U.S. patent application number 15/396356 was filed with the patent office on 2017-04-20 for methods for solubilizing cells and/or tissue.
The applicant listed for this patent is THE REGENTS OF THE UNIVERSITY OF CALIFORNIA, SKINCENTIAL SCIENCES, INC.. Invention is credited to Byeong Hee Hwang, Russell M. Lebovitz, Samir Mitragotri.
Application Number | 20170105673 15/396356 |
Document ID | / |
Family ID | 46928161 |
Filed Date | 2017-04-20 |
United States Patent
Application |
20170105673 |
Kind Code |
A1 |
Mitragotri; Samir ; et
al. |
April 20, 2017 |
Methods for Solubilizing Cells and/or Tissue
Abstract
Solubilizing compositions are provided. The compositions
comprise at least one zwitterionic surfactant and at least one
nonionic surfactant. In one embodiment, the compositions may be
useful for solubilizing and remodeling and/or removing tissue on or
beneath a patient's skin, optionally in conjunction with the
application of energy to a region of interest on the skin. In one
embodiment, at least one analyte may be collected and analyzed from
the solubilized tissue.
Inventors: |
Mitragotri; Samir; (Santa
Barbara, CA) ; Lebovitz; Russell M.; (Oakland,
CA) ; Hwang; Byeong Hee; (Goleta, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE REGENTS OF THE UNIVERSITY OF CALIFORNIA
SKINCENTIAL SCIENCES, INC. |
OAKLAND
SAN FRANCISCO |
CA
CA |
US
US |
|
|
Family ID: |
46928161 |
Appl. No.: |
15/396356 |
Filed: |
December 30, 2016 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
14138090 |
Dec 22, 2013 |
|
|
|
15396356 |
|
|
|
|
13473261 |
May 16, 2012 |
8642664 |
|
|
14138090 |
|
|
|
|
13095639 |
Apr 27, 2011 |
8389582 |
|
|
13473261 |
|
|
|
|
12664994 |
Jun 29, 2010 |
|
|
|
PCT/US2008/072384 |
Aug 6, 2008 |
|
|
|
13095639 |
|
|
|
|
13126105 |
Apr 26, 2011 |
8945482 |
|
|
PCT/US10/24010 |
Feb 12, 2010 |
|
|
|
13095639 |
|
|
|
|
60963773 |
Aug 6, 2007 |
|
|
|
61152585 |
Feb 13, 2009 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/205 20130101;
A61K 31/765 20130101; A61B 2018/00458 20130101; A61K 31/08
20130101; A61K 9/0009 20130101; A61B 10/02 20130101; A61B 18/06
20130101; C07J 9/00 20130101; A61B 5/14546 20130101; A61K 2300/00
20130101; C07K 2317/52 20130101; A61P 17/00 20180101; C07K 2317/94
20130101; A61K 9/127 20130101; A61K 9/0014 20130101; C12Q 1/32
20130101; C07K 16/18 20130101; A61K 31/205 20130101; A61K 9/06
20130101; A61K 31/765 20130101; A61B 5/443 20130101; A61K 2300/00
20130101 |
International
Class: |
A61B 5/00 20060101
A61B005/00; A61B 10/02 20060101 A61B010/02; A61B 18/06 20060101
A61B018/06; A61B 5/145 20060101 A61B005/145 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under
federal grant number W81XWH-06-01-00400 awarded by the United
States Army. The United States Government has certain rights in
this invention.
Claims
1. A method for one or more of solubilizing, remodeling, and
removing a portion of tissue on or beneath a patient's skin, the
method comprising: contacting a region of interest on the skin with
a tissue solubilizing composition, the tissue solubilizing
composition comprising: at least one zwitterionic surfactant,
represented by the following structural formula: ##STR00003## and
at least one non-ionic surfactant, represented by the following
structural formula: H(CH.sub.2).sub.aO(CH.sub.2CH.sub.2O).sub.bH,
wherein: n is 10, 12, 14, 16, or 18; a is 12 or 16; and b is 2, 4,
10, 20, or 23, provided that n, a, and b are not respectively: 10,
12, and 4; or 14, 16, and 10; and applying energy to the region of
interest on the skin effective to cause the one or more of:
solubilizing, remodeling, and removing.
3. The method of claim 1, further comprising: removing at least a
portion of the region of interest to leave an exposed area on the
skin; and applying to the exposed area at least one of a
therapeutic composition and a cosmetic composition.
3. The method of claim 2, at least one of: the therapeutic
composition comprising at least one of: a DNA-based drug, an
RNA-based drug, a protein-based drug, a peptide-based drug, a
lipid-based drug, a carbohydrate-based drug, a small molecule drug,
a nanoparticle-based drug, and a liposome-encapsulated drug; and
the cosmetic composition comprising at least one of: elastin, an
elastin-based peptide, collagen, a collagen-based peptide,
resveratrol, idebenone, co-enzyme Q10, acetyl hexapeptide-3,
glycosaminoglycans, palmitoyl pentapeptide-4, sodium hyaluronate,
and combinations thereof.
4. The method of claim 1, wherein: n is 12, a is 16, and b is 2; n
is 12, a is 16, and b is 10; n is 12, a is 12, and b is 23; n is
14, a is 12, and b is 4; n is 14, a is 16, and b is 2; n is 16, a
is 12, and b is 4; n is 16, a is 16, and b is 2; n is 16, a is 16,
and b is 10; n is 18, a is 12, and b is 4; n is 18, a is 16, and b
is 2; n is 18, a is 16, and b is 10; or n is 18, a is 16, and b is
20.
5. The method of claim 1, wherein: n is 12, a is 16, and b is 10; n
is 14, a is 12, and b is 4; n is 14, a is 16, and b is 2; n is 16,
a is 12, and b is 4; n is 16, a is 16, and b is 2; n is 16, a is
16, and b is 10; n is 16, a is 16, and b is 20; n is 18, a is 12,
and b is 4; n is 18, a is 16, and b is 2; n is 18, a is 16, and b
is 10; or n is 18, a is 16, and b is 20.
6. The method of claim 1, wherein a+b-n is less than 21.
7. The method of claim 1, wherein: a is 12 and b is 4; a is 16 and
b is 2; or a is 16 and b is 10.
8. The method of claim 1, wherein n is 12, 14, 16, or 18.
9. The method of claim 1, wherein n is 14, 16, or 18.
10. The method of claim 1, the tissue solubilizing composition
further comprising a buffer solution effective to buffer the tissue
solubilizing composition to a pH value, the pH value being one or
more of: more basic than about 7.0; between about 7.0 and about
9.0; between about 7.4 and about 9.0; between about 7.4 and about
8.0; and between about 7.2 and about 7.6.
11. The method of claim 1, the tissue solubilizing composition
further comprising a buffer solution, the tissue solubilizing
composition characterized by a total percent (w/v) concentration of
the zwitterionic surfactant and the non-ionic surfactant of one or
more of: about 0.01% to about 10%; about 0.01% to about 5%; about
0.1% to about 2%; and about 0.1% to about 0.5%.
12. The method of claim 1, the tissue solubilizing composition
further comprising a buffer solution that dissolves the
zwitterionic surfactant and the non-ionic surfactant, the
zwitterionic surfactant and the non-ionic surfactant each being
present in an amount of at least about 0.25%) w/v with respect to
the buffer solution.
13. A method for recovering analytes from mucosal membrane, skin,
or other tissue, the method comprising: applying energy to a region
of interest on the mucosal membrane, skin, or other tissue
containing at least one analyte; contacting the region with a
tissue solubilizing composition, thereby solubilizing at least some
of the mucosal membrane, skin, or other tissue containing at least
one analyte; and collecting the at least one analyte from the
solubilized mucosal membrane, skin, and other tissue, wherein the
tissue solubilizing composition comprises: at least one
zwitterionic surfactant, represented by the following structural
formula: ##STR00004## and at least one non-ionic surfactant,
represented by the following structural formula:
H(CH.sub.2).sub.aO(CH.sub.2CH.sub.2O).sub.bH, wherein: n is 10, 12,
14, 16, or 18; a is 12 or 16; and b is 2, 4, 10, 20, or 23,
provided that n, a, and b are not respectively: 10, 12, and 4; or
14, 16, and 10.
14. The method of claim 13, the analyte comprising one or more of:
a protein, a cancer biomarker, an antibody, a peptide, a lipid, a
nucleic acid, a small molecule, a microbe, a warfare agent, an
environmental contaminant, and a drug.
15. The method of claim 13, wherein: n is 12, a is 16, and b is 2;
n is 12, a is 16, and b is 10; n is 12, a is 12, and b is 23; n is
14, a is 12, and b is 4; n is 14, a is 16, and b is 2; n is 16, a
is 12, and b is 4; n is 16, a is 16, and b is 2; n is 16, a is 16,
and b is 10; n is 18, a is 12, and b is 4; n is 18, a is 16, and b
is 2; n is 18, a is 16, and b is 10; or n is 18, a is 16, and b is
20.
16. The method of claim 13, wherein: n is 12, a is 16, and b is 10;
n is 14, a is 12, and b is 4; n is 14, a is 16, and b is 2; n is
16, a is 12, and b is 4; n is 16, a is 16, and b is 2; n is 16, a
is 16, and b is 10; n is 16, a is 16, and b is 20; n is 18, a is
12, and b is 4; n is 18, a is 16, and b is 2; n is 18, a is 16, and
b is 10; or n is 18, a is 16, and b is 20.
17. The method of claim 13, the tissue solubilizing composition
further comprising a buffer solution effective to buffer the tissue
solubilizing composition to a pH value, the pH value being one or
more of: more basic than about 7.0; between about 7.0 and about
9.0; between about 7.4 and about 9.0; between about 7.4 and about
8.0; and between about 7.2 and about 7.6.
18. The method of claim 13, the tissue solubilizing composition
further comprising a buffer solution, the tissue solubilizing
composition characterized by a total percent (w/v) concentration of
the zwitterionic surfactant and the non-ionic surfactant of one or
more of: about 0.01% to about 10%; about 0.01% to about 5%; about
0.1% to about 2%; and about 0.1% to about 0.5%.
19. The method of claim 13, the tissue solubilizing composition
further comprising a buffer solution that dissolves the
zwitterionic surfactant and the non-ionic surfactant, the
zwitterionic surfactant and the non-ionic surfactant each being
present in an amount of at least about 0.25% w/v with respect to
the buffer solution.
20. The method of claim 13, wherein: a is 12 and b is 4; a is 16
and b is 2; or a is 16 and b is 10.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part application of
U.S. patent application Ser. No. 13/473,261, filed on May 16, 2012,
which is a continuation-in-part application of U.S. Pat. Appl. Ser.
No. 13/095,639, filed on Apr. 27, 2011. U.S. patent application
Ser. No. 13/095,639 is a continuation-in-part application of U.S.
patent application Ser. No. 12/664,994, which was filed on Jun. 29,
2010 as a U.S. National Stage filing of PCT/US2008/072384, filed on
Aug. 6, 2008, which, in turn, claims priority to U.S. Prov. Pat.
Appl. Ser. No. 60/963,773, filed on Aug. 6, 2007, and now expired.
U.S. patent application Ser. No. 13/095,639 is also a
continuation-in-part application of U.S. patent application Ser.
No. 13/126,105, which was filed on Apr. 26, 2011 as a U.S. National
Stage filing of PCT/US2010/024010, filed on Feb. 12, 2010, which,
in turn, claims priority to U.S. Prov. Pat. Appl. Ser. No.
61/152,585, filed on Feb. 13, 2009, and now expired. All of these
related applications are incorporated by reference herein in their
entireties.
BACKGROUND
[0003] Skin is the "window" to the body. Skin is unique among the
body's organs for several reasons: (1) skin is the largest organ of
the human body; (2) skin is directly exposed to the environment;
(3) skin is an excellent excretory organ; (4) skin is the most
visible and accessible organ of the body; and (5) skin is a highly
active immune organ of the body,
[0004] Skin has another important quality: The molecular profile of
skis has information that is valuable for physiological monitoring
of, among other things, small organic molecules, proteins, DNA,
RNA, and lipids. Much can be learned from skin's molecular
profiling. Far example, pathogens (e.g., bacteria) that grow on
skis may allow for forensic identification. Skin's molecular
profile may reveal environmental factors to which the body has been
passively exposed. These environmental factors may range from the
mundane, e.g., allergens, toxins, and cosmetic products, to the
industrial and/or agricultural, e.g., industrial solvents,
fertilizers, and pesticides, to the dangerous, e.g., explosives and
other warfare agents.
[0005] Skin's molecular profile may also reveal factors to which
the body has been actively exposed. More particularly, skirts
molecular profile may reveal what the body has consumed. For
example, abused substances (e.g., illegal drugs or narcotics) and
therapeutic drugs (e.g., tramadol, fluconazole, barbitals, and
anabolic steroids) may be found in skin weeks after
consumption.
[0006] Skin's molecular profile may also aid diagnosis of
conditions and diseases. For example, skin cholesterol is a proxy
of the extent of arterial blocks, Glycation of skin collagen is an
indicator of a history of diabetes. Skin deposition of amyloids may
indicate the existence and extent of Alzheimer's disease. And skin
globular proteins (e.g., IgE) may indicate allergies to specific
allergens.
[0007] Several methods exist for sampling biomolecules from skin.
For example, one current method is skin biopsy. However, skin
biopsy is invasive and analysis is difficult. Practically speaking,
skin biopsy is designed for well-equipped experts and, thus, its
use in a point-of-care setting is limited. Another current method
for sampling biomolecules from skin, tape stepping, suffers from
these same limitations and is generally unacceptable because of
variability in results. Yet smother current method for sampling
biomolecules from skin is taking a skin, swab. While desirable
because of is simplicity, a skin swab is superficial in its depth
of inspection, and qualitative in its results. Finally, tissue has
been subjected to ultrasound in the presence of surfactants such as
sorbitans ("SPANs"), polyoxyethelene sorbitans combined with fatty
acids ("tweens"), cetyl trimethylammonium bromide ("CTAB"), and
their mixtures. See U.S. Pat. No. 6,589,173 to Mitragotri et al.
However, SPANs, tweens, and CTAB, individually and collectively,
have been found to be unsuitable to recover skin constituents.
Sorbitans and tweens, which are nonionic surfactants, are mild and
non-denaturing in character, but are ineffective to solubilize skin
tissue, CTAB, a cationic surfactant, is effective to solubilize
skin tissue, but unsuitably denatures proteins, profoundly changing
properties of biomolecules in solution, rendering them unusable for
functional purposes.
[0008] Along with providing a cornucopia of information, skin can
also be a host to myriad undesirable cosmetic conditions, such as
age spots, skin tags, seborrheic keratosis, sear tissues,
xanthomas, non-cancerous hyperproliferative conditions, surface
bumps, and scaly patches; and therapeutic conditions such as skin
tumors, actinic keratosis, leukoplakia, and surface cancers
relating to Barrett's esophagus and right-colon pre-cancer plaque.
For these conditions, solubilization and remodeling or removal may
be the primary concern, with or without subsequent diagnostic
processing.
[0009] A need exists for compositions for skin sampling, as well as
for mucosal membrane and other tissue sampling, which, when used in
conjunction with applied energy, at least partially solubilize such
skin, mucosal membrane, and other tissue. A further need exists to
preserve the functionality and structural integrity of analytes,
including biomolecules, obtained from the solubilized skin, mucosal
membrane, and other tissue.
SUMMARY
[0010] In one embodiment, a composition is provided, the
composition comprising: [0011] a zwitterionic surfactant,
comprising:
[0011] ##STR00001## [0012] wherein n=10, 12, 14, 16, or 18; and
[0013] a non-ionic surfactant, comprising:
[0013] H(CH.sub.2).sub.nO(CH.sub.2CH.sub.2O).sub.bH [0014] wherein
a=12 or 16, and wherein b=2, 4, 10, 20, or 23.
[0015] In one embodiment, n=12 (corresponding to
N-dodecyl-N,N-dimethyl-3ammonio-1-propanesulfonate or "DDPS") a=16,
and b=10 (corresponding to polyoxyethylene (10) cetyl ether or
"Brij C10").
[0016] In one embodiment, n=14 (corresponding to
N-tetradecyl-N,N-dimethyl-3ammonio-1-propanesulfonate or "TPS"),
a=12, and b=4 (corresponding to polyoxyethylene (4) lauryl ether or
"Brij 30"). In another embodiment, n=14 (TPS), a=16, and b=2
(corresponding to polyoxyethylene (2) cetyl ether or "Brij 52"). In
another embodiment, n=14 (TPS), a=16, and b.ltoreq.10 (Brij
C10).
[0017] In one embodiment, n=16 (corresponding to
N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate or "HPS"),
a=12, and b=4 (Brij 30). In another embodiment, n=16 (HPS), a=16,
and b=2 (Brij 52). In another embodiment, n=16 (HPS), a=16, and
b=10 (Brij C10).
[0018] In one embodiment, n=18 (corresponding to
N-octadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate or "OPS"),
a=12, and b=4 (Brij 30). In another embodiment, n=18 (OPS), a=16,
and b=2 (Brij 52). In another embodiment, n=18 (OPS), a=16, and
b=10 (Brij C10). In another embodiment, n=18 (OPS), a=16, and b=20
(corresponding to polyoxyethylene (20) cetyl ether or "Brij
58").
[0019] In one embodiment, a method is provided for solubilizing and
remodeling and/or removing tissue on or beneath a patient's skin,
comprising optionally applying energy to a region of interest on
the skin; and contacting the region with a tissue solubilizing
composition,
[0020] In another embodiment, a method for recovering analytes from
mucosal membrane, skin, or other tissue is provided, the method
comprising: optionally applying energy to a region of interest on
the mucosal membrane, skin, or other tissue containing at least one
analyte; contacting the region with a tissue solubilizing
composition, thereby solubilizing at least some of the mucosal
membrane, skin, or other tissue containing at least one analyte;
and collecting the at least one analyte from the solubilized
mucosal membrane, skin, and other tissue.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] In the accompanying figures, experimental data are given
that, together with the detailed description provided below,
describe example embodiments of the claimed invention.
[0022] FIG. 1 illustrates in graphical form the total protein
(mg/ml) recovered from porcine skin when the porcine skin is
contacted with various combinations of zwitterionic and nonionic
surfactants in the presence of ultrasound.
[0023] FIG. 2 illustrates in graphical form the soluble protein
(mg/ml) recovered from porcine skin when the porcine skin is
contacted with various combinations of zwitterionic and nonionic
surfactants in the presence of ultrasound.
[0024] FIG. 3 illustrates the dependence on concentration of the
protein extraction efficacy from porcine skin of TPS:Brij C10.
[0025] FIG. 4 illustrates in graphical form the preserved activity
of the intracellular enzyme glyceraldehyde 3-phosphate
dehydrogenase (GAPDH) in various combinations of zwitterionic and
nonionic surfactants.
[0026] FIG. 5 illustrates in graphical form the combined dependence
of soluble protein extraction efficacy and GAPDH stability in
various combinations of zwitterionic and nonionic surfactants.
[0027] FIG. 6 illustrates the total protein recovery from Human
Epidermal Keratinocyte (HEK) cells at t=0 (solid bars), t=4 hours
at 4.degree. C. (diagonal bars), and t=4 hours at RT (cross-hatched
bars), using various reagents.
[0028] FIG. 7 illustrates the GAPDH activity measured in HEK cells
solubilized in various reagents, at t=0 (solid bars), t=4 hours at
4.degree. C. (diagonal bars), and t=4 hours at RT (cross-hatched
bars).
[0029] FIG. 8 illustrates the GAPDH activity measured in HEK cells
solubilized in various reagents, at room temperature at t=4 hours
(solid bars), t=24 hours (diagonal bars), 72 hours (cross-hatched
bars), and 7 days (horizontal bars).
[0030] FIG. 9 illustrates the total protein recovered from
homogenized mouse skin using various reagents.
[0031] FIG. 10 illustrates the specific GAPDH activity measured in
homogenized mouse skin using various reagents.
DETAILED DESCRIPTION
[0032] In one embodiment, a composition is provided, the
composition comprising: [0033] a zwitterionic surfactant,
comprising:
[0033] ##STR00002## [0034] wherein n=10, 12, 14, 16, or 18; and
[0035] a non-ionic surfactant, comprising;
[0035] H(CH.sub.2).sub.aO(CH.sub.2CH.sub.2O).sub.bH [0036] wherein
a=12 or 16, and wherein b=2, 4, 10, 20, or 23.
[0037] In one embodiment, the zwitterionic surfactant comprises DPS
and the nonionic surfactant comprises at least one of Brij 30, Brij
35, Brij 52, Brij C10, and Brij 58. In one embodiment, the
combination of DPS:Brij 30 is excluded.
[0038] In another embodiment, the zwitterionic surfactant comprises
DDPS and the nonionic surfactant comprises at least one of Brij
301, Brij 35, Brij 52, Brij C10, and Brij 58.
[0039] In another embodiment, the zwitterionic surfactant comprises
TPS and the nonionic surfactant: comprises at least one of Brij 30,
Brij 35, Brij 52, Brij C10, and Brij 58.
[0040] In another embodiment, the zwitterionic surfactant comprises
HPS and the nonionic surfactant comprises at least one of Brij 30,
Brij 35, Brij 52, Brij C10, and Brij 58. In one embodiment, the
combination of HPS:Brij 35 is excluded.
[0041] In another embodiment, the zwitterionic surfactant comprises
OPS and the nonionic surfactant comprises at least one of Brij 30,
Brij 35, Brij 52, Brij C10, and Brij 58.
[0042] The zwitterionic surfactant and the nonionic surfactant may
be dissolved in a buffer solution. The buffer solution may
comprise, for example, one or more of phosphate-buffered saline
(PBS) (pH=7.2-7.6), tris-buffered saline (pH=7.4-8.0),
tris-hydrochloride (7.0-9.0), and ethylenediaminetetraacetic acid
(EDTA) (pH=7.4-9.0). Thus, the surfactant combination may have a pH
of greater than about 7.0 in buffer solution, between about 7.0 and
9.0 in buffer solution, between about 7.4 and 9.0 in buffer
solution, between about 7.4 and 8.0 in buffer solution, and between
about 7.2 and 7.6 in buffer solution..
[0043] The zwitterionic surfactant and the nonionic surfactant may
be present In a total concentration of between about 0.01% and
about 10% (w/v) in the buffer solution. For example, the
zwitterionic surfactant and the nonionic surfactant may be present
in a total concentration of about 0.01% to about 5% (w/v) in the
buffer solution, including total concentrations of about 0.1% (w/v)
to about 2% (w/v) in the butler solution, about 1% (w/v) in the
buffer solution, and about 0,1% (v/v) to about 0.5% (w/v) in the
buffer solution. In one embodiment, the zwitterionic surfactant and
the nonionic surfactant are present in a total concentration of
about 0.5% (w/v) in the buffer solution. In another embodiment, the
zwitterionic surfactant and the nonionic surfactant may be present
in a ratio of about 3:1 to about 1:3 or about 3:2 to about 2:3. In
one embodiment, the zwitterionic surfactant and the nonionic
surfactant may be present in a ratio of about 1:1.
[0044] The zwitterionic surfactant:nonionic surfactant composition
may have several applications.
[0045] For example, in one embodiment, a method is provided for
solubilizing and remodeling and/or removing tissue on or beneath a
patient's skin, comprising applying energy to a region of interest
on the skin; and contacting the region with a tissue solubilizing
composition comprising at least one of: (1) DPS and at least one of
Brij 30, Brij 35, Brij 52, Brij C10, and Brij 58; (2) DDPS and at
least one of Brij 30, Brij 35, Brij 52, Brij C10, and Brij 58; (3)
TPS and at least one of Brij 30, Brij 35, Brij 52, Brij C10, and
Brij 58; (4) HPS and at least one of Brij 30, Brij 35, Brij 52,
Brij C10, and Brij 58; and (5) OPS and at least one of Brij 30,
Brij 35, Brij 52, Brij C10, and Brij 58.
[0046] In another embodiment, a method for recovering analytes from
mucosal membrane, skin, or other tissue is provided, the method
comprising; applying energy to a region of interest on the mucosal
membrane, skin, or other tissue containing at least one analyte;
contacting the region with a tissue solubilizing composition, the
tissue solubilizing composition comprising at least one of: (1) DPS
and at least one of Brij 30, Brij 35, Brij 52, Brij C10, and Brij
58; (2) DDPS and at least one of Brij 30, Brij 35, Brij 52, Brij
C10, and Brij 38; (3) TPS and at least one of Brij 30, Brij 35,
Brij 52, Brij C10, and Brij 58; (4) HPS and at least one of Brij
30, Brij 35, Brij 52, Brij C10, and Brij 58; and (5) OPS abd at
least one of Brij 30, Brij 35, Brij 52, Brij C10, and Brij 58,
thereby solubilizing at least some of the mucosal membrane, skin,
or other tissue containing: at least: one analyte; and collecting
the at least one analyte from the solubilized mucosal membrane,
skin, and other tissue.
[0047] In some embodiments, "other tissue" may include breast,
prostate, eye, vagina, bladder, nail, hair, colon, testicles,
intestine, lung, brain, pancreas, liver, heat, bone, or aorta
wall.
[0048] In some embodiments, an "analyte" may include any
biomolecule, drag, small molecule, warfare agent, environmental
contaminant, microbe, and the like that is present in or on the
tissue and can be extracted from the tissue of interest.
[0049] In some embodiments, "biomolecules" may include proteins
(e.g., disease biomarkers such as cancer biomarkers, antibodies:
IgE, IgG, IgA, IgD, or IgM, and the like), peptides, lipids (e.g.,
cholesterol, ceramides, and fatty acids), nucleic acids (e.g., RNA
and DNA), small molecules (e.g., glucose, urea, and creatine),
small molecule drugs or metabolites of small molecule drugs,
microbes, inorganic molecules, elements, and ions (e.g., iron,
Ca.sup.2+, K.sup.+, Na.sup.+, and the like). In some embodiments,
the bimolecule is exclusive of glucose and cancer markers.
[0050] In some embodiments, "drugs" may include abused drags, such
as. for example, cocaine, heroin, methyl amphetamine, and
prescription drugs taken in excess of dosage, or taken without a
prescription (e.g., painkillers such as opioids); and therapeutic
drugs, such as, for example, tramadol, fluconazole, barbitals, and
anabolic steroids.
[0051] In some embodiments, "warfare agents" may include any
molecule, compound, or composition of either biological or chemical
origin that may be used as a weapon. Non-limiting examples of
warfare agents include explosives, nerve gases (e.g., VX and
Sarin), phosgene, toxins, spores (e.g., anthrax), and the like.
[0052] In some embodiments, "environmental contaminants" may
include any molecule, compound, or composition that can be
detrimental to an individual, e.g., when at concentrations elevated
above a risk threshold. Examples include water pollutants (e.g.,
fertilizers, pesticides, fungicides, insecticides, herbicides,
heavy metals, and halides), soil pollutants (e.g., fertilizers,
pesticides, fungicides, insecticides, herbicides, heavy metals, and
halides), and air pollutants (e.g., NO.sub.x, SO.sub.x, greenhouse
gases, persistent organic pollutants, particulate matter, and
smog).
[0053] In some embodiments, solubilizing the target cells and
tissue includes the application of energy. In some embodiments, the
energy may be applied by any number of suitable methods, including
mechanical (e.g., abrasion, shear, vacuum, pressure, suction,
ultrasound), optical (e.g., laser), thermal, and electrical energy.
However, in one embodiment, the energy does not include externally
supplied thermal energy (i.e., heat). Suitable energy applicators
are disclosed in U.S. patent application Ser. Nos. 12/664,994,
13/126,105, and 13/095,771, each of which is incorporated by
reference herein in its entirety.
[0054] The compositions may be used for solubilizing cells for in
vitro protein recovery. In addition to effective dissolution of
cells, the compositions may provide a benefit of preservation of
bioactivity. The compositions may also possess the ability to
quickly solubilize various tissues, including those with durable
mechanical properties, such as skin. To aid in the preservation of
bioactivity, a protease inhibitor may be included in the
compositions. However, with or without the addition of protease
inhibitors, the compositions may be able to preserve the biological
activity of proteins. The compositions are also applicable in vivo.
In particular, the compositions may be able to recover labile
phosphoproteins with RPPA, thus opening the possibility of quickly
and noninvasively probing multiple signaling pathways.
[0055] In one embodiment, the compositions may be useful as
antibacterial compositions. Thus, a method for inhibiting the
growth and reproduction of bacteria and/or treating a bacterial
infection is provided, the method comprising applying an
antibacterial composition to an area that is subject to attack by
the bacteria, the antibacterial composition comprising at least one
of: (1) DPS and at least one of Brij 30, Brij 35, Brij 52, Brij
C10, and Brij (2) DPPS and at least one of Brij 30, Brij 35, Brij
52, Brij C10, and Brij 58; (3) TPS and at ketone of Brij 30, Brij
35, Brij 52, Brij C10, and Brij 58; (4) HPS and at least one of
Brij 30, Brij 35, Brij 52, Brij C10, and Brij 58; and (5) OPS and a
least one of Brij 30, Brij 35, Brij 52, Brij C10, and Brij 58. The
method may further include the application of energy.
[0056] In another embodiment, a method is provided for solubilizing
cells and/or tissues, the method comprising contacting the cells
and/or tissues with a composition, the composition comprising at
least one of: (I) DPS and at least one of Brij 30, Brij 35, Brij
52, Brij C10, and Brij 58; (2) DDPS and at least one of Brij 30,
Brij 35, Brij 52, Brij C10, and Brij 58; (3) TPS and at least one
of Brij 30, Brij 35, Brij 52, Brij C10, and Brij 58; (4) HPS and at
least one of Brij 38, Brij 35, Brij 52, Brij C10, and Brij 58; and
(5) OPS and at least one of Brij 30, Brij 35, Brij 52, Brij C10,
and Brij 58, In one embodiment, proteins, including cytosolic
proteins, nuclear proteins, and surface proteins may be recovered
from the solubilized cells and/or tissues. In some embodiments,
such as, for example, embodiments where it is desirable to preserve
biological activity of the proteins, the composition may further
optionally comprise a protease inhibitor.
[0057] In one embodiment, the compositions may be used to probe
protein functional states and related skin cell signaling pathways.
Skin cell signaling pathways may be stress-induced, and may change
over minutes to hours. Phosphorylation is a highly labile
post-translational modification that regulates many aspects of
protein function. The ability to probe these functional states in
the epidermis necessitates a fast and efficient method to
solubilize and isolate phosphoproteins. Thus, in another
embodiment, a method is provided for recovering signaling proteins
from skin cells, the method comprising: contacting the skin cells
with a composition comprising at least one of (1) DPS and at least
one of Brij 30, Brij 35, Brij 52, Brij C10, and Brij 58; (2) DDPS
and at least one of Brij 30, Brij 35, Brij 52, Brij C10, and. Brij
58; (3) TPS and at least one of Brij 30, Brij 35, Brij 52, Brij
C10, and Brij 58; (4) HPS and at least one of Brij 30, Brij 35,
Brij 52, Brij C10, and Brij 58; and (5) OPS and at least one of
Brij 30, Brij 35, Brij 52, Brij C10, and Brij 58, to provide
solubilized signaling proteins; a mi subjecting the solubilized
signaling proteins to reverse phase protein array.
[0058] In one embodiment, the compositions comprising at least one
of: (1) DPS and at least one of Brij 30, Brij 35, Brij 52, Brij
C10, and Brij 58; (2) DDPS and at least one of Brij 30, Brij 35,
Brij 52, Brij C10, and Brij 58; (3) TPS and at least one of Brij
30, Brij 35, Brij 52, Brij C10, and Brij 58; (4) HPS and at least
one of Brij 30, Brij 33, Brij 52, Brij C10, and Brij 58; and (5)
OPS and at least one of Brij 30, Brij 35, Brij 52, Brij C10, and
Brij 58, may be useful for, among other things, disaggregating,
solubilizing, and stabilizing cell components to be used as disease
biomarkers, forensic biornarkers, or both, in one embodiment, the
compositions are useful to disaggregate, solubilize, and stabilize
components from living tissues in situ, from freshly resected
tissues, frozen resected tissues, preserved paraffin embedded
tissues, tissue and cell extracts and cultured cells derived from
cell fines or resected tissues, and from exogenous agents such as
viruses, bacteria, and prions.
[0059] In one embodiment, the compositions comprising at least one
of: (1) DPS and at least one of Brij 30, Brij 35, Brij 52, Brij
C10, and Brij 58; (2) DDPS and at least one of Brij 30, Brij 35,
Brij 52, Brij C10, and Brij 58; (3) TPS and at least one of Brij
30, Brij 35, Brij 52, Brij C10, and Brij 58; (4) HPS and at least
one of Brij 30, Brij 35, Brij 52, Brij C10, and Brij 58; and (5)
DPS and at least one of Brij 30, Brij 35, Brij 52, Brij C10, and
Brij 58, may be useful to solubilize, remodel, and remove diseased
tissue on or beneath the surface of the skin, or elsewhere within
the body. For example, the compositions may be useful to
solubilize, remodel, and remove tissue hosting precancerous
conditions such as actinic keratosis, leukoplakia, Barretts
esophagus, and right-colon pre-cancer plaque, and surface cancers
arising from-any of these precancerous conditions. Other
therapeutic uses may include solubilizing and removing tumors from
a variety of surface or deep sites, or treating tumor surgical
margins to remove any residual tumor ceils at these sites. In some
instances, after treating tumors with the compositions and
solubilizing the constituent tumor markers, the immune system may
detect the dissolved tumor markers and initiate a potent anti-tumor
immune response against these markers, leading to regression of the
local tumor, as well as destruction of any systemic tumor cells
carrying the detected tumor markers.
[0060] In one embodiment, the compositions comprising at least one
of: (1) DPS and at least one of Brij 30, Brij 35, Brij 52, Brij
C10, and Brij (2) DDPS and at least one of Brij 30, Brij 35, Brij
52, Brij C10, and Brij 58; (3) TPS and at least one of Brij 30,
Brij 35, Brij 52, Brij C10, and Brij: 58; (4) HPS and at least one
of Brij 30, Brij 35, Brij 52, Brij C10, and Brij 58; and (5) OPS
and at least one of Brij 30, Brij 35, Brij 52, Brij C10, and Brij
58, may be useful for treating skin lesions and damaged skin with
therapeutic molecules and drugs that are unable to penetrate an
intact outer skin harrier. More particularly, the compositions
described herein may be useful to enhance absorption of topical
therapeutics by removing diseased tissue, inflammatory cells, and
thickened, hyper-keratinized skin that may block access to
otherwise effective topical therapies. An example of such a use is
as a pre-treatment for psoriasis topical therapies, since psoriasis
lesions typically have a hardened top layer or hyperkeratosis that
inhibits absorption. In one embodiment, such treatment may Include
perturbation of the outer skin barrier using the compositions, and
in some circumstances applied energy, to disrupt the barrier by
disaggregation and solubilization of barrier cells and tissues,
followed by application of the therapeutic molecules and drugs
directly to the surface of the barrier-perturbed skin. Example
therapeutic molecules and drugs may include, for example, DNA-based
drugs, RNA-based drugs, protein-based drugs, peptide-based drugs,
lipid-based drugs, carbohydrate-based drugs, small molecule drugs,
nanoparticle based drugs, liposome-encapsulated drags, and
combinations of such classes of drugs.
[0061] In one embodiment, the compositions comprising at least one
of: (1) DPS and at least one of Brij 30, Brij 35, Brij 52, Brij
C10, and Brij 58; (2) DDPS and at least one of Brij 30, Brij 35,
Brij 52, Brij C10, and Brij 58; (3) TPS and at least one of Brij
30, Brij 35, Brij 52, Brij C10, and Brij 58; (4) HPS and at least
one of Brij 30, Brij 35, Brij 52, Brij C10, and Brij 58; and (5)
OPS and at least one of Brij 30, Brij 35, Brij 52, Brij C10, and
Brij 58, may be useful for introducing therapeutic or diagnostic
molecules and drugs into the body and bloodstream by disrupting the
outer skin layer. In one embodiment, the introducing may include
perturbation of the outer skin barrier using the compositions, and
in some circumstances applied energy, to disrupt the barrier by
disaggregation and solubilization of barrier cells and tissues,
followed by application of the therapeutic or diagnostic molecules
directly to the surface of the barrier-perturbed skin.
[0062] In another embodiment, the compositions comprising at least
one of: (1) DPS and at least one of Brij 30, Brij 35, Brij 52, Brij
C10, and Brij 58; (2) DDPS and at least one of Brij 30, Brij 35,
Brij 52, Brij C10, and Brij 58; (3) TPS and at least one of Brij
30, Brij 35, Brij 52, Brij C10, and Brij 58; (4) HPS and at least
one of Brij 30, Brij 35, Brij 52, Brij C10, and Brij 58; and (5)
OPS and at least one of Brij 30, Brij 35, Brij 52, Brij C10, and
Brij 58, may be useful to remove malignant and benign growths and
obstructions in other organs in the body or within the central and
peripheral nervous systems, including the eye, middle ear, brain,
spinal cord, nerve roots, and ganglia. Since the compositions may
dissociate and dissolve diseased tissue directly after injection
through a thin needle or catheter, the compositions may allow
ablative surgery in areas that are not accessible to either open
surgery or even to minimally invasive surgical instruments (such as
in the vascular system. Including arteries and coronary
arteries).
[0063] In another embodiment* the compositions comprising at least
one of: (1) DPS and at least one of Brij 30, Brij 35, Brij 52, Brij
C10, and Brij 58; (2) DDPS and at least one of Brij 30, Brij 35,
Brij 52, Brij C10, and Brij SB; (3) TPS and at least one of Brij
30, Brij 35, Brij 52, Brij C10, and Brij 58: (4) HPS and at least
one of Brij 30, Brij 35, Brij 52, Brij C10, and Brij 58; and (5)
OPS and at least one of Brij 30, Brij 35, Brij 52, Brij C10, and.
Brij 58, may be useful to diminish or reduce intra-abdominal and
peritoneal adhesions by dissolving specific bands of adherent
tissue between intra-abdominal tissues and organs.
[0064] In one embodiment, the compositions comprising at least one
of: (1) DPS and at least one of Brij 30, Brij 35, Brij 52, Brij
C10, and Brij 58; (2) DDPS and at least one of Brij 30, Brij 35,
Brij 52, Brij C10, and Brij 58; (3) TPS and at least one of Brij
30, Brij 35, Brij 52, Brij C10, and Brij 58; (4) HPS and at least
one of Brij 30, Brij 35, Brij 52, Brij C10, and Brij 58; and (5)
OPS and at least one of Brij 30, Brij 35, Brij 52, Brij C10, and
Brij 51, may be useful for wound debridement. In one embodiment,
the treatment may include contacting the compositions, and in some
circumstances applied energy, to a wound's surrounding tissue to
remove unhealthy tissue, including, for example, necrotic eschar
and fibrinous slough.
[0065] In one embodiment, the compositions comprising at least one
of: (1) DPS and at least, one of Brij 30, Brij 35, Brij 52, Brij
C10, and Brij 58; (2) DDPS and at least one of Brij 30, Brij 35,
Brij 52, Brij C10, and Brij 58; (3) TPS and at least one of Brij
30, Brij 35, Brij 52, Brij C10, and Brij 58; (4) HPS and at least
one of Brij 30, Brij 35, Brij 52, Brij C10, and Brij 58; and (5)
OPS and at least one of Brij 30, Brij 35, Brij 52, Brij C10, and
Brij 58, may be useful as a bio-glue to enhance post-operation
healing.
[0066] In one embodiment, the compositions comprising at least one
of: (1) DPS and at least one of Brij 30, Brij 35, Brij 52, Brij
C10, and Brij 58; (2) DDPS and at least one of Brij 30, Brij 35,
Brij 52, Brij C10, and Brij 58; (3) TPS and at least one of Brij
30, Brij 35, Brij 52, Brij C10, and Brij 58; (4) HPS and at least
one of Brij 30, Brij 35, Brij 52, Brij C10, and Brij 58; and (5)
OPS and at least one of Brij 30, Brij 35, Brij 52, Brij C10, and
Brij 58, may be useful to promote oral and dental hygiene. For
example, in one embodiment, the compositions may be useful to
soften and/or dissolve hard, and soli deposits on teeth and
dentures.
[0067] In one embodiment, the compositions comprising at least one
of: (1) DPS and at least one of Brij 30, Brij 35, Brij 52, Brij
C10, and Brij 58; (2) DDPS and at least one of Brij 30, Brij 35,
Brij 52, Brij C10, and Brij 58; (3) TPS and at least one of Brij
30, Brij 35, Brij 52, Brij C10, and Brij 58; ( 4) HPS and at least
one of Brij 30, Brij 35, Brij 52, Brij C10, and Brij 58; and (5)
OPS and at least erne of Brij 30, Brij 35, Brij 52, Brij C10, and
Brij 58, may be useful to solublilize, remodel, and remove
cosmetically relevant structures on or beneath the surface of the
skin. For example, is one embodiment, the compositions may be
useful for treating aged, scarred, and UV-damaged skin, and
removing and/or remodeling age spots, skin tags, seborrheic
keratosis, scar tissues, xanthomas, noncancerous hyperproliferative
conditions, surface bumps, and scaly patches. In one embodiment,
the use may include perturbation of the outer skin barrier using
the compositions, and in some circumstances applied energy, to
disrupt the harrier by disaggregation and solubilization of barrier
cells and tissues. In one embodiment, the compositions may be
introduced to deeper layers of skin to facilitate disaggregation,
solubilization, and removal of structures associated with
wrinkling, scarring, or both, of the skin surface. Other cosmetic
uses include dermal peel or skin bleaching. The compositions may be
used to remove discoloration of the skin associated with previous
injury, UV-damage, or aging. In some embodiments, the perturbation
may be followed by application of therapeutic drags or natural
products and other cosmetic compositions that are believed or known
to increase the smoothness, elasticity, and resilience of skin.
Such cosmetic compositions may include, for example, elastin or its
peptides (e.g., V-V-P-Q), collagen or its peptides, resveratrol,
idebenone, co-enzyme Q10, acetyl hexapeptide-3, glycosaminoglycans,
palmitoyl pentapeptide-4, sodium hyaluronate, and the like, and
combinations thereof.
[0068] In one embodiment, cell components recovered using the
compositions may be Introduced into biochemical assays to detect,
quantify, and identify specific biomarkers associated with specific
diseases. The biochemical assays may Include all molecular
diagnostic, assays for detecting DNA, RNA, proteins, peptides,
lipids, carbohydrates, and small molecules, both endogenous and
exogenous. The biochemical assays to be used include PCR, ELISA,
chromatography, gel analysis, electrophoresis. Western Blots,
Southern Blots, Northern Blots, and other methods used in clinical
laboratories for identification of molecular biomarkers of
disease.
[0069] In one embodiment, the compositions comprising at least one
of: (1) DPS and at least one of Brij 30, Brij 35, Brij 52, Brij
C10, and Brij 58; (2) DDPS and at least one of Brij 30, Brij 35,
Brij 52, Brij C10, and Brij 58; (3) TPS and at least one of Brij
30, Brij 35, Brij 52, Brij C10, and Brij 58; (4) HPS and at least
one of Brij 30, Brij 35, Brij 52, Brij C10, and Brij 58; and (S)
OPS and at least one of Brij 30, Brij 35, Brij 52, Brij C10, and
Brij 58, may be useful in the treatment of mucosal and skin
lesions. Such use may involve impregnating an abrasive and
absorbent swab (similar to a for-daily-use facial exfoliating
sponge) with one or more of the compositions, and applying the swab
to the region of interest with medium pressure in a twisting or
back-and-forth motion onto target tissue for a predetermined time.
The depth may be controlled by the amount of pressure, as well as
application time. After the lesion is dissolved and the released
biomarkers are absorbed into the pad, the swab and the dissolved
tissue may be collected and sent to, e.g., a pathology lab, for
biomarker and other analyte analysis.
EXAMPLES
[0070] Certain embodiments axe described below in the form of
examples. It is impossible to depict every potential application of
the invention. Thus, while the embodiments are described in
considerable detail, it is not the intention to restrict or in any
way limit the scope of the appended claims to such detail, or to
any particular embodiment.
Example 1
Materials
[0071] DPS, DDPS, TPS, HPS, and OPS were procured from
Sigma-Aldrich as zwitterionic surfactants, Brij 35, Brij 52, Brij
C10, and Brij 58 were procured from Sigma-Aldrich as nonionic
surfactants. Brij 30 was procured from TCI America. All solutions
were prepared by adding 025% w/v zwitterionic surfactant and 0.25%
w/v nonionic surfactant (0.25% v/v B30), for a total concentration
of 0.5% w/v in PBS (pH=7.4). A positive control was prepared by
placing 1% v/v Triton X-100 (TX-1) in PBS.
Example 2
Protein Recovery from Porcine Skin
[0072] Protein was recovered from porcine skin as a model tissue.
Skin was procured in frozen form from Lampire Biological
Laboratories Inc., and stored at 0.degree. C. Two hours before use,
skin was thawed at room temperature (RT) and cot into small pieces
(2.5 cm.times.2.5 cm). Skin pieces were stripped, off from
subcutaneous fat and used without visible scratches or abrasions.
Protein recovery was carried out by mounting the skin piece on a
Franz diffusion cell (FDC) assembly (tissue exposure area of 1.77
cm.sup.2; Permegear). The receiver chamber of the FDC was filled
with PBS and the donor chamber was filled with 1 mL of surfactant
combination as a sampling buffer. This buffer also acted as the
coupling fluid between the ultrasound transducer and the tissue.
Protein recovery was performed at RT with a 600-W probe sonicator
(Sonics Materials) operating at a frequency of 20 kHz. The
ultrasound transducer was placed at a distance of 5 mm from the
tissue surface and an ultrasonic intensity of 2.4 W/cm.sup.2 at 50%
duty cycle was applied for 3 min. The sampling buffer, now
containing solubilized tissue constituents, was aspirated and kept
at -70.degree. C. until analysis,
[0073] The solubilization ability for each surfactant formulation
disclosed herein was quantified by the concentrations of total
protein and solubilized protein (mg/ml). Supernatants were isolated
from the samples using a centrifuge operating at 10,000.times.g and
4.degree. C. for 15 min. The solubilized protein amount was
measured in the sample supernatant by using a colorimetric
defection kit (Micro BCA Protein Assay Kit; Pierce).
[0074] Table 1 illustrates the total protein (mg/ml) recovered from
porcine skin when the porcine skin is contacted with various
combinations of zwitterionic and nonionic surfactants in the
presence of ultrasound. FIG. 1 illustrates the same results in
graphical form.
TABLE-US-00001 TABLE 1 Brij 30 Brij 35 Brij 52 Brij C10 Brij 58 DPS
1.47 .+-. 0.26 1.67 .+-. 0.20 1.33 .+-. 0.46 1.44 .+-. 0.23 1.69
.+-. 0.33 DDPS 1.68 .+-. 0.25 1.90 .+-. 0.43 1.95 .+-. 0.44 2.11
.+-. 0.29 1.84 .+-. 0.23 TPS 1.95 .+-. 0.26 1.79 .+-. 0.05 2.23
.+-. 0.25 2.33 .+-. 0.36 1.52 .+-. 0.19 HPS 2.19 .+-. 0.05 1.68
.+-. 0.26 2.32 .+-. 0.29 1.98 .+-. 0.31 1.64 .+-. 0.39 OPS 2.15
.+-. 0.54 1.71 .+-. 0.19 2.22 .+-. 0.23 2.42 .+-. 0.36 1.88 .+-.
0.32
[0075] Table 2 illustrates the soluble protein (mg/ml) recovered
from porcine skin when the porcine skin is contacted with those
same combinations of zwitterionic and nonionic surfactants in the
presence of ultrasound. FIG. 2 illustrates the results in graphical
form.
TABLE-US-00002 TABLE 2 Brij 30 Brij 35 Brij 52 Brij C10 Brij 58 DPS
0.56 .+-. 0.04 0.99 .+-. 0.30 0.65 .+-. 0.03 0.91 .+-. 0.13 1.15
.+-. 0.07 DDPS 1.02 .+-. 0.12 1.27 .+-. 0.19 1.10 .+-. 0.12 1.44
.+-. 0.16 1.39 .+-. 0.22 TPS 1.40 .+-. 0.26 1.31 .+-. 0.15 1.63
.+-. 0.16 1.64 .+-. 0.33 1.16 .+-. 0.25 HPS 1.34 .+-. 0.27 1.29
.+-. 0.26 1.55 .+-. 0.13 1.45 .+-. 0.18 1.15 .+-. 0.29 OPS 1.27
.+-. 0.28 1.09 .+-. 0.14 1.06 .+-. 0.25 1.34 .+-. 0.20 1.14 .+-.
0.16
Example 3
Concentration Dependence
[0076] TPS:Brij C10 was investigated for concentration effect. The
surfactant concentration was tested at total concentrations of
0.5%, 1%, and 2% of TPS:Brij C10, at a 1:1 ratio. FIG. 3
illustrates the dependence on concentration of TPS:Brij C10 on the
soluble and total protein extraction efficacy from porcine
skin,
Example 4
Enzyme Protection
[0077] GAPDH from rabbit was procured as a model enzyme from
Sigma-Aldrich. GAPDH powder was dissolved in ddH.sub.2O (Milipore)
to 90 U/ml and distributed into an E-tube, then stored at
-80.degree.C. until use. Enzyme protection was carried out by
incubation of GAPDH in each, combination disclosed herein, compared
to that in PBS as a positive control The concentration of GAPDH was
fixed to 0.5 U/ml. GAPDH was incubated at 37.degree. C. for 10 min.
The activity of the GAPDH enzyme (U/ml) was assessed by KDalert.TM.
GAPDH Assay Kit (Ambion, Inc, TX, USA). FIG. 4 illustrates in
graphical form the preserved activity of GAPDH In each of the
various combinations of zwitterionic and nonionic surfactants.
[0078] GAPDH activities were multiplied by soluble protein
recovery. FIG. 5 illustrates in graphical form the combined
dependence of soluble protein, extraction efficacy and GAPDH
stability in each of the various combinations of zwitterionic and
nonionic surfactants.
Example 5
Cell Solubilization
[0079] HEK cells (HEKa-APF, Invitrogen, CA, USA) were cultured in a
Corning.RTM. cell culture treated flask with a Vent Cap (75
cm.sup.2 Rectangular Canted Neck, Corning). The HEK cells were
grown between passage 3 to 8 in the EpiLife.RTM. Medium with 60
.mu.M calcium added by Human Keratinocyte Growth Supplement FIG. 6
illustrates the total protein recovery from HEK cells at t=0 (solid
bars), t=4 hours at 4.degree. C. (diagonal bars), and t=4 hours at
RT (cross-hatched bars), using DPS:Brij 30, TPS:Brij C10, HPS:Brij
52, OPS:Brij 52, OPS:Brij C10, and DPS:Brij 58, as compared to
TX-1.
Example 6
In Vitro Assay
[0080] Total protein in solubilized cells was analyzed using a
Micro BCA Protein Assay Kit (Fisher Scientific, PA, USA).
Trypsinized cells were counted by hemocytometer (Fisher Scientific,
PA, USA) and added to 96-well plates. After overnight growth, the
medium w removed and replaced by 100 .mu.l of lysis agents (TX-1,
DPS:Brij 30, TPS:Brij C10, HPS:Brij 52, OPS:Brij 52, OPS:Brij C10,
and OPS:Brij 58). FIG. 7 illustrates the GAPDH activity measured in
HEK cells solubilized in those same reagents, at t=0 (solid bars),
t=4 hours at 4.degree. C. (diagonal bars), and t=4 hours at RT
(cross-hatched bars).
[0081] For further study of stability, the enzymes were incubated
for one day at RT, three days at RT, and seven days at RT. FIG. 8
illustrates the GAPDH activity measured at RT, at t=4 hours (solid
bars), t=24 hours (diagonal bars), t=72 hours(cross-hatched bars),
and 7 days (horizontal bars).
Example 7
Tissue Solubilization
[0082] Skin tissue was collected from euthanized mice and stored at
-80.degree. C. until use. About 10 mg of each tissue was placed in
a 2 ml tube. 400 .mu.l of chilled lysis boiler (TX-1, TPS:Brij C10,
HPS:Brij 52, and OPS:Brij C10) and protease inhibitor cocktail
tablets (Roche Applied Science, IN, USA) were added to the tube
(one tablet per 50 ml; one tablet contains Antipain-dihydrochloride
3 mg, Aprotinin 0.5 mg, Bestatin 0.5 mg, Chymostatin 1 mg, E-64 3
mg, EDTA-Na.sub.2 10 mg, Leupeptin 0.5 mg, Pefabloc SC 20 mg,
Pepstatin 0.5 mg, and Phosphoramidon 3 mg). Tissue was homogenized
using: a homogenizer (IKA, NC, USA) at 13,000 rpm for 1 min. FIG. 9
illustrates the total protein recovered from homogenized mouse skin
using TPS:Brij C10, HPS:Brij 52, and OPS:Brij C10, as compared to
TX-1, FIG. 10 Illustrates the specific GAPDH activity measured in
the homogenized mouse skin.
[0083] To the extent that the term "includes" or "including" is
used in the specification or the claims, it is intended to be
inclusive in a manner similar to the term "comprising" as that term
is interpreted when employed as a transitional word in a claim, As
used in the specification and the claims, the singular forms "a,"
"an," and "the" include the plural. Furthermore, to the extent that
the term "or" is employed (e.g., A or B), it is intended to mean "A
or B or both." Finally, where the term "about" is used in
conjunction with a number, it is intended to include .+-.10% of the
number. In other words, "about 10" may mean from 9 to 11.
[0084] As stated above, while the present application has been
illustrated by the description of embodiments thereof, and while
the embodiments have been described in considerable detail, it is
not the intention of the applicants to restrict or in my way limit
the scope of the appended claims to such detail. Additional
advantages and modifications will readily appear to those skilled
in the art, with the benefit of the disclosure provided in this
application. Therefore, the application, in its broader aspects, is
not limited to the specific details, illustrative examples shown,
or arty apparatus referred to. Departures may be made from such
details, examples, and apparatuses without departing from the
spirit or scope of the general inventive concept.
* * * * *