U.S. patent application number 14/253030 was filed with the patent office on 2014-10-09 for porous nanoparticle supported lipid nanostructures.
The applicant listed for this patent is Carlee Ashley, C. Jeffrey Brinker, Eric C. Carnes, Juewen Liu. Invention is credited to Carlee Ashley, C. Jeffrey Brinker, Eric C. Carnes, Juewen Liu.
Application Number | 20140301951 14/253030 |
Document ID | / |
Family ID | 42310639 |
Filed Date | 2014-10-09 |
United States Patent
Application |
20140301951 |
Kind Code |
A1 |
Liu; Juewen ; et
al. |
October 9, 2014 |
POROUS NANOPARTICLE SUPPORTED LIPID NANOSTRUCTURES
Abstract
Various exemplary embodiments provide protocell nanostructures
and methods for constructing and using the protocell
nanostructures. In one embodiment, the protocell nanostructures can
include a core-shell structure including a porous particle core
surrounded by a shell of lipid bilayer(s). The protocell can be
internalized in a bioactive cell. Various cargo components, for
example, drugs, can be loaded in and released from the porous
particle core of the protocell(s) and then delivered within the
bioactive cell.
Inventors: |
Liu; Juewen; (Kitchener,
CA) ; Brinker; C. Jeffrey; (Albuquerque, NM) ;
Ashley; Carlee; (Albuquerque, NM) ; Carnes; Eric
C.; (Albuquerque, NM) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Liu; Juewen
Brinker; C. Jeffrey
Ashley; Carlee
Carnes; Eric C. |
Kitchener
Albuquerque
Albuquerque
Albuquerque |
NM
NM
NM |
CA
US
US
US |
|
|
Family ID: |
42310639 |
Appl. No.: |
14/253030 |
Filed: |
April 15, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13143164 |
Jul 1, 2011 |
8734816 |
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PCT/US2010/020096 |
Jan 5, 2010 |
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14253030 |
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61142495 |
Jan 5, 2009 |
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Current U.S.
Class: |
424/9.6 ;
424/172.1; 424/450; 424/600; 424/617; 424/649; 424/682; 514/21.2;
514/44R |
Current CPC
Class: |
A61K 9/127 20130101;
A61K 9/1272 20130101; A61K 39/3955 20130101; A61K 33/06 20130101;
A61K 49/005 20130101; A61K 38/16 20130101; A61K 33/00 20130101;
A61P 43/00 20180101; A61K 33/24 20130101; A61P 35/00 20180101; A61K
31/713 20130101; A61K 9/5123 20130101; A61K 9/5115 20130101 |
Class at
Publication: |
424/9.6 ;
424/450; 514/21.2; 424/172.1; 514/44.R; 424/600; 424/649; 424/682;
424/617 |
International
Class: |
A61K 9/127 20060101
A61K009/127; A61K 39/395 20060101 A61K039/395; A61K 33/06 20060101
A61K033/06; A61K 49/00 20060101 A61K049/00; A61K 33/00 20060101
A61K033/00; A61K 33/24 20060101 A61K033/24; A61K 38/16 20060101
A61K038/16; A61K 31/713 20060101 A61K031/713 |
Goverment Interests
GOVERNMENT RIGHTS
[0002] This invention was made with Government support under
Contract No. PHS 2 PN2 EY016570B awarded by the National Institutes
of Health (NIH). The U.S. Government has certain rights in the
invention.
Claims
1. A protocell nanostructure comprising: a porous particle core
comprising a plurality of pores; and at least one lipid bilayer
surrounding the porous particle core to form a protocell, wherein
the protocell is capable of loading one or more cargo components to
the plurality of pores of the porous particle core and releasing
the one or more cargo components from the porous particle core
across the surrounding lipid bilayer.
2. The nanostructure of claim 1 further comprising a negatively
charged porous particle core, a non-negatively charged lipid
bilayer and a negatively charged cargo component.
3. The nanostructure of claim 1, wherein each of the one or more
cargo components comprises peptide, protein, antibody, DNA, RNA,
fluorescent dye, inorganic nanoparticle cargo component,
chemotherapeutic drug, or hydrophobic anti-cancer drug, wherein the
inorganic nanoparticle cargo component comprises a gold
nanoparticle, a magnetic nanoparticle or a quantum dot.
4. The nanostructure of claim 1, wherein the porous particle core
comprises a polymer hydrogel particle, or an inorganic
particle.
5. The nanostructure of claim 1, wherein the porous particle core
is made of a material comprising polystyrene, silica, alumina,
titania, or zirconia.
6. The nanostructure of claim 1, wherein the porous particle core
has a mean pore size ranging from about 2 nm to about 30 nm.
7. The nanostructure of claim 1, wherein the porous particle core
has a particle diameter ranging from about 30 nm to about 3000
nm.
8. The nanostructure of claim 1, wherein the at least one lipid
bilayer comprises a phospholipid comprising
1,2-Dioleoyl-3-Trimethylammonium-Propane (DOTAP),
1,2-Dioleoyl-sn-Glycero-3-Phosphocholine (DOPC) or a combination
thereof.
9. The nanostructure of claim 1, wherein the at least one lipid
bilayer comprises a fluidic interface for a ligand display or for a
multivalent targeting.
10. A method for forming a loaded protocell comprising: providing a
porous particle core, a lipid bilayer, and a cargo component; and
fusing the lipid bilayer to surround the porous particle core and
synergistically loading the cargo component into one or more pores
of the porous particle core to form a loaded protocell.
11. The method of claim 10 further comprising tuning a composition
of the lipid bilayer to control the synergistic loading of the
cargo component.
12. The method of claim 10 further comprising controlling one or
more of an electrostatic charge, a surface wettability or a pore
size of the porous particle core for the synergistic loading of the
cargo component.
13. The method of claim 10 further comprising using excess amount
of the lipid bilayer to improve colloidal stability of the loaded
protocell.
14. The method of claim 10 further comprising forming the porous
particle core by mixing water, HCl, ethanol, cetyltrimethylamonium
bromide (CTAB), and tetraethyl orthosilicate (TEOS).
15. The method of claim 10 further comprising treating the porous
particle core with ammonium hydroxide and hydrogen peroxide to
provide a more hydrophilic surface.
16. A method for delivering a cargo component using a protocell
comprising: providing a porous particle core, a lipid bilayer, and
one or more cargo components; wherein the lipid bilayer is fused
onto the porous particle core and the one or more cargo components
are synergistically loaded into one or more pores of the porous
particle core to form a loaded protocell; incubating a bioactive
cell with the loaded protocell to internalize the loaded photocell
within the bioactive cell; and rupturing the lipid bilayer of the
loaded photocell by applying a surfactant in preparation for
transporting the one or more cargo components from the porous
particle core into the bioactive cell.
17. The method of claim 16 further comprising: transporting the one
or more cargo components into a targeted bioactive cell, wherein
the lipid bilayer of the loaded protocell is modified with a
targeting active species corresponding to the targeted bioactive
cell.
18. The method of claim 16 further comprising releasing the cargo
components from the porous particle core according to a pH value or
an interaction of the porous particle core with the lipid
bilayer.
19. The method of claim 16 further comprising releasing a
doxocubicin cargo component into the bioactive cell through
dissolution of a porous silica particle core.
20. The method of claim 16 further comprising releasing a calcein
cargo component from the porous particle core by adjusting a pH
value.
21. The method of claim 16 further comprising transporting a
negatively charged DNA or a calcein dye into the bioactive
cell.
22. A delivery system according to the method of claim 16.
Description
RELATED APPLICATIONS
[0001] This application claims priority from U.S. Provisional
Patent Application Ser. No. 61/142,495, filed Jan. 5, 2009, which
is hereby incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0003] This invention relates generally to nanostructures and, more
particularly, to protocells having a porous particle supported
lipid bilayer, and methods for forming and using the
protocells.
BACKGROUND OF THE INVENTION
[0004] One of the challenges in nanomedicine is to engineer
nanostructures and materials that can efficiently encapsulate
cargo, for example, drugs, at high concentration, cross the cell
membrane, and controllably release the drugs at the target site
over a prescribed period of time. Recently, inorganic nanoparticles
have emerged as a new generation of drug or therapy delivery
vehicles in nanomedicine. More recently, gating methods that employ
coumarin, azobenzene, rotaxane, polymers, or nanoparticles have
been developed to seal a cargo within a particle and allow a
triggered release according to an optical or electrochemical
stimulus.
[0005] While liposomes have been widely used in drug delivery due
to their low immunogenicity and low toxicity, they still need to be
improved in several aspects. First, the loading of cargo can only
be achieved under the condition in which liposomes are prepared.
Therefore, the concentration and category of cargo may be limited.
Second, the stability of liposomes is relatively low. The lipid
bilayer of the liposomes often tends to age and fuse, which changes
their size and size distribution. Third, the release of cargo in
liposomes is instantaneous upon rupture of the liposome which makes
it difficult to control the release.
[0006] Thus, there is a need to overcome these and other problems
of the prior art and to provide a nanostructure including a porous
core and methods for forming and using the nanostructure.
SUMMARY OF THE INVENTION
[0007] According to various embodiments, the present teachings
include a protocell nanostructure. The protocell nanostructure can
include a porous particle core and at least one lipid bilayer
surrounding the porous particle core to form a protocell. The
protocell can be capable of loading one or more cargo components to
the pores of the porous particle core and releasing the one or more
cargo components from the porous particle core across the
surrounding lipid bilayer.
[0008] According to various embodiments, the present teachings also
include a method of forming a loaded protocell. The method can
begin with providing a porous particle core, a lipid bilayer, and a
cargo component. The lipid bilayer can then be fused to surround
the porous particle core and the cargo component can be
synergistically loaded into one or more pores of the porous
particle core. A loaded protocell can thus be formed.
[0009] According to various embodiments, the present teachings
further include a method for delivering a cargo component using a
protocell. In this method, a porous particle core, a lipid bilayer,
and one or more cargo components can be provided to fuse the lipid
bilayer onto the porous particle core and to synergistically load
the one or more cargo components into one or more pores of the
porous particle core to form a loaded protocell. A bioactive cell
can then be incubated with the loaded protocell to internalize the
loaded photocell within the bioactive cell. Following the
internalization, the lipid bilayer of the loaded photocell can be
ruptured by applying a surfactant in preparation for transporting
the one or more cargo components from the porous particle core into
the bioactive cell. Various embodiments can thus include a delivery
system according to the method of using protocells.
[0010] It is to be understood that both the foregoing general
description and the following detailed description are exemplary
and explanatory only and are not restrictive of the invention, as
claimed.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] The accompanying drawings, which are incorporated in and
constitute a part of this specification, illustrate several
embodiments of the invention and together with the description,
serve to explain the principles of the invention.
[0012] FIG. 1 depicts a schematic of an exemplary protocell
nanostructure as well as its formation in accordance with the
present teachings.
[0013] FIG. 2 depicts an exemplary protocell delivery system in
accordance with the present teachings.
[0014] FIG. 3 is a schematic showing surface re-organization of
targeting ligands on a protocell to bind a targeted bioactive cell
in accordance with the present teachings.
[0015] FIGS. 4A-4B depict release profiles of exemplary calcein
loaded protocells in accordance with the present teachings.
DESCRIPTION OF THE EMBODIMENTS
[0016] Reference will now be made in detail to the exemplary
embodiments of the invention, examples of which are illustrated in
the accompanying drawings. Wherever possible, the same reference
numbers will be used throughout the drawings to refer to the same
or like parts. In the following description, reference is made to
the accompanying drawings that form a part thereof, and in which is
shown by way of illustration specific exemplary embodiments in
which the invention may be practiced. These embodiments are
described in sufficient detail to enable those skilled in the art
to practice the invention and it is to be understood that other
embodiments may be utilized and that changes may be made without
departing from the scope of the invention. The following
description is, therefore, merely exemplary.
[0017] Various embodiments provide nanostructures and methods for
constructing and using the nanostructures. In one embodiment, the
nanostructures can include, for example, a core-shell structure
including a porous particle core surrounded by a shell of lipid
bilayer(s). The porous particle core can include, for example, a
porous nanoparticle made of an inorganic or organic material. In
various embodiments, such nanostructures can also be referred to as
"protocells" or "functional protocells," since the "protocells" can
mimic bioactive cells (or real cells) to have a supported lipid
bilayer membrane structure.
[0018] In embodiments, the porous particle core of the protocells
can be loaded with various desired species, which are also referred
to herein as "cargo" or "cargo components". In embodiments, the
cargo components can include, but are not limited to, chemical
molecules, nucleic acids, therapeutic agents, and/or other
nanoparticles, which are useful for a wide range of applications,
such as, for example, biomedical diagnostics, imaging, disease
treatment, drug delivery, and anti-bacteria applications.
[0019] In embodiments, the lipid bilayer of the protocells can
provide biocompatibility and can be modified to possess targeting
species including, for example, targeting peptides, antibodies,
aptamers, and PEG (polyethylene glycol) to allow, for example,
further stability of the protocells and/or a targeted delivery into
a bioactive cell.
[0020] FIG. 1 depicts a schematic of an exemplary protocell and its
formation in accordance with the present teachings. As shown, the
exemplary protocell 100 can include, for example, at least one
lipid bilayer 120 surrounding a porous particle core 110 to form a
supported lipid bilayer on the porous particle core.
[0021] In embodiments, the porous particle core 110 can include
porous nanoparticles having at least one dimension, for example, a
width or a diameter of about 3000 nm or less, or about 1000 nm or
less, or about 100 nm or less. For example, the porous particle
core 110 can have a particle diameter ranging from about 30 nm to
about 3000 nm. In embodiments, the porous particle core 110 can
have various cross-sectional shapes including a circular,
rectangular, square, or any other shape.
[0022] In embodiments, the porous particle core 110 can have pores
with a mean pore size ranging from about 2 nm to about 30 nm,
although the mean pore size and other properties (e.g., porosity of
the porous particle core) are not limited in accordance with
various embodiments of the present teachings.
[0023] In embodiments, the porous particle core 110 can be made of
various materials, inorganic or organic, such as, for example,
silica, alumina, titania, zirconia, polymers (e.g., polystyrene),
or combinations thereof. In embodiments, the porous particle core
110 can include inorganic particles, polymer hydrogel particles or
other suitable particles.
[0024] In embodiments, the porous particle core 110 can be
biocompatible. Drugs and other cargo components can be loaded by
adsorption and/or capillary filling of the pores of the particle
core. In embodiments, the loaded cargo can be released from the
porous surface of the particle core 110, wherein the release
profile can be determined or adjusted by, for example, the pore
size, the surface chemistry of the porous particle core, the pH
value of the system, and/or the interaction of the porous particle
core with the surrounding lipid bilayer(s).
[0025] In various exemplary embodiments, the porous particle core
110 can include, for example, mesoporous silica particles that can
provide biocompatibility and precisely defined nanoporosity. In
embodiments, the mesoporous silica particles can be prepared, for
example, by mixing HCl, ethanol, cetyltrimethylamonium bromide
(CTAB), and/or tetraethyl orthosilicate (TEOS). In embodiments, the
mesoporous silica particles can be prepared by surfactant templated
aerosol-assisted self-assembly method as described in a journal
paper from Nature 1999, vol. 398, page 223-226, entitled
"Aerosol-Assisted Self-Assembly of Mesostructure Spherical
Nanoparticles," which is hereby incorporated by reference in its
entirety. In this example, after removal of surfactant templates,
hydrophilic nanoparticles characterized by a uniform, ordered, and
connected mesoporosity can be prepared with a specific surface area
of, for example, about 935 m.sup.2/g.
[0026] In embodiments, the porous particle core 110 can be
hydrophilic and can be further treated to provide a more
hydrophilic surface. For example, mesoporous silica particles can
be further treated with ammonium hydroxide and hydrogen peroxide to
provide a high hydrophilicity.
[0027] Referring back to FIG. 1, the lipid bilayer 120 can be fused
onto the porous particle core 110 to form the protocell 100. In
embodiments, the lipid bilayer 120 can include a phospholipid
including, but not limited to,
1,2-Dioleoyl-3-Trimethylammonium-Propane (DOTAP),
1,2-Dioleoyl-sn-Glycero-3-Phosphocholine (DOPC) or a combination
thereof.
[0028] In embodiments, the lipid bilayer 120 can be prepared, for
example, by extrusion of hydrated lipid films through a filter with
pore size of, for example, about 100 nm, using standard protocols.
The filtered lipid bilayer films can then be fused with the porous
particle cores, for example, by a pipette mixing.
[0029] In embodiments, excess amount of lipid bilayers 120 or the
exemplary lipid bilayer films can be used to form the protocell 100
in order to improve the protocell colloidal stability.
[0030] In embodiments, various dyes or fluorescences can be
attached to the porous particle core 110 and/or the lipid bilayer
120 for analyzing the formed protocell nanostructure. For example,
the porous particle core can be a silica core and can be covalently
labeled with FITC (green fluorescence), while the lipid bilayer can
be labeled with Texas red (red fluorescence). The porous particle
core, the lipid bilayer and the formed protocell can then be
observed by, for example, confocal fluorescence microscopy.
[0031] In various embodiments, the protocell 100 can be a
synergistic system where the lipid bilayer fusion or liposome
fusion (i.e., on the porous particle core) can load and seal
various cargo components 130 into the pores of the particle core
110, creating a loaded protocell useful for cargo delivery across
the cell membrane of the lipid bilayer. For example, FIG. 2 depicts
an exemplary delivery system and its method in accordance with
various embodiments of the present teachings. In embodiments, in
addition to fusing a single lipid (e.g., phospholipids) bilayer,
multiple bilayers with opposite charges can be successively fused
onto the porous particle core to further influence cargo loading
and/or sealing.
[0032] At 210 of FIG. 2, a fusion and synergistic loading mechanism
can be included for the exemplary cargo delivery. For example,
cargo 130, can be loaded, encapsulated, or sealed, synergistically
through liposome fusion on the exemplary porous particles 110. The
cargo 130 can include, for example, peptides, proteins, antibodies,
DNAs, RNAs, fluorescent dyes, inorganic nanoparticles that include
gold nanoparticles, magnetic nanoparticles or quantum dots, and/or
drugs such as chemotherapeutic drugs, hydrophobic anti-cancer drugs
or other types of drugs.
[0033] In embodiments, the cargo 130 can be loaded into the pore of
porous particle cores 110 to form the loaded protocell 205, which
is different from bioactive cells that have an aqueous interior
area but do not include particle nanopores. In various embodiments,
any conventional technology that is developed for liposome-based
drug delivery, for example, targeted delivery using PEGylation, can
be transferred and applied to the disclosed porous particle
supported lipid bilayers, i.e., the protocells. In an exemplary
embodiment, versatile loading with improved bilayer stability can
be achieved for the protocell 100.
[0034] In various embodiments, electrostatics and pore size can
play a role in cargo loading. For example, porous silica
nanoparticles can carry a negative charge and the pore size can be
tunable from about 2 nm to about 10 nm. Such negatively charged
nanoparticles can have a natural tendency to adsorb positively
charged molecules. In an exemplary embodiment, a chemotherapeutic
drug doxocubicin (red fluorescence) that carries a positive charge
can be adsorbed by porous silica. After fusion with an NBD (green
fluorescence)-labeled DOPC liposome, the red-in-green core-shell
structure can be observed under fluorescence microscopy. In various
embodiments, other properties such as surface wettability (e.g.,
hydrophobicity) can also affect loading cargo with different
hydrophobicity.
[0035] In various embodiments, the cargo loading can be a
synergistic lipid-assisted loading by tuning the lipid composition.
For example, if the cargo component is a negatively charged
molecule, the cargo loading into a negatively charged silica can be
achieved by the lipid-assisted loading. In an exemplary embodiment
that is absent of lipid, the silica particle can not adsorb any
exemplary calcein molecule, which is also negatively charged.
However, in the presence of DOTAP lipid (e.g., labeled with Texas
red, the red fluorescence dye), the negatively charged calcein dye
(green fluorescence) can be loaded into the pores of the negatively
charged silica particle when the lipid bilayer is fused onto the
silica surface showing a fusion and synergistic loading
mechanism.
[0036] In this manner, fusion of a non-negatively charged (i.e.,
positively charged or neutral) lipid bilayer or liposome on a
negatively charged mesoporous particle can serve to load the
particle core with a negatively charged dye or other negatively
charged cargo components. The negatively charged cargo components
can be concentrated in the loaded protocell having a concentration
exceed about 100 times as compared with the charged cargo
components in a solution.
[0037] At 220, the loaded protocells 205 can have a cellular uptake
for cargo delivery into desirable site. For example, the
cargo-loaded protocells 205 can be incubated with a desirable
bioactive cell 207 and can be internalized or uptaken by the
bioactive cell 207, for example, a mammalian cell.
[0038] Due to the internalization of cargo-loaded protocells 205 in
the bioactive cell 207, cargo components 130 can then be delivered
into the bioactive cell 207. For example, when free calcein cargo
is mixed with Chinese Hamster Ovary (CHO) cells, the CHO cells can
not take the green dye calcein because calcein is a membrane
impermeable dye. However, calcein can be taken into the CHO cells
through the internalized protocells that is loaded with green dye
calcein. The CHO cells can then be observed green (not shown) due
to the delivery of the green dye calcein.
[0039] In another example, negatively charged DNA itself can not be
directly delivered or internalized into the bioactive cells.
However, the negatively charged DNA can be loaded first into a
protocell and then into bioactive cells through the internalization
of the loaded protocells. As such, loaded protocells can deliver
the exemplary calcein or negatively charged DNA into bioactive
cells, e.g., CHO.
[0040] In various embodiments, the protocells and/or the loaded
protocells can provide a targeted delivery methodology for
selectively delivering the protocells or the cargo components to
targeted bioactive cells (e.g., cancer cells). For example, a
surface of the lipid bilayer can be modified by a targeting active
species that corresponds to the targeted bioactive cell 207.
[0041] For example, FIG. 3 depicts a schematic showing
re-organization of an exemplary targeting active species to bind a
targeted bioactive cell 350 in accordance with the present
teachings. In one embodiment, by conjugating an exemplary targeting
peptide SP94 (see 330 of FIG. 3) that targets cancer liver cells to
the lipid bilayer 120 of the protocells 100 or the loaded
protocells 205 (see FIG. 2), a large number of protocells can be
recognized and internalized by this specific cancer cells (see 350
of FIG. 3) due to the specific targeting of the exemplary SP94
peptide with the liver cancer cells. In some cases, if the
protocells are not conjugated with the targeting peptide, if the
peptide is a non-targeting peptide to the targeted liver cancer
cell, or if the liver cancer cells are normal liver cells, there
can be no significant association, uptake or internalization of the
protocells or the loaded protocells by the cancer cells.
[0042] In various embodiments, the protocells and/or the loaded
protocells can provide a ligand display and/or a multivalent
targeting on a fluidic interface of the lipid bilayer. For example,
displaying multiple copies of the targeting peptide on the
protocell surface can allow multivalent targeting. Different from
other displaying platforms, protocells can have a unique fluidic
phospholipid bilayer surface, which allows re-organization of
ligands in response to targets. Such re-organization can allow high
affinity multivalent binding at low overall ligand density, which
may decrease the immune response of host cells.
[0043] Referring back to FIG. 2, at 230, the cellular uptaken
protocells 205 can release cargo components 130 from the porous
particle 110 and transport the released cargo components into the
bioactive cell 207. For example, sealed within the protocell by the
liposome fusion on the porous particle core, the cargo components
130 can be released from the porese, transported across the
protocell membrane of the lipid bilayer 120 and delivered within
the bioactive cell 207.
[0044] In embodiments, the release profile of cargo components in
protocells can be more controllable as compared with when only
using liposomes as known in the prior art. The cargo release can be
determined by, for example, interactions between the porous core
and the lipid bilayer and/or other parameters such as pH value of
the system. For example, the release of doxocubicin cargo can be
achieved through dissolution of porous silica; while the release of
calcein cargo in the synergistically loaded protocells can be
pH-dependent as shown in FIGS. 4A-4B.
[0045] In embodiments, the pH value for releasing calcein cargo can
be of about pH 14 or less. As shown in FIG. 4A, lower pHs can
facilitate the release of calcein cargo more as compared with high
pHs. This is advantageous because the endosomal compartments inside
cells can be at low pHs. FIG. 4B shows a calcein cargo release at a
specific pH value of about 7.4, wherein the release can span for
about 20 days.
[0046] In embodiments, surfactants can be applied to rupture the
lipid bilayer or the liposome, transporting the cargo components
across the liposome within the bioactive cell. In exemplary
embodiments, the phospholipid bilayer of the protocells can be
ruptured by applying a surfactant of sodium dodecyl sulfate (SDS).
In embodiments, the rupture of the lipid bilayer can in turn induce
immediate and complete release of the cargo components from the
pores of the particle core of the protocells.
[0047] In this manner, the protocell platform can provide versatile
delivery systems as compared with other delivery systems in the
art. For example, when compared to delivery systems using
nanoparticles only, the disclosed protocell platform can provide a
simple system and can take advantage of the low toxicity and
immunogenicity of liposomes or lipid bilayers along with their
ability to be PEGylated or to be conjugated to extend circulation
time and effect targeting. In another example, when compared to
delivery systems using liposome only, the protocell platform can
provide a more stable system and can take advantage of the
mesoporous core to control the loading and/or release profile.
[0048] In addition, the lipid bilayer and its fusion on porous
particle core can be fine-tuned to control the loading, release,
and targeting profiles. Further, the lipid bilayer can provide a
fluidic interface for ligand display and multivalent targeting,
which allows specific targeting with relatively low surface ligand
density due to the capability of ligand re-organization on the
fluidic lipid interface. Furthermore, the disclosed protocells can
readily enter bioactive cells while empty liposome without the
support of porous particles cannot be internalized by the
cells.
[0049] The following examples are illustrative of the invention and
its advantageous properties, and are not to be taken as limiting
the disclosure or claims in any way. In the examples, as well as
elsewhere in this application, all parts and percentages are by
weight unless otherwise indicated.
EXAMPLES
Example 1
Materials
[0050] Exemplary phospholipids were obtained from Avanti Polar
Lipids Inc. (Alabaster, Ala.). Exemplary cholesterol was obtained
from Sigma (St. Louis, Mo.). Texas Red-labeled DHPE lipid and
fluorescein isothiocyanate (FITC) were obtained from Invitrogen
(Carlsbad, Calif.). All silanes and calcein were obtained from
Aldrich Sigma (St. Louis, Mo.). Chinese Hamster Ovary (CHO) cells
and cell culture related chemicals and media were obtained from
American Type Culture Collection (ATCC).
[0051] All UV-vis absorption data were collected on a Perkin-Elmer
spectrophotometer; all fluorescence data were obtained on a Horiba
Jobin Yvon Fluoromax-4 fluorometer; and all light scattering data
were collected on a Zetasizer Nano dynamic light scattering
instrument (Malvern).
[0052] Lipids and other chemicals used in the examples included the
following:
##STR00001##
Example 2
Preparation of Mesoporous Silica Nanoparticles
[0053] Mesoporous silica nanoparticles were prepared by the
aerosol-assisted self-assembly method, wherein silica/surfactant
aerosols were generated using a commercial atomizer (Model 9302A,
TSI, Inc., St Paul, Minn.) operated with nitrogen as a
carrier/atomization gas. The reaction was started with a
homogeneous solution of soluble silica precursor tetraethyl
orthosilicate (TEOS), HCl, and surfactant prepared in an
ethanol/water solution with an initial surfactant concentration
much less than the critical micelle concentration. The pressure
drop at the pinhole was about 20 psi. The temperature for the
heating zones was kept at about 400.degree. C. Particles were
collected on a durapore membrane filter maintained at about
80.degree. C. cetyltrimethylamonium bromide (CTAB) was selected as
the structure directing template.
[0054] In a typical synthesis of mesoporous silica nanoparticles,
about 55.9 mL H.sub.2O, about 43 mL ethanol, about 1.10 mL 1N HCl,
about 4.0 g CTAB, and about 10.32 g TEOS were mixed. The mixture
was also referred to as water/ethanol/HCl/CTAB/TEOS mixture.
[0055] To prepare FITC-labeled particles, 18 mg FITC and 100 .mu.L
3-aminopropyltriethoxysilane (APTES) were reacted in about 1 mL 200
proof ethanol for about four hours in dark. The resultant solution
along with about 36 .mu.L of 12N HCl were then added to the
water/ethanol/HCl/CTAB/TEOS mixture to make FITC-labeled
particles.
[0056] The reaction between FITC and APTES is presented below.
##STR00002##
Example 3
Preparation of Liposomes
[0057] Phospholipids were dissolved in chloroform at concentrations
of about 10 to about 25 mg/mL. Aliquots were dispensed into
scintillation vials so that each vial contained 2.5 mg lipids. For
mixed lipids, the total amount of lipids was also controlled to be
about 2.5 mg per vial. Some lipids were mixed with a small fraction
(2-5%) of Texas Red-labeled DHPE. The chloroform in the vials was
evaporated under a nitrogen flow in a fume hood and lipid films
were formed. The vials were then stored in a vacuum oven at room
temperature overnight to remove any residual chloroform. The
samples were frozen at about -20.degree. C. before use.
[0058] To prepare lipid bilayers or liposomes, the vials were
brought to room temperature and rehydrated by adding 1 mL of
0.5.times.PBS with occasional shaking for at least 1 hr, forming a
cloudy lipid suspension. The suspension was extruded with a
mini-extruder purchased from Ananti Polar Lipids. A membrane with
pore diameter of 100 nm was used and at least ten extrusion cycles
were performed. The resulting clear lipid bilayers or liposomes
were stored in a new vial at 4.degree. C. Light scattering
experiments showed that the as-prepared liposomes have a mean
hydrodynamic diameter of about 140 nm and the size distribution did
not change after storing at about 4.degree. C. for a week.
Example 4
Preparation of Supported Bilayers, the Protocells
[0059] The silica nanoparticles were weighed (about 25 to about 50
mg) and transferred into a scintillation vial. About 20 mL of 200
proof ethanol with 1% HCl was added and the solution was sonicated
for at least 30 min to extract the CTAB surfactant from the pores.
The particles were collected by centrifugation and removal of the
supernatants. The washing process was repeated twice with ethanol
and twice with water. To make the surface more hydrophilic, the
particles were then treated with 4% ammonium hydroxide and 4%
hydrogen peroxide at about 80.degree. C. for about 10 min. After
washing with water, the particles were further treated with 0.4 M
HCl and 4% hydrogen peroxide at about 80.degree. C. for about 10
min and washed with water. The final concentration of silica
nanoparticles were made to be about 25 mg/mL in water.
[0060] Equal volumes (e.g., about 50 .mu.L) of the above prepared
silica nanoparticles and liposomes (e.g., about 2.5 mg/mL) were
mixed by pipetting the mixture several times. The mixture was
allowed to sit at room temperature for about 20 min with occasional
agitation. Extra lipids were removed by centrifugation of the
mixture at 4000 rpm for about 1 minute, and removal of the
supernatant. The supported bilayers were subsequently washed with
200 .mu.L of 0.25.times.PBS and finally dispersed in 200 .mu.L
0.25.times.PBS.
[0061] To prepare supported bilayers that encapsulate calcein, the
silica nanoparticles were first mixed with 250 .mu.M calcein and
liposomes were subsequently added. The remaining procedures were
the same as described above. Because DOTAP lipids showed the
highest calcein encapsulation efficiency, most examples herein
included the supported bilayers with DOTAP lipids.
Example 5
Cellular Uptake of Supported Bilayers (Protocells)
[0062] Cell Culture:
[0063] Chinese Hamster Ovary cells (CHO) were obtained from the
American Type Culture Collection (ATCC) and maintained in K-12
media supplemented with about 10% fetal bovine serum, about 1%
penicillin and about 1% streptomycin. The media were changed every
two to three days and the cells were passaged by trypsinization. To
prepare samples for confocal imaging, round glass cover slips were
used for cell growth. The glass slides were treated with 0.1 M KOH
for at least 24 hours before use. Cells in the media were dropped
onto the cover slips and the slips were kept in Petri dishes. The
cells were kept in an incubator at 37.degree. C. with 5% CO.sub.2
and 95% humidity.
[0064] Cellular Uptake of Supported Bilayers (Protocells):
[0065] about 1 mL of serum free media was warmed to about
37.degree. C. and about 10 .mu.L of the above prepared supported
bilayers were added and vortexed. To study the uptake of supported
bilayers by CHO cells, the cells were grown to .about.70%
confluence. The old media was removed and fresh media with
supported bilayers were introduced. The cells were incubated for
about four hours at about 37.degree. C. and free particles were
washed away with PBS and media before imaging.
Example 6
Effect of Supported Bilayers on CHO Cell Viability
[0066] CHO cells were incubated with supported bilayers as
described above. The media was removed and 300 .mu.L viability dyes
(0.5 .mu.L calcein-AM and 2 .mu.L ethidium homodimer dissolved in
about 2 mL serum free media) were added. The cells were incubated
at 37.degree. C. for about 30 min. Fluorescence was monitored under
an inverted fluorescence microscope. Viability assays indicated
that more than 97% of the cells were viable.
[0067] Confocal Fluorescence Microscopy:
[0068] A Bio-Rad Radiance 2100 confocal fluorescence microscope
system was used for imaging cells. Argon 488 nm line was used for
imaging FITC and calcein; green HeNe (543 nm) was used for imaging
Texas Red; and Red Diode (633 nm) was used for DIC imaging. All
images were collected with a 60.times. oil immersion objective. To
image supported lipid bilayers, 3 .mu.L of dilute protocells were
spotted on a glass slide and sealed with a cover slip by super
glue.
Example 7
Quantification of Calcein Encapsulated by Different Lipids
[0069] Mesoporous silica nanoparticles (about 50 mg, no FITC
modification) were dispersed in 2 mL water. About 5 .mu.L of 100 mM
calcein was added so the final dye concentration was .about.250
.mu.M. The solution was divided into 50 .mu.L aliquots and equal
volumes of liposomes of different compositions were mixed to form
supported bilayers. The supported bilayers were centrifuged and
washed three times with 200 .mu.L of 0.25.times.PBS to remove free
calcein. Finally, about 50 .mu.L of 1% SDS buffer solution was
added to the precipitated supported bilayers to disrupt the lipid
bilayer and release the calcein dye. About 150 .mu.L of
0.25.times.PBS was then added to make the final volume to be about
200 .mu.L and the tubes were centrifuged at 15000 rpm for about 2
minutes to precipitate silica nanoparticles. About 10 .mu.L from
the supernatant was transferred into a quartz microcuvette with a
path length of about 1 cm to measure the absorbance at a wavelength
of about 500 nm, which is proportional to the amount of calcein dye
retained in the mesoporous silica nanoparticle.
Example 8
Quantification of Calcein Release Profile
[0070] With the method described above, 200 .mu.L of supported
lipid bilayers (DOTAP lipids) with calcein encapsulated inside were
prepared. At designated time points, about 20 .mu.L aliquots were
taken out into another tube and centrifuged at about 15000 rpm for
about 2 minutes. About 10 .mu.L of the supernatant was taken out
and transferred into another tube and its fluorescence intensity is
denoted to be F.sub.1. The fluorescence intensity of the remaining
10 .mu.L is denoted to be F.sub.2, which included the other 10
.mu.L of the supernatant and the silica precipitant. The fraction
of release was calculated to be 2.times.F.sub.1/(F.sub.1+F.sub.2).
To measure fluorescence, the dye was released by using 20 .mu.L of
2.5% SDS and the solution was finally dispersed in 500 .mu.L PBS
and centrifuged at 15000 rpm for about 5 minutes to precipitate all
the silica nanoparticles. About 400 .mu.L of the supernatant was
transferred into a fluorescence cuvette and the calcein
fluorescence was measured by exciting at a wavelength of about 467
nm and collecting emission at a wavelength of about 517 nm. All
experiments were run in triplicate.
[0071] As a result, .about.90% of the calcein dye was released in
18 days and the rate of releasing gradually decreased with time. A
pH-dependent study was also performed and the release was measured
after 12 hours. The measured results (see FIG. 4A) indicated that
the fraction of calcein dye release significantly increased at
lower pH.
Example 9
Concentration Estimation of Calcein Inside Mesoporous Silica
Nanoparticles
[0072] When 100% DOTAP lipids were used to form supported bilayers,
the retained calcein had an absorbance of about 2.5, which
corresponded to a concentration of about 45 .mu.M (the extinction
coefficient of calcein is .about.55,000 M.sup.-1 cm.sup.-1 at
wavelength of about 500 nm). Because the volume of this final
solution was about 200 .mu.L, the retained calcein in silica was
then about 9 nmol. The silica mass was about 1.25 mg (i.e., 50
.mu.L of 25 mg/mL). The density of mesoporous silica nanoparticles
was estimated to be about 1.07 g/cm. Therefore, the volume of the
silica was about 1.17.times.10.sup.-3 cm.sup.3, and the
concentration of calcein inside silica was about 7.7 mM.
[0073] The initial calcein concentration in solution was about 250
.mu.M and the final concentration was about 70 .mu.M, with the
remaining calcein being inside the silica nanoparticles. Therefore,
.about.72% of the dye was encapsulated in the particles, and the
concentration inside silica was .about.110 times higher than that
in solution.
Example 10
Lipid Association with Mesoporous Silica Nanoparticles
[0074] To measure the amount of lipid associated with silica
nanoparticles as a function of lipid concentration, about 20 .mu.L
aliquots of 25 mg/mL silica nanoparticles were mixed with 1, 2, 3,
5, 7, 10, 20, and 30 .mu.L of 2.5 mg/mL lipids. The lipids tested
included DOPC, DOPS and DOTAP, all containing 5% DHPE-Texas Red
labels. The mixtures were centrifuged. The Texas Red absorbance
from the supernatant and the silica nanoparticles was measured. As
a result, positively charged DOTAP and neutral DOPC liposomes
almost quantitatively associated with silica nanoparticles when
<20 .mu.g of liposome was used for 0.5 mg silica particles,
suggesting a high binding affinity. Further addition of liposomes
did not increase association, possibly due to the saturation of the
silica surface. Negatively charged DOPS did not associate with
silica, which can be attributed to the electrostatic repulsion
between them at neutral pH.
Example 11
Colloidal Stability of the Silica/lipid Mixture as a Function of
Lipid Concentration
[0075] Depending on the relative amount of liposome added, silica
particles first aggregated at low lipid concentrations to form
large aggregates, which disappeared upon adding more liposomes. As
characterized by dynamic light scattering, for both DOTAP and DOPC,
there was a significant increase in the average size of particles
at low liposome contents. Similar observations were also reported
for polystyrene beads, where aggregation was attributed to liposome
mediated nanoparticle assembly at low lipid concentrations.
Therefore, to form supported bilayers with good colloidal
stability, excess amount of liposomes (50 .mu.g liposome per 0.5 mg
silica) were used.
[0076] Notwithstanding that the numerical ranges and parameters
setting forth the broad scope of the invention are approximations,
the numerical values set forth in the specific examples are
reported as precisely as possible. Any numerical value, however,
inherently contains certain errors necessarily resulting from the
standard deviation found in their respective testing measurements.
Moreover, all ranges disclosed herein are to be understood to
encompass any and all sub-ranges subsumed therein. For example, a
range of "less than 10" can include any and all sub-ranges between
(and including) the minimum value of zero and the maximum value of
10, that is, any and all sub-ranges having a minimum value of equal
to or greater than zero and a maximum value of equal to or less
than 10, e.g., 1 to 5. In certain cases, the numerical values as
stated for the parameter can take on negative values. In this case,
the example value of range stated as "less that 10" can assume
negative values, e.g. -1, -2, -3, -10, -20, -30, etc.
[0077] Other embodiments of the invention will be apparent to those
skilled in the art from consideration of the specification and
practice of the invention disclosed herein. It is intended that the
specification and examples be considered as exemplary only, with a
true scope and spirit of the invention being in dicated by the
following claims.
* * * * *