5 hr. Moreover, an accelerated release pattern
of the nanoencapsulated trehalose can be achieved by a cold shock
treatment of the trehalose loaded nanocapsules at 22 for a short
period of time (15 min) followed by heating back to 37.degree. C.
in aqueous solution.
[0070] Besides the thermal responsiveness of the nanocapsule in
size and surface charge, the capability of the Pluronic F127-PEI
nanocapsule for encapsulation and controlled release of trehalose
indicates a temperature dependent wall permeability of the
nanocapsule: it is high at 22.degree. C. to allow free diffusion of
trehalose in and out of the nanocapsule whereas it is so low at
37.degree. C. that trehalose can be withheld in the nanocapsule for
at least 5 hr with minimum release. In other words, it is the
thermal responsiveness of the nanocapsule wall permeability that
makes it possible to encapsulate the small molecular weight
trehalose in the nanocapsule post its synthesis for controlled
release. It has been shown that both hydrophilic and hydrophobic
therapeutic agents with a molecular weight greater than 1 kDa can
be effectively withheld in appropriately designed Pluronic based
hydrogel or nanocapsule with minimum release for up to 2 days at a
temperature above its LCST. The results from this study demonstrate
that the Pluronic F127 based hydrogel nanocapsule is capable of
withholding even smaller molecular weight molecules (i.e.,
trehalose, MW=342 Da) at 37.degree. C. However, drugs or
therapeutic agents were directly encapsulated in the hydrogel or
nanocapsule during the synthesis steps in the previous studies.
Successful nanoencapsulation and controlled release of drugs or
therapeutic agents utilizing the temperature dependent wall
permeability of the Pluronic based nanocapsule post its synthesis
has never been reported until this study. This finding is
significant because the direct contact between sensitive drugs or
therapeutic agents and the organic solvents for nanocapsule
synthesis during direct encapsulation may significantly compromise
their activity, which can be avoided by the approach developed in
this study utilizing the temperature dependent wall permeability of
the nanocapsules. Moreover, the new approach allows the two
processes of nanocapsule synthesis and drug nanoencapsulation being
performed at different times, which can significantly reduce the
shipping and maintenance cost. Therefore, we believe, this novel
approach for nanoencapsulation opens a whole new avenue for
controlled delivery of small molecular weight drugs and therapeutic
agents.
[0071] 3.5. Nanocapsule Assisted Intracellular Delivery of
Trehalose
[0072] FIG. 7 shows the intracellular trehalose concentration of
3T3 fibroblasts after incubating the cells with extracellular
trehalose at 37.degree. C. in the presence and absence of
trehalose-loaded nanocapsules. The extracellular trehalose
concentration for all the loading conditions is given along the
bottom horizontal axis. The corresponding nanocapsule (excluding
nanoencapsulated trehalose) concentration for the loading
conditions with trehalose-loaded nanocapsules in the incubation
medium is given along the top horizontal axis. For samples without
nanocapsules, the figure shows that the measured intracellular
trehalose concentration increased slightly with the extracellular
trehalose concentration, but was always less than .about.0.05 M
under all the experimental conditions. For the samples with
nanocapsules, the intracellular trehalose concentration was also
negligible (<0.05 M) when the nanocapsule concentration in the
extracellular medium is not higher than 0.25 mg/ml (the
corresponding extracellular trehalose concentration is 0.11 M),
However, when the medium nanocapsule concentration was increased to
0.375 mg/ml (the corresponding extracellular trehalose
concentration is 0.165 M), the resultant intracellular trehalose
concentration is significantly higher than that in the
corresponding control samples without nanocapsules (0.13 vs. 0.02
M). Moreover, the intracellular trehalose concentration (.about.0.3
M) was even much higher than the corresponding trehalose
concentration (0.22 M) in the extracellular medium when the medium
nanocapsule concentration was 0.5 mg/ml. However, the intracellular
trehalose concentration does not always increase with the increase
of extracellular trehalose and nanocapsule concentration. When the
extracellular nanocapsule concentration was 1 mg/ml (the
corresponding extracellular trehalose was 0.44 M), the
intracellular trehalose concentration was measured to be only
.about.0.12 M with big variation and is not significantly different
from that of control. This result probably is because of the high
concentration of extracellular trehalose (0.44 M) used that results
in significant damage to the cells (or more specifically, their
plasma membrane), since a significant amount of cells were observed
to detach from the substrate during washing after the loading step.
If the cell membrane is comprised, the cells cannot withhold
trehalose during washing and a low intracellular trehalose
concentration is assured. In summary, intracellular delivery of a
significant amount of trehalose into mammalian cells sufficient for
biostabilization of mammalian cells (0.1-0.3 M) is achievable with
the aid of the Pluronic F127-PEI nanocapsules. Nevertheless, the
extracellular trehalose and nanocapsule concentrations should be
carefully controlled.
[0073] The data in FIG. 7 clearly demonstrates that a significant
amount of trehalose could be internalized by 3T3 fibroblasts in a
short incubation period of 40 minutes with an appropriate amount of
extracellular trehalose and trehalose-loaded nanocapsules. However,
cellular uptake of trehalose dissolved in solution by fluid phase
endocytosis (or pinocytosis) has been shown to be much less
effective. For example, it has been shown that more than 5 hours
and even days are required for platelets and human mesenchymal stem
cells to uptake even a much smaller amount of trehalose
(<.about.0.05 M) by pinocytosis. Our results from the control
samples are consistent with this observation in the literature. The
high efficiency of trehalose uptake by the 3T3 fibroblasts in the
presence of the Pluronic F127-PEI nanocapsules is presumably due to
two reasons: 1), the positively charged surface of the nanocapsule
results in an accelerated endocytotic (i.e., adsorptive endocytosis
discussed previously) pathway rather than that of the slow fluid
phase endocytosis; and 2), the high trehalose encapsulation
capability of the nanocapsule results in a much higher amount of
trehalose being enwrapped in each endosome comparing with
pinocytosis for which the contents in the endosome is dominantly
water. Based on the discussions here and previously in Section 3.2,
a schematic representation of the nanocapsule assisted trehalose
loading process is proposed in FIG. 8. The highly positively
charged trehalose-loaded nanocapsule floating in culture medium at
37.degree. C. (1) is attracted onto the negatively charged plasma
membrane and enwrapped in a clathrin-coated pit on the plasma
membrane (2). The coated pit then buds into the cytoplasm to form
the early endosome (.about.150 nm in size, (3)). A cold shock
treatment at 22.degree. C. results in breaking the early endosome
by the swollen nanocapsule to release trehalose into the cytoplasm
slowly by diffusion (4). A quick release of the encapsulated
trehalose can be achieved by heating the cells back to 37.degree.
C. to squeeze the dissolved trehalose out of the nanocapsule as a
result of the more than 15 times of volume contraction (5).
[0074] 3.6. Immediate Cell Viability, Proliferation, and Collagen
Production of Trehalose Loaded Cells
[0075] The immediate cell viability was measured to be 96.8.+-.1.6%
after loading the cells with trehalose under the condition that
results in .about.0.3 M intracellular trehalose according to FIG.
7. The 3 day cell proliferation and collagen production data of
fresh control cells (Control), cells exposed to extracellular
trehalose without nanoencapsulated trehalose (Trehalose w/o NE),
and cells loaded with .about.0.3 M intracellular trehalose
(Trehalose w/ NE) are shown in panels A and B of FIG. 9,
respectively. No significant differences are observable between the
three groups in terms of both cell proliferation and collagen
production. Therefore, the 3T3 cells can survive well and function
normally after being loaded with exogenous trehalose at least when
the intracellular trehalose concentration is not higher than 0.3
M.
[0076] Although a significant amount of intracellular trehalose
might result in apparent osmotic stress on the cells, it is not
surprising to observe that 3T3 fibroblasts can survive well after
being loaded with 0.3 M intracellular trehalose. This is because a
variety of sensitive mammalian cells including hepatocytes and
renal cells have been reported to be capable of controlling its
volume under osmotic stress by activating a regulatory volume
control mechanism. Moreover, trehalose should not interrupt the
biochemical processes in cells in view of its non-reducing nature.
No evidence is better to support these arguments than the fact that
even the osmotically sensitive mammalian oocytes have been shown to
survive well after microinjection with 0.15 M intracellular
trehalose. Trehalose was observed to be eliminated rapidly from the
cells during their embryonic development. Moreover, the trehalose
loaded oocytes were found to survive well post cryopreservation
using trehalose as the sole cryoprotectant. Further studies are
ongoing in our lab to cryo- and lyo-preserve the NIH 3T3
fibroblasts loaded with 0.1-0.3 M trehalose.
[0077] 4. Summary and Conclusions
[0078] In this study, thermally responsive Pluronic F127-PEI
nanocapsules were synthesized and characterized. They have a small
size (.about.95 nm in diameter), positively charged surface, and
low wall permeability at 37.degree. C. whereas they are larger
(>.about.250 nm in diameter), neutral to negatively charged and
highly permeable at or below 22.degree. C. It was shown that a
significant amount of the nanocapsules can be easily internalized
by fibroblasts in 40 min at 37.degree. C. via absorptive
endocytosis, a much faster endocytotic pathway in comparison to
fluid phase endocytosis that is normally used by cells for
self-feeding. This is because the positively charged nanocapsules
have a high affinity with the negatively charged cell membrane
presumably as a result of electrostatic interaction. In addition,
the small size (.about.95 nm) of the nanocapsule at 37.degree. C.
allows them being easily entrapped in endosomes (.about.150 nm)
during endocytosis. It was further demonstrated that the
nanocapsules can mechanically break and escape the
endosome/lysosome system into cytosol in response to a cold shock
treatment at or below room temperature when their diameter is more
than .about.250 nm (more than 1.5 times greater than that of the
early endosome, .about.150 nm). Results from immediate cell
viability and long-term cell proliferation studies indicate that
the nanocapsules are not toxic to mammalian cells at the dose used
for intracellular delivery of trehalose. Trehalose can be loaded
into the nanocapsules by simply incubating the nanocapsules with
trehalose in water at room temperature when the permeability of the
nanocapsule wall is high followed by freeze-drying to remove water.
It was further found trehalose can be withheld in the nanocapsule
dissolved in aqueous solution for hours at 37.degree. C. when the
nanocapsule wall permeability is low. A quick release of the
nanoencapsulated trehalose can be achieved by thermally cycling the
trehalose-loaded nanocapsules between 22 and 37.degree. C.
Moreover, a significant amount of trehalose (up to 0.3 M) can be
loaded into the cytosol of NIH 3T3 fibroblasts by a short (40 min)
incubation of the trehalose loaded nanocapsules with the cells at
37.degree. C. followed by a short (20 min) cold shock treatment at
or below room temperature. An intracellular trehalose concentration
of 0.1-0.3 M is generally believed to be sufficient to protect
mammalian cells from damage under the stress of both cryo and
lyopreservation. Therefore, loading trehalose into mammalian cells
using the thermally responsive nanocapsules should provide an
enabling approach to achieve long-term stabilization of important
mammalian cells for future use particularly at ambient
temperatures, which is critical to the eventual success of modern
cell-based medicine.
EXAMPLE 2
[0079] Activation of Pluronic F127 and preparation of Pluronic
F127-Chitosan Nanocapsules
[0080] 6.3 g Pluronic F 127 and 122.17 mg 4-dimethylaminopyridine
(DMAP) were dissolved in 15 ml anhydride 1,4-dioxane solution with
139 .mu.l triethylamine (TEA). After stirring for 30 min under N2
atmosphere, 125 mg succinic anhydride in anhydride 5 ml 1,4-dioxane
was added dropwisely. The mixture was stirred under N2 atmosphere
for 24 h at room temperature. Then the solvent was removed with a
rotary evaporator and the residue was filtered and precipitated in
ice-cold diethyl ether for three times. Finally, the precipitate
was dried under vacuum overnight to get the white powder of
di-carboxylated Pluronic F127.
[0081] The thermally responsive Pluronic F127-chitosan nanocapsule
was prepared using a modified emulsification/solvent evaporation
method. 100 mg carboxylated Pluronic F127 and 5 mg
1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC)
were dissolved in 1 ml CH2Cl2 for 15 minutes. The solution was
added drop-wise into 10 ml aqueous solution of chitosan
oligosaccharide (7.5 mg/ml) with a pH of 5. The oil-in-water
mixture was emulsified for 4 min using a Branson 450 Sonifier
(Danbury, Conn.). Then the solution was stirred gently for 24
hours. Organic solvents in the emulsion were then removed by
evaporation using a rotary evaporator until the solution became
clear. The sample was then dialyzed against DI water with a
Spectra/Por (Spectrum Labs, Rancho Dominguez, Calif.) dialysis tube
(MWCO, 50 kD).
[0082] Characterization of Nanocapsule Morphology, Size, Surface
Charge and Pluronic/chitosan Ratio in the Nanocapsule
[0083] The morphology of the synthesized nanocapsules was studied
using transmission electron microscopy (TEM). For TEM analysis, one
drop (2 .mu.l) of the aqueous nanocapsule solution (2 mg/ml) was
dried on a copper TEM grid for 10 min. The dried nanocapsule
specimen was then stained by adding a drop (.about.2 .mu.l) of 2%
(w/v) uranyl acetate solution followed by drying for 10 min. The
sample was then examined using a Hitachi H-800 transmission
electron microscope. All the procedures were performed at room
temperature. Typical TEM images of the nanocapsules are shown in
FIG. 10. The empty-core shell structure is clearly visible if no
EDC was used as the catalyst (EDC: 0 hr) while the shell was
thickened and tightened after 24 hr EDC catalysis and the empty
core-shell structure is not clear anymore.
[0084] The size (FIG. 11, top panel) and surface charge (FIG. 11
bottom panel, represented by the surface .zeta. potential) of the
synthesized nanocapsule at various temperatures from 4-45.degree.
C. was further measured using a Wyatt dynamic light scattering
(Santa Barbara, Calif.) and Brookhaven Zeta potential analyzer
(Holtsville, N.Y.) respectively, for which the nanocapsule was
dissolved in 1.times. phosphate-buffered saline (PBS) at a
concentration of 1 mg/ml. The data in FIG. 11 clearly shows the
thermal responsiveness of the nanocapsule in both size and surface
charge.
[0085] FT-IR was used to characterize the polymer structure.
CH.sub.2Cl.sub.2 was used to dissolve the polymer or help form a
suspension. The samples were tested at room temperature with
CH.sub.2Cl.sub.2 as control and the spectrums were recorded and
further processed using OrignPro software. The FTIR data shown in
FIG. 12 clearly indicates the activation of the Pluronic 127 and
the crosslinking reaction between the Pluronic F127 and chitosan
that results in the formation of the Pluronic F127-chitosan
nanocapsule.
[0086] Cytotoxicity of the Synthesized Pluronic F127-Chitosan
Nanocapsules
[0087] Immediate cell viability, long-term cell proliferation and
cell differentiation were studied to test the cytotoxicity of the
nanocapsules synthesized. For immediate cell viability study (i.e.,
short-term toxicity), C3H10T1/2 and MCF-7 cells were seeded in 33
mm Petri dishes at a density of 2.5.times.10.sup.5 and
5.times.10.sup.5cells/dish in 1 ml medium respectively. After 24 h,
the cell culture medium was replaced with warm (37.degree. C.)
serum-free medium containing nanocapsules of various
concentrations. After incubating for 40 min at 37.degree. C., cells
were washed three times using warm (37.degree. C.) 1.times. PBS to
remove any extracellular nanocapsules followed by a cold shock
treatment in fresh culture medium for 15 min at 22.degree. C. Cell
viability of the cells immediately after cold shock was determined
using the standard live/dead assay kit purchased from Invitrogen.
The cells were examined using an Olympus BX 51 microscope equipped
with fluorescent cubes and a QICAM CCD digital camera (Qlmaging,
Surrey, BC, Canada). At least 10 representative images were taken
and processed using the Linksys 32 software (Linkam, UK) to count
viable (green fluorescence) and dead (red fluorescence) cells.
Immediate cell viability was calculated as the ratio of the number
of viable cells to the total number of cells, which were at least
1200 for each sample.
[0088] For long-term cell proliferation studies, C3H10T1/2 and
MCF-7 cells were seeded in 33 mm Petri dishes at a relatively low
density of 0.5.times.10.sup.5 and 1.times.10.sup.5 cells/dish in 1
ml medium respectively. At 24 h, the cells were exposed to
nanocapsules in the same way as that described above for immediate
cell viability studies. After cold shock, the cells were further
cultured for 3 days to monitor their proliferation (long-term
toxicity). This was done by taking at least 10 representative
images of the samples every day including the day (taken as day 0)
when the cells were exposed to nanocapsules. The total number of
cells in each image were counted using NIH ImageJ.
[0089] The results of immediate viability and cell proliferation
are shown in FIG. 13. Cellular uptake of the nanocapsule doesn't
appear to affect the immediate and long-term survival of the
cells.
[0090] For adipogenic differentiation study, C3H10T1/2 cells were
seeded in 33 mm Petri dishes at a relatively low density of
2.5.times.10.sup.5 cells/dish in 1 ml medium. At 24 h, the cells
were exposed to nanocapsules in the same way as that described
above for immediate cell viability studies. After cold shock, the
cells were further cultured for 2 days before inducing adipogenic
differentiation. Three days later, change the adipogenic induction
medium to maintenance medium by completely replacing the spent
induction medium. Two days later, change the medium back to
induction medium. The adipogenic induction medium consisted of DMEM
high glucose supplemented with 1 mM dexamethasone, 0.2 mM
indomethacin, 0.01 mg/ml insulin, 0.5 mM
3-isobutyl-1-methyl-xanthine, and 10% FCS. The adipogenic
maintenance medium was composed of DMEM high glucose with 0.01
mg/ml insulin and 10% FCS. Adipogenic potential was assessed by Oil
Red O staining. The cells were washed with PBS and fixed with 4%
paraformaldehyde for 30 min at room temperature after 4 cycle
induction/maintenance. Then the cells were washed with 60%
isopropanol and incubated with filtered 0.3% Oil Red O
(Sigma-Aldrich, St. Louis, Mo.) in 60% isopropanol for 30 min.
After one wash in 60% isopropanol and three washes in PBS, images
of the cells were taken.
[0091] The results are shown in FIG. 14 and cellular uptake of the
nanocapsule does not appear to significantly affect the capability
of adipogenic differentiation of the cells.
[0092] Cellular Uptake and Intracellular Distribution of the
Nanocapsule
[0093] To study cellular uptake and the subsequent intracellular
distribution of the Pluronic F127-chitosan nanocapsules,
nanocapsules were first labeled with the fluorescent probe FITC. A
total of 30 mg of the freeze-dried nanocapsules was dissolved in
2.3 ml of 0.1 M sodium carbonate buffer at pH 9, followed by adding
dropwise a total of 110 .mu.l of 26 mM FITC solution (in DMSO).
FITC labeling of the nanocapsules was done by allowing the
solutions to react for 8 h at 4.degree. C. under gentle and
continuous shaking in the dark. A total of 6.1 mg ammonium chloride
was then added into the solution for 2 h at 4.degree. C. to quench
the reaction. The FITC labeled nanocapsules were further purified
by dialysis against deionized water in the dark for 24 hours with
the water being replaced every 3-5 hours.
[0094] To study cellular uptake of the FITC labeled nanocapsules,
MCF-7 cells were seeded in 33 mm Petri dishes at a density of
5.times.10.sup.5 cells/dish in 1 ml medium. After 24 h, the culture
medium was replaced with serum-free medium containing FITC-labeled
nanocapsules (500 .mu.g/ml) and LysoTracker Red DND-99 (55 nM). The
latter is a fluorescent probe that can permeate cell plasma
membrane and accumulates in sub-cellular organelles with an acidic
internal environment such as the endosome and lysosome. After
incubation for 40 min at 37.degree. C., cells were washed three
times using warm 1.times. phosphate-buffered saline (PBS). The
cells were then fixed using 4% warm paraformaldehyde with 5
.mu.g/ml Hoechst 33342 for 20 min either immediately or after a
cold shock treatment by incubating the cells in 1.times. PBS for 15
min at 22.degree. C. After fixation, the cells were washed using
1.times. PBS and intracellular distribution of FITC-labeled
nanocapsules in the cells was examined using a confocal microscope
(LSM 510, Carl-Zeiss Inc, Oberkochen, Germany) with fluorescent
capability.
[0095] Confocal images show cell uptake of the labeled nanocapsule
as illustrated in FIG. 15. Without cold shock treatment, the
nanocapsule co-located with the early endosome (yellowish spotted
appearance) while with cold shock, co-localization of the
nanocapsule and early endosome is not clearly visible.
[0096] Encapsulation and Intracellular Delivery of Fluorescence
Dye
[0097] Encapsulation of fluorescence dye, ethidium bromide (EB),
was done in two steps: 1), incubating the nanocapsules (7.5 mg/ml)
with EB (150 .mu.M final concentration) in water overnight
(.about.12 hr) at 4.degree. C. when the nanocapsules were swollen
and their wall permeability was high; and 2), freeze-drying the
sample to remove water both inside and outside the nanocapsules.
EB, which diffused into the nanocapsule during the incubating step,
should remain in the nanocapsule after freeze drying. For the
samples with the step of removing free EB, high EB concentration,
10 mg/ml, was used. After incubating at 4.degree. C. over night and
freeze drying, the samples were dissolved in 37.degree. C. DI
water. And the free EB was removed during dialysis process at
37.degree. C. for 5 hours with one time change of dialysis
water.
[0098] For intracellular delivery of EB, the freeze-dried mixture
of EB and EB-loaded nanocapsules or EB-loaded nanocapsules (after
removing of free EB) were preheated to 37.degree. C. and dissolved
in warm (37.degree. C.) serum-free culture medium. The procedures
performed for cellular uptake of the EB loaded nanocapsules was the
same as that used for the uptake of empty nanocapsules. After cold
shock at 22.degree. C. for 15 min, the cells were further incubated
for 30 minutes for dye fully release.
[0099] Cellular uptake of the encapsulated EB either in the
presence or absence of free extracellular EB is shown in FIGS.
16A-E. All the data show that the nanocapsule can be used to
encapsulate the dye and facilitate its uptake by MCF-7 and
C3H10T1/2 cells. Moreover, cold shock treatment can be used to
control the release of the dye so that they can bind with
chromosome in the nucleus to give brighter red fluorescence.
[0100] eGFP Plasmid Transfection Using Pluronic/PEI
Nanocapsules
[0101] 16.4 .mu.l eGFP plasmid (0.46 mg/ml) was complexed with 50
.mu.l nanocapsules (1 mg/ml) at an N/P ratio of about 45 at
37.degree. C. for 1 hour. MCF-7 cells were seeded in 33 mm Petri
dishes at a density of 2.times.10.sup.5 cells/dish in 1 ml medium.
After 24 h, the culture medium was replaced with serum-free medium
containing eGFP plasmid/nanocapsules complexes with a final
nanocapsule concentration of 0.5 mg/ml. After incubation for 5
hours at 37.degree. C., cells were washed three times using warm
1.times. phosphate-buffered saline (PBS). The cells were then
cultured for 48 hours or after a cold shock treatment by incubating
the cells in 1.times. PBS for 15 min at 22.degree. C. After
fixation, the cells were washed using 1.times. PBS and was examined
using a fluorescence microscope to check the GFP expression. The
data in FIG. 17 clearly shows that the nanocapsule can be used to
condense DNA plasmids to transfect cells and a cold shock treatment
can significantly enhance the transfection efficiency.
* * * * *