U.S. patent application number 15/804013 was filed with the patent office on 2018-03-01 for ionically crosslinked polyelectrolytes as underwater adhesives and controlled release vehicles.
This patent application is currently assigned to The University of Toledo. The applicant listed for this patent is The University of Toledo. Invention is credited to Yan Huang, Yakov Lapitsky.
Application Number | 20180055942 15/804013 |
Document ID | / |
Family ID | 55453738 |
Filed Date | 2018-03-01 |
United States Patent
Application |
20180055942 |
Kind Code |
A1 |
Lapitsky; Yakov ; et
al. |
March 1, 2018 |
Ionically Crosslinked Polyelectrolytes as Underwater Adhesives and
Controlled Release Vehicles
Abstract
Underwater adhesive materials, methods of making the same, and
methods of using the same are described.
Inventors: |
Lapitsky; Yakov; (Toledo,
OH) ; Huang; Yan; (Toledo, OH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The University of Toledo |
Toledo |
OH |
US |
|
|
Assignee: |
The University of Toledo
Toledo
OH
|
Family ID: |
55453738 |
Appl. No.: |
15/804013 |
Filed: |
November 6, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
14639759 |
Mar 5, 2015 |
9814778 |
|
|
15804013 |
|
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|
61948907 |
Mar 6, 2014 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61L 2400/06 20130101;
A61L 26/0019 20130101; C09J 177/04 20130101; A61K 47/32 20130101;
A61L 24/0031 20130101; A61L 24/046 20130101; C08L 79/02 20130101;
C08G 73/0266 20130101; C09J 139/08 20130101; C09J 139/02 20130101;
A61L 26/008 20130101; A61L 24/0015 20130101; A61K 9/0024 20130101;
C09J 179/02 20130101; C08K 2003/321 20130101; C08K 3/32 20130101;
A61K 47/34 20130101; A61L 26/0066 20130101; C08G 73/0206 20130101;
A61L 26/0019 20130101; C08L 79/02 20130101; A61L 24/046 20130101;
C08L 79/02 20130101; C09J 179/02 20130101; C08K 3/32 20130101; C09J
177/04 20130101; C08K 3/32 20130101; C09J 139/02 20130101; C08K
3/32 20130101; C09J 139/08 20130101; C08K 3/32 20130101 |
International
Class: |
A61K 47/32 20060101
A61K047/32; C09J 179/02 20060101 C09J179/02; A61K 9/00 20060101
A61K009/00; C09J 139/08 20060101 C09J139/08; C09J 139/02 20060101
C09J139/02; C08L 79/02 20060101 C08L079/02; C09J 177/04 20060101
C09J177/04; A61K 47/34 20060101 A61K047/34; A61L 24/00 20060101
A61L024/00; C08K 3/32 20060101 C08K003/32; A61L 26/00 20060101
A61L026/00; A61L 24/04 20060101 A61L024/04 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with government support under Grant
Number 1133795 awarded by the National Science Foundation. The
government has certain rights in this invention.
Claims
1. A method for controllably releasing a small molecule comprising:
adding a small molecule to either (i) a first solution comprising
non-polysaccharide polymer having two or more amine groups, or (ii)
a second solution comprising a multivalent phosphate crosslinker;
mixing the first solution with the second solution, at a pH ranging
from about 4 to about 10, to form an adhesive gel; and applying the
adhesive gel to a target location, wherein the small molecule is
released from the adhesive gel over a period of time.
2. A method of delivering an adhesive to a target location
comprising: mixing a polyamine with a multivalent phosphate
crosslinker in acidic or basic solution to obtain a mixture;
injecting the mixture to a target location; and adjusting the pH of
the mixture to ambient pH to form an adhesive gel-like complex in
the target location.
3. The method of claim 2, further comprising adjusting the pH to an
acidic or basic pH to dissolve the adhesive gel.
4. A method of delivering an adhesive to a target location
comprising: mixing a polyamine with a multivalent phosphate
crosslinker in a solution to obtain a mixture; injecting the
mixture to a target location; and adjusting the ionic strength of
the solution to form an adhesive gel-like complex in the target
location.
5. The method of claim 4, further comprising adjusting the pH to an
acidic or basic pH to dissolve the adhesive gel.
6. A composition comprising: a non-polysaccharide polymer having
two or more amine groups; and a multivalent phosphate crosslinker;
the composition comprising an ionically crosslinked network that
forms a gel-like coacervate at a pH in the range of from about 4 to
about 10.
7. The composition of claim 6, wherein the multivalent phosphate
crosslinker is selected from the group consisting of a tetravalent
phosphate and a pentavalent phosphate.
8. The composition of claim 6, wherein the multivalent phosphate
crosslinker is selected from the group consisting of pyrophosphate
(PPi) and tripolyphosphate (TPP).
9. The composition of claim 6, wherein the polymer having two or
more amine groups comprises poly(vinylamine).
10. The composition of claim 6, wherein the polymer having two or
more amine groups is selected from the group consisting of:
poly(vinylamine), polylysine, polyarginine, polyhistidine,
polyethyleneimine, polyaminostyrene, polyvinylpyrrolidone,
polymethylvinylamine, polyaniline, and poly(vinylpyridine).
11. The composition of claim 6, wherein the composition has a
crosslinker:polymer molar ratio above about 0.12:1.
12. The composition of claim 6, wherein the composition has a
crosslinker:polymer molar ratio ranging from about 0.10:1 to about
0.25:1.
13. The composition of claim 6, wherein the polymer is present at a
concentration ranging from about 0.1 wt % to about 40 wt %.
14. The composition of claim 6, wherein the composition contains
water at a concentration ranging from about 20 wt % to about 50 wt
%.
15. The composition of claim 6, wherein the composition has a
storage modulus of greater than 10.sup.5 Pa.
16. The composition of claim 6, wherein the composition exhibits a
self-healing capability when torn.
17. The composition of claim 6, wherein the composition has a
tensile adhesion strength of greater than about 350 kPa.
18. The composition of claim 6, wherein the composition is capable
of adhering to hydrophobic and hydrophilic surfaces under
water.
19. The composition of claim 6, wherein the composition is capable
of adhering to human skin.
20. The composition of claim 6, further comprising an active
payload for long-term release.
Description
RELATED APPLICATIONS
[0001] This application is a divisional application of U.S. Ser.
No. 14/639,759 filed Mar. 5, 2015, now U.S. Pat. No. 9,814,778
issued Nov. 14, 2017, which claims priority to U.S. Provisional
Application Ser. No. 61/948,907 filed under 35 U.S.C. .sctn. 111(b)
on Mar. 6, 2014, the disclosure of which is incorporated herein by
reference in its entirety.
BACKGROUND OF THE INVENTION
[0003] Underwater adhesion has several potential medical,
household, and industrial applications. Underwater adhesives have
been prepared by methods that typically rely on in situ
polymerization, covalent crosslinking, or the use of highly
specialized biological or biomimetic polymers. There has been a
focus in the literature on biomimetic materials inspired by sessile
marine organisms such as zebra mussels, tubeworms, and barnacles.
Currently used strategies include the use of extracted or
recombinant proteins, reactive dopamine derivatives, or
coacervation and subsequent crosslinking of custom-designed
polymers, which mimic the molecular structure and, consequently,
the underwater adhesion properties of natural adhesive proteins.
Some biomimetic adhesion strategies have included mixing
polyelectrolytes with catechol-based crosslinkers, which convert
the polymer solutions into adhesive gels. Other strategies involve
synthesizing polyelectrolytes with crosslinkable catechol-based
sidechain groups, or designing biomimetic polyelectrolyte complexes
that undergo coacervation followed by either ionic or covalent
crosslinker (which converts the liquid coacervates into adhesive
solids).
[0004] Despite the current strategies, many challenges have limited
the yield and application of biomimetic adhesives. The use of
biological and biomimetic adhesion remains somewhat limited by the
high cost of natural proteins, inefficient recombinant protein
production, complicated syntheses, the use of potentially harmful
oxidants to induce crosslinking, and the need for
highly-specialized polymer structures Similarly, older adhesion
strategies such as epoxides, acrylic adhesives, and cyanoacrylate
gels, suffer from deficiencies such as relying on chemical
reactions to set the adhesive, being limited to a specific set of
adhesion substrates, forming underwater bonds that are permanent
and/or not self-healing upon failure, or requiring in situ
polymerization. There is a need in the art for additional and
improved underwater adhesives.
SUMMARY OF THE INVENTION
[0005] Provided herein is a composition comprising a
non-polysaccharide polymer having two or more amine groups, and a
multivalent phosphate crosslinker. The composition comprises an
ionically crosslinked network that forms a gel-like coacervate at a
pH ranging from about 4 to about 10. In certain embodiments, the
polymer is substantially linear. In certain embodiments, the
polymer is present at a concentration up to about 40 wt %. In
certain embodiments, the multivalent phosphate crosslinker is
selected from the group consisting of a tetravalent phosphate and a
pentavalent phosphate. In certain embodiments, the multivalent
phosphate crosslinker is not a single phosphate ion,
PO.sub.4.sup.3-, or salt thereof. In certain embodiments, the
multivalent phosphate crosslinker does not have more than 10
repeating units. In certain embodiments, the polymer having two or
more amine groups comprises poly(allylamine hydrochloride) (PAH).
In certain embodiments, the PAH has a molecular weight ranging from
about 5 kDa to about 800 kDa. In certain embodiments, the PAH has a
molecular weight ranging from about 1 kDa to about 2,500 kDa. In
certain embodiments, the PAH has a molecular weight ranging from
about 120 kDa to about 200 kDa. In certain embodiments, the
multivalent phosphate crosslinker is selected from the group
consisting of pyrophosphate (PPi) and tripolyphosphate (TPP). In
certain embodiments, the polymer having two or more amine groups
comprises poly(vinylamine). In certain embodiments, the polymer
having two or more amine groups is selected from the group
consisting of poly(vinylamine), polylysine, polyarginine,
polyhistidine, polyethyleneimine, polyaminostyrene,
polyvinylpyrrolidone, polymethylvinylamine, polyaniline, and
poly(vinylpyridine).
[0006] In certain embodiments, the composition has a
crosslinker:polymer molar ratio ranging from about 0.12:1 to about
0.33:1. In certain embodiments, the composition has a
crosslinker:polymer molar ratio ranging from about 0.10:1 to about
0.25:1. In certain embodiments, the polymer is present at a
concentration ranging from about 0.1 wt % to about 40 wt %. In
certain embodiments, the polymer is present at a concentration up
to about 10 wt %. In certain embodiments, the polymer is present at
a concentration up to about 30 wt %. In certain embodiments, the
polymer is present at a concentration up to about 40 wt %.
[0007] In certain embodiments, the polymer having one or more amine
groups consists essentially of PAH, and the multivalent phosphate
crosslinker consists essentially of PPi. In particular embodiments,
the composition has a PPi:PAH molar ratio greater than about
0.12:1. In particular embodiments, the composition has a PPi:PAH
molar ratio greater than about 0.25:1. In particular embodiments,
the composition has a PPi:PAH molar ratio of about 0.33:1. In
particular embodiments, the composition has a zeta potential of
below +23 mV. In particular embodiments, the composition has a zeta
potential of below +15 mV. In particular embodiments, the
composition has a zeta potential of below +11 mV.
[0008] In certain embodiments, the polymer having two or more amine
groups consists essentially of PAH, and the multivalent phosphate
crosslinker consists essentially of TPP. In particular embodiments,
the composition has a TPP:PAH molar ratio greater than about
0.10:1. In particular embodiments, the composition has a TPP:PAH
molar ratio greater than about 0.20:1. In particular embodiments,
the composition has a TPP:PAH molar ratio ranging from about 0.02:1
to about 10:1. In particular embodiments, the composition has a
TPP:PAH molar ratio ranging from about 0.19:1 to about 0.25:1. In
particular embodiments, the composition has a TPP:PAH molar ratio
of about 0.20:1. In particular embodiments, the concentration of
PAH monomer is above 1.7 mM. In particular embodiments, the
concentration of PAH monomer is above 6 mM. In particular
embodiments, the composition has a zeta potential of below +20 mV.
In particular embodiments, the composition has a negative zeta
potential. In particular embodiments, the composition is at a pH of
from about 6 to about 8. In particular embodiments, the composition
is at a pH of from about 4 to about 10.
[0009] In certain embodiments, the composition contains water at a
concentration ranging from about 20 wt % to about 50 wt %. In
certain embodiments, the composition contains water at a
concentration ranging from about 24 wt % to about 38 wt %. In
certain embodiments, the composition contains water at a
concentration ranging from about 25 wt % to about 30 wt %. In
certain embodiments, the composition has a storage modulus of
greater than about 400 kPa. In certain embodiments, the composition
exhibits a self-healing capability when torn. In certain
embodiments, the composition has a tensile adhesion strength of
greater than about 350 kPa.
[0010] In certain embodiments, the composition is capable of
adhering to hydrophobic surfaces. In certain embodiments, the
composition is capable of adhering to hydrophilic surfaces. In
certain embodiments, the composition is capable of adhering to
human skin. In certain embodiments, the composition is a soft gel.
In certain embodiments, the composition has an underwater adhesion
strength of greater than 10.sup.5 Pa. In certain embodiments, the
composition further comprises an active payload (such as a drug)
for long-term release.
[0011] Further provided herein is a method of making an adhesive
material comprising the steps of mixing a polyamine with a
multivalent phosphate crosslinker to obtain a mixture, allowing the
mixture to coagulate over a period of time to form a gel-like
complex and a supernatant, and separating the gel from the
supernatant to obtain an adhesive material. In certain embodiments,
the period of time is about 3 days. In certain embodiments, the
period of time is less than an hour. In certain embodiments, the
multivalent phosphate crosslinker consists essentially of a PPi
solution with a concentration ranging from about 5.4 mM to about
54.1 mM. In certain embodiments, the multivalent phosphate
crosslinker consists essentially of a TPP solution with a
concentration ranging from about 4.4 mM to about 43.5 mM. In
certain embodiments, the polyamine consists essentially of a PAH
solution with a concentration ranging from about 1.7 mM to about
17.1 mM. In certain embodiments, the multivalent phosphate
crosslinker has a concentration of about 7.5 wt %. In certain
embodiments, the polyamine has a concentration of about 10 wt %. In
certain embodiments, the method further comprises stirring the
mixture. In certain embodiments, the method further comprises
adjusting the pH to dissolve the adhesive material.
[0012] Further provided herein is a method of treating a wound
comprising the steps of applying a composition described herein to
a wound of a subject in need thereof, allowing the composition to
set in the wound for a period of time, and contacting the
composition with an agent having a pH of greater than 10 to
dissolve the composition.
[0013] Further provided herein is a method for controllably
releasing a drug (or other active ingredient) comprising the steps
of encapsulating a drug in a composition described herein, and
applying the composition to a target in need thereof.
[0014] Further provided herein is a method for controllably
releasing a small molecule, the method comprising the steps of:
adding a small molecule to either (i) a first solution comprising a
non-polysaccharide polymer having two or more amine groups, or (ii)
a second solution comprising a multivalent phosphate crosslinker;
mixing the first solution with the second solution, at a pH ranging
from about 4 to about 10, to form an adhesive gel; and applying the
adhesive gel to a target location, where the small molecule is
released from the adhesive gel over a period of time. In certain
embodiments, the method further comprises adjusting the pH of the
target location to an acidic or basic pH in order to dissolve the
adhesive gel.
[0015] Further provided herein is a method of delivering an
adhesive to a target location comprising the steps of mixing a
polyamine with a multivalent phosphate crosslinker in acidic or
basic solution to obtain a mixture, injecting the mixture to a
target location, and adjusting the pH of the mixture to ambient pH
to form an adhesive gel-like complex in the target location. In
certain embodiments, the method further comprises adjusting the pH
to an acidic or basic pH to dissolve the adhesive gel. In certain
embodiments, the method further comprises removing supernatant
solution from the target location.
[0016] Further provided herein is a method of delivering an
adhesive to a target location comprising the steps of mixing a
polyamine with a multivalent phosphate crosslinker in a solution to
obtain a mixture, injecting the mixture to a target location, and
adjusting the ionic strength of the solution to form an adhesive
gel-like complex in the target location. In certain embodiments,
the method further comprises adjusting the pH to an acidic or basic
pH to dissolve the adhesive gel. In certain embodiments, the method
further comprises removing supernatant solution from the target
location.
[0017] Further provided herein is a method of delivering an
adhesive to a target location comprising the steps of injecting a
polyamine to a target location, and injecting a multivalent
phosphate crosslinker to the target location to form an adhesive
gel. In certain embodiments, the method further comprises removing
supernatant solution from the target location.
[0018] Further provided herein is a kit for preparing an adhesive
gel comprising a first container housing a polymer having two or
more amine groups, and a second container housing a multivalent
phosphate crosslinker. In certain embodiments, the kit further
comprises a syringe.
[0019] Further provided herein is a kit for preparing an adhesive
gel comprising a first container housing a dispersion comprising a
polymer having two or more amine groups and a multivalent phosphate
crosslinker, and a second container housing a pH modifier. In
certain embodiments, the kit further comprises a syringe.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] The patent or application file may contain one or more
drawings executed in color and/or one or more photographs. Copies
of this patent or patent application publication with color
drawing(s) and/or photograph(s) will be provided by the U.S. Patent
and Trademark Office upon request and payment of the necessary
fees.
[0021] FIGS. 1A-1B: Phase maps of PAH/PPi (FIG. 1A) and PAH/TPP
(FIG. 1B) mixtures at pH 7.0 showing compositions where clear
molecular solutions (S), translucent colloidal dispersions (D), and
macroscopic adhesive gels (G) form. The solid lines are phase
boundaries, while the dotted lines represent counterion:monomer
molar ratios of 0.1:1, 0.2:1, and 0.3:1. The points near the solid
lines show the last data point before, and the first data point
after, each state transition. The visual appearance of each
aggregation state is illustrated in the panel at the bottom.
[0022] FIGS. 1C-1F: Photographs of the adhesive gels.
[0023] FIGS. 2A-2B: The evolution in PAH/PPi complex (FIG. 2A) and
PAH/TPP complex (FIG. 2B) .zeta.-potentials plotted versus the
ion:monomer molar ratio. The error bars are standard deviations,
and the line is a guide to the eye. The shaded region indicates the
composition where the colloidal complexes coagulate into
macroscopic gels.
[0024] FIGS. 3A-3B: Frequency sweep data comparing the G'
(.box-solid.) and G'' (.quadrature.) of the PAH/PPi (FIG. 3A) and
PAH/TPP (FIG. 3B) gels.
[0025] FIGS. 4A-4B: Representative dynamic rheology data
illustrating the self-healing properties of the PAH/PPi (FIG. 4A)
and PAH/TPP (FIG. 4B) gels.
[0026] FIGS. 5A-5B: Average tensile bond strengths of PAH/PPi and
PAH/TPP gels bonded to glass, PMMA, and Teflon.RTM. adhesion
substrates (FIG. 5A), and the percentage of their initial bond
strength retained plotted as a function of the adhesion/debonding
cycle number obtained using glass substrates (FIG. 5B). The error
bars show the standard deviations (n=6).
[0027] FIGS. 6A-6B: Frequency sweep data comparing the G'
(.box-solid.) and G'' (.quadrature.) of the PAH/citrate (FIG. 6A)
and PAH/phosphate (FIG. 6B) complexes.
[0028] FIGS. 7A-7B: Sustained release over time demonstrated with
Fast Green FCF (FIG. 7A) and Rhodamine B (FIG. 7B) dyes.
[0029] FIG. 8A: Exemplary illustration of an adhesive gel formed
from a mixture of PAH and TPP.
[0030] FIG. 8B: Exemplary illustration of the preparation of an
adhesive formed from a mixture of PAH and either PPi or TPP.
[0031] FIGS. 9A-9D: Photographs showing a 1.times.PBS and (blue)
magnetic stir bar in a test tube prior to injection of the
dispersion (FIG. 9A), the dispersion being injected into the
1.times.PBS (FIG. 9B), the dispersion/PBS mixture immediately after
injection (FIG. 9C), and the test tube inverted 5 minutes after the
injection with the magnetic stir bar adhered to the glass with a
thin layer of gel (FIG. 9D).
[0032] FIGS. 10A-10B: State diagrams at pH 7 showing the effect of
NaCl concentration on PAH/PPi (FIG. 10A) and PAH/TPP (FIG.
10B).
[0033] FIGS. 11A-11B: State diagrams showing the effect of pH on
PAH/PPi (FIG. 11A) and PAH/TPP (FIG. 11B).
[0034] FIGS. 12A-12D: Plots comparing the z-average hydrodynamic
diameters (FIGS. 12A-12B) and PDI-values (FIGS. 12C-12D) of the
PAH/PPi and PAH/TPP colloidal complexes (plotted as a function of
the ion:monomer molar ratio) at various PAH monomer
concentrations.
[0035] FIG. 13: Temporal size evolution of PAH/TPP complexes formed
at 0.26:1 (.box-solid.), 0.32:1 ( ), and 0.4:1 (.tangle-solidup.)
TPP:PAH molar ratios. The lines are guides to the eye.
[0036] FIGS. 14A-14D: State diagrams for PAH/PPi (FIG. 14A) and
PAH/TPP (FIG. 14B) mixtures plotted in terms of the parent PAH,
PPi, and TPP solution pH, and state diagrams for PAH/PPi (FIG. 14C)
and PAH/TPP (FIG. 14D) mixtures plotted in terms in terms of the pH
after mixing. The lines indicate the boundaries between the (S)
solution, (D) dispersion, and (G) gel-like aggregation states
recorded after 1 month of equilibration.
[0037] FIGS. 15A-15B: .zeta.-potentials of colloidal complexes
plotted as a function of the ion:monomer molar ratio for the
PAH/PPi (FIG. 15A) and PAH/TPP (FIG. 15B) mixtures prepared from
PAH, PPi, and TPP solutions at initial pH levels of (black circles)
6.0, (blue squares) 7.0, and (red triangles) 8.0. The lines are
guides to the eye.
[0038] FIGS. 16A-16B: G' (closed symbols) and G'' (open symbols)
for PAH/PPi (FIG. 16A) and PAH/TPP (FIG. 16B) adhesives prepared at
initial pH-levels of (black circles) 6.0, (blue squares) 7.0, and
(red triangles) 8.0 and final pH-levels summarized in Table 3.
[0039] FIGS. 17A-17B: Average tensile bond strengths of PAH/PPi
(FIG. 17A) and PAH/TPP (FIG. 17B) complexes prepared from PAH, PPi,
and TPP solutions at different pH levels and bonded to glass and
Teflon adhesion substrates. The error bars are standard
deviations.
[0040] FIG. 18: Average bond longevity achieved by the PAH/PPi and
PAH/TPP adhesives prepared using PAH, PPi, and TPP solutions at
various pH levels under a 17.8-kPa tensile stress when adhered to
glass. The error bars are standard deviations (n=3).
[0041] FIGS. 19A-19B: State diagrams for PAH/PPi (FIG. 19A) and
PAH/TPP (FIG. 19B) mixtures at NaCl concentrations between 0 and
500 mM after 1 month of equilibration. The data points show the
compositions of the samples tested, while the lines indicate the
boundaries between the solution (S), dispersion (D), and gel-like
aggregation states (G).
[0042] FIGS. 20A-20B: .zeta.-potentials of colloidal complexes
plotted as a function of the ion:PAH monomer molar ratio for the
PAH/PPi (FIG. 20A) and PAH/TPP (FIG. 20B) mixtures at (blue
squares) 0, (black circles) 150, and (red triangles) 500 mM NaCl
concentrations. The lines are guides to the eye.
[0043] FIGS. 21A-21B: G' (closed symbols) and G'' (open symbols)
for PAH/PPi (FIG. 21A) and PAH/TPP (FIG. 21B) adhesives prepared in
(blue squares) 0 mM NaCl, (black circles) 150 mM NaCl, and (red
triangles) 300 mM NaCl.
[0044] FIGS. 22A-22B: Average tensile bond strengths of the PAH/PPI
(FIG. 22A) and the PAH/TPP (FIG. 22B) complexes prepared at
different NaCl concentrations bonded to glass and Teflon adhesion
substrates. The error bars are standard deviations (n=6).
[0045] FIG. 23: The average bond longevity achieved by the PAH/PPi
and the PAH/TPP adhesives prepared at different NaCl concentrations
under a 17.8 kPa tensile stress when adhered to glass. The error
bars are standard deviations (n=3).
[0046] FIG. 24: The mass of the (.box-solid.) PAH/PPi and ( )
PAH/TPP adhesives, expressed as a percentage of their original mass
and plotted as a function of time. The lines are guides to the eye.
The error bars are standard deviations (n=3).
[0047] FIGS. 25A-25B: G' (closed symbols) and G'' (open symbols)
for PAH/PPi (FIG. 25A) and PAH/TPP (FIG. 25B) complexes tested ( ,
.smallcircle.) immediately after preparation in DI water and
(.box-solid., .quadrature.) after being stored in PBS for 2
weeks.
[0048] FIGS. 26A-26D: ITC data for Fast Green titrated into the
PAH/PPi (FIG. 26A) and PAH/TPP (FIG. 26B) dispersions, and
Rhodamine B titrated into the PAH/PPi (FIG. 26C) and PAH/TPP (FIG.
26D) dispersions.
[0049] FIG. 27: The LEs of the Rhodamine B-loaded (.box-solid.)
PAH/PPi and ( ) PAH/TPP adhesives plotted versus the PAH
concentration. The line is a guide to the eye. The error bars are
standard deviations (n=3).
[0050] FIGS. 28A-28D: Release profiles obtained from PAH/PPi (FIGS.
28A, 28C) and PAH/TPP (FIGS. 28B, 28D) complexes loaded with
(.box-solid.) 1 mg/ml and ( ) 4 mg/ml Fast Green dye plotted as (a,
b) percent of dye released (FIGS. 28A-28B) and total mass of
releases dye (FIGS. 28C-28D). The lines are guides to the eye. The
error bars are standard deviations (n=3).
[0051] FIGS. 29A-29B: The mass of the Fast Green FCF-loaded PAH/PPi
(FIG. 29A) and PAH/TPP (FIG. 29B) adhesives at initial Fast Green
FCF concentrations of (.box-solid.) 1 mg/ml and ( ) 4 mg/ml,
expressed as a percentage of their original mass and plotted as a
function of time. The lines are guides to the eye. The error bars
are standard deviations (n=3).
[0052] FIGS. 30A-30B: The mass of the Rhodamine B-loaded PAH/PPi
(FIG. 30A) and PAH/TPP (FIG. 30B) adhesives at initial Rhodamine B
concentrations of (.box-solid.) 1 mg/ml and ( ) 4 mg/ml, expressed
as a percentage of their original mass and plotted as a function of
time. The lines are guides to the eye. The error bars are standard
deviations (n=3).
[0053] FIGS. 31A-31D: Release profiles obtained from PAH/PPi (FIGS.
31A, 31C) and PAH/TPP (FIGS. 31B, 31D) complexes loaded with
(.box-solid.) 1 mg/ml and ( ) 4 mg/ml Rhodamine B dye plotted as
percent of dye released (FIGS. 31A-31B) and total mass of releases
dye (FIGS. 31C-31D). The lines are guides to the eye. The error
bars are standard deviations (n=3).
[0054] FIGS. 32A-32D: Release profiles obtained in tap water at
room temperature from PAH/PPi (FIGS. 32A, 32C) and PAH/TPP (FIGS.
32B, 32D) complexes loaded with (.box-solid.) 1 mg/ml and ( ) 4
mg/ml Fast Green FCF dye plotted as percent of dye released (FIGS.
32A-32B) and total mass of released dye (FIGS. 32C-32D). The lines
are guides to the eye. The error bars are standard deviations
(n=3).
[0055] FIGS. 33A-33D: Release profiles obtained in tap water at
room temperature from PAH/PPi (FIGS. 33A, 33C) and PAH/TPP
complexes (FIGS. 33B, 33D) loaded with (.box-solid.) 1 mg/ml and (
) 4 mg/ml Rhodamine B dye plotted as percent of dye released (FIGS.
33A-33B) and (c, d) total mass of released dye (FIGS. 33C-33D). The
lines are guides to the eye. The error bars are standard deviations
(n=3).
[0056] FIGS. 34A-34B: Release profiles obtained from 1-2 mm thick
(.box-solid.) PAH/PPi and ( ) PAH/TPP gel-like plugs loaded with 4
mg/ml Fast Green FCF (FIG. 34A) or Rhodamine B (FIG. 34B) plotted
as percent of dye released. The lines are guides to the eye. The
error bars are standard deviations (n=3).
DETAILED DESCRIPTION OF THE INVENTION
[0057] Throughout this disclosure, various publications, patents
and published patent specifications are referenced by an
identifying citation. The disclosures of these publications,
patents and published patent specifications are hereby incorporated
by reference into the present disclosure in their entirety to more
fully describe the state of the art to which this invention
pertains.
Definitions
[0058] For convenience, various terms used herein are defined prior
to further description of the various embodiments of the present
disclosure.
[0059] The term "self-healing" as used herein refers to the ability
of a material to repair damage caused by mechanical stress or usage
without external stimuli like heat, solvents, or plasticizers.
[0060] The term "polyphosphate" refers to a compound composed of
phosphate units linked by phosphoanhydride bonds.
[0061] The term "polyamine" refers to a compound having two or more
amine groups. As used herein, the term "polyamine" includes
polymers having monomer units with one amine group in each
monomer.
[0062] The term "polysaccharide" refers to a polymeric
carbohydrate.
[0063] The term "gel" as used herein includes gels, gel-like
complexes, gel-like adhesives, and gel-like coacervates.
General Description
[0064] Described herein are stiff gel-like complexes, also referred
to as adhesive gels, that adhere to dissimilar substrates
underwater, prepared from mixtures of readily-available
polyelectrolytes and multivalent counterions. Specifically, the
adhesive gels of the present disclosure are prepared by the
ionotropic gelation of polyamines with phosphate-bearing
multivalent anions. The adhesive gels form spontaneously and are
soft materials that are adhesive in both wet and dry environments.
Because the adhesive gels demonstrate a rheology in the presence of
salt that is not conventionally associated with a gel, the term
"gel-like coacervate" is sometimes used herein to refer to the
adhesive gels. Thus, it is understood that the present disclosure
is not limited by use of the term "gel."
[0065] In certain embodiments, the gel-like complexes deliver
underwater adhesion strengths that are comparable to those of zebra
mussels and barnacle adhesives, exhibit self-healing functionality,
and form through spontaneous self-assembly. In certain embodiments,
the gel-like complexes exhibit very high storage moduli
(G'.sub.28.about.400 kPa), self-heal when torn, and adhere to both
hydrophilic and hydrophobic substrates under water, with short-term
tensile adhesion strengths of 350-450 kPa. Surprisingly, these
gel-like complexes adhere to both hydrophilic and hydrophobic
substrates under water with tensile adhesive strength considerably
greater than that of Scotch Permanent Double Sided Tape (up to
.about.400 kPa vs. .about.85 kPa when used as a pressure-sensitive
adhesives). In certain embodiments, the gel-like complexes deliver
underwater adhesion strengths that exceed 10.sup.5 Pa. Furthermore,
the gel-like complexes can be dissolved on demand by changing the
ambient pH, which controls the ionization state of the
polyelectrolyte and ionic crosslinker These properties enable the
gel-like complexes to provide a simple, cost-effective, and
scalable platform for underwater adhesion.
[0066] When multivalent ions are mixed with oppositely charged
polyelectrolytes in aqueous solutions, the multivalent ions can
crosslink the polyeletrolyte chains into a variety of
self-assembled, three-dimensional ionic networks. These range from
colloidal particles to macroscopic coacervates and gels. The
gel-like complexes of the present disclosure are prepared from
mixing a polycation with a strongly-binding multivalent anion. More
specifically, the gel-like complexes include a non-polysaccharide
polymer backbone having two or more amine groups, and a multivalent
phosphate crosslinker These mixtures can self-assemble into
remarkably stiff gels (or gel-like coacervates) with adhesive and
self-healing properties. The two oppositely charged species undergo
associative phase separation (e.g., through ionic crosslinking) to
form a dense coacervate phase (rich in both the polymer and
multivalent ion) and a dilute supernatant phase. In particular
embodiments, the polycation that makes up the polymer backbone with
amine groups is poly(allylamine hydrochloride) (PAH), and the
phosphate crosslinker that makes up the multivalent anion is either
pyrophosphate (PPi) or tripolyphosphate (TPP). An illustration of a
non-limiting example of such a mixture between PAH and TPP is shown
in FIG. 8A. These mixtures exhibit strong ionic bonding and can
self-assemble into remarkably stiff gels with adhesive and
self-healing properties. The adhesive gels can deliver short-term
underwater adhesion strengths that exceed 10.sup.5 Pa, and can be
dissolved on demand by altering the ambient pH.
[0067] The adhesive gels are prepared from simple methods involving
the mixing of a polycation and phosphate crosslinker. An
illustration of a non-limiting example of the process for preparing
an adhesive gel from a mixture of PAH and either TPP or PPi is
shown in FIG. 8B. In certain embodiments, the crosslinker is added
dropwise to a dilute polycation solution. The cationic amine groups
are then ionically crosslinked into gel-like complexes. A method of
making the adhesive gels can further involve separating the
gel-like complexes from supernatant solution.
[0068] The rheological and adhesive properties of these
phosphate-crosslinked polyamine gels are distinct from those of
other ionically-crosslinked or polyelectrolyte structures. Calcium
alginate and chitosan/TPP gels, for example, are not easily
moldable and (unlike the gels described herein) are not usually
adhesive. Conversely, the macroscopic complexes that form through
the crosslinking of PAH with succinate, citrate, or
ethylenediaminetetraacetic acid (EDTA) ions are viscous liquids.
The viscous liquid-like rheology of PAH/citrate and PAH/phosphate
complexes are shown in FIGS. 6A-6B. Despite their viscosity, these
complexes have low cohesive strength and, like the alginate gels,
make poor adhesives. Notably, the G' values of both PAH/citrate and
PAH/phosphate complexes were significantly lower than those of
PAH/PPi and PAH/TPP gels, as well as significantly more sensitive
to the oscillation frequency, which further reflects weaker
PAH/citrate and PAH/phosphate binding.
[0069] The adhesive gels of the present disclosure demonstrate that
underwater adhesion can be achieved through the ionic crosslinking
of common synthetic polyelectrolytes, which are widely available
and have simple molecular structures. For example, PAH is a
commercially-available polyelectrolyte that is being explored for
drug and gene delivery, as well as cell encapsulation. PAH is a
weak polycation with an effective pK.sub.a of 8.5. The molecular
structure of PAH, in its hydrochloride salt form, is shown below as
Formula I:
##STR00001##
where n is any integer. PAH is prepared by the polymerization of
allylamine, C.sub.3H.sub.5NH.sub.2.
[0070] Likewise, both PPi and TPP are biocompatible tetra- and
pentavalent polyprotic acids that are used as food additives and
generally recognized as safe by the U.S. Food and Drug
Administration. PPi and TPP are commercially available and
inexpensive. The molecular structure of PPi, in its sodium salt
form, is shown below as Formula II:
##STR00002##
[0071] The molecular structure of TPP, in its sodium salt form, is
shown below as Formula III:
##STR00003##
[0072] While PPi and TPP mixtures with PAH are described for
exemplary purposes, PPi and TPP mixtures with poly(vinylamine)
indicate that similar adhesive materials can be prepared from a
broader range of polyelectrolytes and ionic crosslinkers.
Therefore, the skilled practitioner will understand that the
present disclosure is by no means limited to PAH/PPi and PAH/TPP
mixtures, but rather encompasses a wide variety of mixtures between
a polyamine and a phosphate-bearing multivalent anion. In general,
the adhesive gels of the present disclosure are compositions that
include a polymer having two or more amine groups, where the
polymer is a non-polysaccharide (as opposed to having sugar ring
monomers), and a multivalent phosphate crosslinker In some
embodiments, the polymer backbone is substantially linear. In other
embodiments, the polymer backbone is made of a branched polymer,
such as, but not limited to, a branched polyethyleneimine It is to
be understood that the amine groups can be pendant groups or in the
polymer backbone itself. Polymers other than PAH which are suitable
in their salt forms for use as the polymer backbone include, but
are not limited to: poly(vinylamine), polylysine, polyarginine,
polyhistidine, and polyethyleneimine Many other polyamines can be
utilized in the polymer backbone. However, while not wishing to be
bound by theory, it is believed that the polymer should have more
than two amine groups. Phosphate crosslinkers other than PPi and
TPP which are suitable for use as a multivalent phosphate
crosslinker include any multivalent anions containing multiple
phosphorus atoms, such as polyphosphates, pyrophosphates,
trimetaphosphates, tripolyphosphates, or hexametaphosphates. Some
non-limiting examples of salt forms of these phosphates include,
but are not limited to: sodium hexametaphosphate (SMHP), sodium
polyphosphate, tetrasodium pyrophosphate, and tetrapotassium
pyrophosphate.
[0073] In the examples discussed herein, mixtures of PAH/PPi and
PAH/TPP were prepared and analyzed via dynamic and electrophoretic
light scattering, rheology, and adhesion tests. These mixtures
showed that the adhesive gels of the present disclosure have many
advantages. Their preparation is simple, inexpensive, and scalable.
Because they form via self-assembly, their use requires no chemical
crosslinking and minimizes the risk of harmful side reactions. They
are extremely stiff, with plateau storage moduli
(G.sub..infin.'-values) near 4.times.10.sup.5 Pa, which helps
immobilize the bonded surfaces. (The high storage moduli of the
adhesive gels is indicative of a very high crosslink density.) The
adhesive gels adhere well to both hydrophilic and hydrophobic
substrates. They are able to self-heal when torn. They are capable
of the encapsulation and long-term release of active molecules.
Also, they can be redissolved on demand by changing the ambient
solution conditions.
[0074] The formation of adhesive PAH/PPi and PAH/TPP complexes
depends on the cross-linking ion:PAH monomer ratios, where PAH
remains solubilized at very low ion:monomer molar ratios and forms
ionically cross-linked complexes when higher ion:monomer ratios are
used. These complexes first form stable colloidal dispersions and,
at even higher multivalent ion concentrations (where their
electrostatic repulsion is diminished), coagulate into macroscopic
adhesives.
[0075] The adhesive gels described herein are sensitive to pH and
ionic strength. Neutral pH is preferred for making the adhesive
gels. However, the optimal pH level is determined by the identity
of the polymer molecule used. For example, above a pH of about 10
or 10.5, the PAH-based gels no longer formed. Without wishing to be
bound by theory, it is believed this is because a high charge
density is important for forming the gel. Because of this, the
adhesion of the gels is controllable. Because the ionization states
of PAH and phosphates are pH-sensitive, the adhesive gels can be
rapidly dissolved on demand by changing the pH. In certain
embodiments, the adhesive gels are easily removed by raising the pH
to about 12, which deprotonates the PAH amines and dissolves the
ionotropic gels. In certain embodiments, at pH 11 or 12, the
adhesive gels simply dissolve. Similarly, the adhesive gels
dissolve when placed in concentrated acid, which reduces the
ionization of the multivalent phosphate crosslinker This stimulus
sensitivity further distinguishes these ionotropic adhesive gels
from covalently crosslinked adhesives. Also, in certain
embodiments, the phosphate crosslinker is leached out of the
complex over a long period of time, on the order of several months,
causing the complex to dissolve, providing further methods of
controllably reversing adhesion.
[0076] Because the polymer-counterion binding is reversible, the
gels also exhibit self-healing properties. In certain embodiments,
when the gels are torn or broken, they are capable of recovering
their entire storage moduli within 10-30 minutes. This property is
significantly advantageous for many industrial, household, and
medical applications.
[0077] Dilute mixtures of the polymer (such as PAH) and crosslinker
(such as TPP or PPi) starting materials can be used, or,
alternatively, much higher concentrations of the starting materials
can be used to form more adhesive gel. However, the coagulation
rates are concentration-dependent. Also, increased concentrations
may decrease the adhesion of the adhesive gel due to an increased
salt concentration resulting from the counterions of the starting
materials. The salt concentration also affects the self-healing of
the adhesive gels. In the presence of salt, the relaxation becomes
faster, so self-healing is also faster. Additionally, when the
polymer binds, it releases a proton, which changes the pH upon
complexation. Thus, the concentrations of the initial polymer and
crosslinker starting materials can be selected based on the desired
characteristics of the resulting adhesive gels, and various
properties of the adhesive gels can be tailored by the choice of
starting material concentration.
[0078] Ionotropic gelation of polyamines with phosphate-bearing
multivalent anions, as described herein, yields remarkably stiff,
self-healing, and adhesive gels, which can adhere to dissimilar
substrates under water. The elastic moduli of these gels
(G'.about.4.times.10.sup.5 Pa) are significantly higher than those
typically obtained from gels formed through spontaneous
self-assembly, and their adhesion strength is comparable to some of
the natural underwater adhesives, such as those produced by zebra
mussels and barnacles. The adhesive gels are prepared from
inexpensive and readily-available ingredients, are easily scalable,
and are useful in a wide variety of applications including, but not
limited to, wound healing, bone repair, drug and gene delivery,
antibacterial coatings, sealants, materials synthesis, surgical
adhesives, and water treatment. The adhesive gels adhere to a wide
variety of surfaces that includes human skin, Teflon.RTM., glass,
acrylic, PMMA, paper, and metal, and the gels likely adhere to a
wide variety of other surfaces such as, but not limited to, wood,
plastics, ceramics, concrete, brick, stone, marble, granite, and
clay.
[0079] In certain embodiments, the adhesive gels described herein
are highly sensitive to both pH and ionic strength. Because PPi and
TPP are polyprotic acids whose charges increase with pH, the
complexes form more readily (i.e., at lower ion:PAH monomer ratios)
from neutral or slightly basic parent PAH, PPi, and TPP solutions
(at pH 7.0 or 8.0) than under acidic conditions. When the parent
solution pH exceeds the effective pK.sub.a of PAH (i.e., pH 8.5),
however, the PAH becomes less charged, though the ionic complexes
still continue to form upon mixing until a pH of about 10.
Conversely, the ionic strength has little impact on the onset of
PAH/PPi and PAH/TPP complex formation, although the coagulation of
these complexes into macroscopic gel-like adhesives occurs over a
broader range of compositions at higher monovalent salt
concentrations.
[0080] The ionic networks have the longest relaxation times when
prepared from PAH, PPi, and TPP solutions at a pH of near 7.0 and
at low ionic strengths. Hence, the adhesive gels' adhesion is
strongest and most durable when prepared under these conditions.
These relaxation times and adhesion strengths/longevities decrease
slightly when the preparation pH is reduced to 6.0, but diminish
sharply when the preparation pH is increased to 8.0 (where the PAH
linear charge density is reduced). Once formed, the ionic networks
prepared from solutions at pH 7.0 maintain their long relaxation
times when the ambient pH is between roughly 6.5 and 9, but become
fluid-like at higher and lower pH-levels. Similarly, the ionic
networks become more fluid-like and deliver weaker adhesion at
higher ionic strengths, with the TPP-based complexes exhibiting
greater salt stability than the PPi-based complexes.
[0081] The adhesive gels' sensitivity to changes in pH and ionic
strength can be utilized to trigger their deposition to surfaces in
response to external stimuli, which enables their use as injectable
adhesives. The adhesive gels also have very low solute
permeabilities, and can therefore serve as adhesive scaffolds for
either pH-triggered or sustained release. In certain embodiments,
the sustained release of small molecules from the gel lasts for
multiple weeks. In certain embodiments, the sustained release of
active payload molecules lasts for many months or even years. In
certain embodiments, the active payload molecules are small
molecules having a molecular weight of up to about 1,000
Daltons.
[0082] While elevated ionic strengths and high/low pH levels weaken
the adhesion of the PAH/PPi and PAH/TPP complexes, the short-term
tensile adhesion strengths still exceed 10.sup.5 Pa under many
conditions. Furthermore, the salt and pH sensitivity of these
mixtures can be exploited for injectable underwater adhesives or to
dissolve the adhesives on demand. This versatility, combined with
the inexpensive ingredients, results in the adhesive gels described
herein being a useful and scalable platform for underwater
adhesion, especially for applications requiring temporary or
reversible attachment.
[0083] Among many other uses, the adhesive gels can be used as
pressure-sensitive adhesives, or injectable formulations for
medical applications such as wound closure. As a pressure-sensitive
adhesive, a gel can be placed between two surfaces, whereupon a
squeezing force between the two surfaces will cause the surfaces to
adhere. For injection applications, the adhesive gels can either be
pre-mixed into a colloidal dispersion, loaded into a syringe, and
injected in dispersion form, or can be injected as a two-part
mixture (of, for instance, polymer and crosslinker) that forms the
colloidal dispersion after injection. Alternatively, an injectable
composition could be prepared by mixing the polymer backbone (such
as PAH) with the phosphate crosslinker (such as PPi or TPP) in an
environment that prevents the adhesive gel from forming, such as a
high or low pH. This mixture could then be injected into or onto a
desired target location, and the pH could then be adjusted to
ambient or near-ambient levels, thereby causing the adhesive gel to
spontaneously form in or on the target location.
[0084] Any of a variety of suitable carriers, excipients, or
adjuvants can be used to prepare an injectable formulation. By way
of non-limiting examples, injectable dispersions may be prepared in
glycerol, liquid polyethylene glycols, and mixtures thereof, or in
oils. Under ordinary conditions of storage and use, these
preparations can contain a preservative to prevent the growth of
microorganisms. In some cases, the form should be sterile and
should be fluid to the extent that easy injectability exists. It
should be stable under the conditions of manufacture and storage
and can be preserved against the contaminating action of
microorganisms, such as bacteria and fungi. A carrier for an
injectable formulation can be a solvent or dispersion medium
containing, for example, water, ethanol, a polyol (i.e., glycerol,
propylene glycol, and liquid polyethylene glycol, and the like),
suitable mixtures thereof, and/or vegetable oils. Proper fluidity
may be maintained, for example, by the use of a coating, such as
lecithin, by the maintenance of the required particle size in the
case of dispersion, and by the use of surfactants. The prevention
of the action of microorganisms can be brought about by various
antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In
some cases, it is preferable to include isotonic agents, such as,
but not limited to, sugars or sodium chloride. Prolonged absorption
of the injectable compositions can be brought about by the use of
agents delaying absorption such as, for example, aluminum
monostearate, or gelatin. For parenteral administration in an
aqueous solution, for example, the formulation can be suitably
buffered if necessary and the liquid diluent can be first rendered
isotonic with sufficient saline or glucose.
[0085] The adhesive gels are particularly useful for medical
applications such as wound treatment. By way of a non-limiting
example, the adhesive gels can be utilized as a surgical adhesive
in wet environments. This is especially advantageous for military
personnel. Because the adhesive gels have very low solute
permeabilities, the adhesive gels are also useful as adhesive
scaffolds for drugs or drug-like molecules. This is due to the
tight network formed in the stiff gels. There is a scaling
relationship between G'.sub..infin. and the ionic crosslink
concentration (i.e., .sub..infin..about.k.sub.BTc.sub.x),
corresponding to a hydrodynamic mesh size
(.zeta..sub.H.about.c.sup.1/3.sub.x) of approximately 1 nm, which
indicates that the adhesive gels are an effective barrier for
controlled release.
[0086] The adhesive gels offer a simple and effective method of
controlled release. A payload can be loaded into these complexes by
simply adding it to the polymer or crosslinker solution. Then, upon
mixing these solutions (to form the adhesive gel) the payload
becomes incorporated into the ionic network during the crosslinking
process. After incorporation, the payload can be slowly released,
with the release rate being dependent on the crosslinking density,
the payload/polymer interaction, and the size of the payload
molecule. Thus, the adhesive gels can be used as bioadhesive drug
carriers or adhesive devices for other underwater controlled
release applications. Long (multiple-month) release timescales can
be achieved while releasing small molecule payloads using the
gel-like coacervates. Without wishing to be bound by theory, it is
believed that multiple-month release results from a densely
crosslinked network acting as a remarkably effective barrier to
diffusion, and not simply payload/polymer binding.
[0087] The encapsulation of active ingredients can be achieved by
(1) incorporation during the gel formation process, where the
active payload spontaneously binds to the polymer during its
gelation, (2) blending the active payloads into the preformed
adhesive gel, (3) simply allowing the payload to diffuse into the
adhesive gel, or (4) pre-mixing the payload in a polymer solution
(such as a concentrated 10 wt % PAH solution), then adding the
premixed polymer/payload solution to a crosslinker solution (such
as PPi or TPP solution) to form the ionic network. As demonstrated
by the examples herein, the payload can be incorporated even when
it is essentially non-binding. Suitable payloads for controlled
release by the adhesive gels include, but are not limited to:
antimicrobial agents, antibiotic agents, antiviral agents,
antifungal agents, antiseptic agents, anti-inflammatory agents, and
combinations thereof. In a similar fashion, the adhesive gels can
also be used in household products to release fragrances,
antibacterial agents, or cleaning agents. By way of a non-limiting
examples, the adhesive gels could be utilized as toilet-bowl
cleaners, where the gels are adhered to a toilet bowl and
controllably release cleaning agents. As another non-limiting
example, the adhesive gels can be adhered to medical implants and
controllably release antibacterial agents. As another non-limiting
example, the adhesive gels can be adhered to catheters and
controllably release agents to prevent the formation of biofilm. As
another non-limiting example, the adhesive gels can be used in a
buccal patch that adheres to the inside of an animal's mouth and
controllably release drugs. The release of drugs or other payload
molecules from the adhesive gels can be pH-triggered or sustained
release, which can last for multiple weeks. A pH-triggered release
of molecules can be effected by altering the pH above about 10 or
below about 4, so as to dissolve the ionically crosslinked adhesive
gels and form dispersions or solutions that release the payload
molecules that were blended with, incorporated into, or diffused
into the adhesive gels.
[0088] Following adhesion to a surface, the non-adhered surface of
an adhesive gel described herein can be coated with a polymer or
other substance so as to make the non-adhered surface non-adhesive.
In this manner, the adhesive gels can be utilized for applications
where a one-sided adhesive is desirable. As one non-limiting
example, the adhesive gels can be adhered to the hull of a boat and
be used to prevent the adhesion of barnacles to the boat hull by
being non-adhesive on the side not adhered to the boat hull.
[0089] Embodiments of the present disclosure further include
methods of determining coverage or denial of health insurance
reimbursement and/or payment for treatments of disease or injury
comprising the adhesive gels described herein. In certain
embodiments, the treatment comprises the use of an adhesive gel
made from a polyamine and a multivalent phosphate crosslinker, and
a provider of health insurance denies coverage or reimbursement for
the treatment.
Kits
[0090] The adhesive gels and methods described herein can be
embodied as parts of a kit or kits. A non-limiting example of such
a kit comprises the ingredients for preparing an adhesive gel,
namely a polyamine and a multivalent phosphate crosslinker, in
separate containers, where the containers may or may not be present
in a combined configuration. Many other kits are possible, such as
kits comprising a pre-mixed dispersion of polyamine and multivalent
phosphate crosslinker in acidic or basic pH in one container and a
pH modifier in another container. In certain embodiments, the kits
further comprise a syringe. The kits may further include
instructions for using the components of the kit to practice the
subject methods. The instructions for practicing the subject
methods are generally recorded on a suitable recording medium. For
example, the instructions may be present in the kits as a package
insert or in the labeling of the container of the kit or components
thereof. In other embodiments, the instructions are present as an
electronic storage data file present on a suitable computer
readable storage medium, such as a flash drive, CD-ROM, or
diskette. In other embodiments, the actual instructions are not
present in the kit, but means for obtaining the instructions from a
remote source, such as via the internet, are provided. An example
of this embodiment is a kit that includes a web address where the
instructions can be viewed and/or from which the instructions can
be downloaded. As with the instructions, this means for obtaining
the instructions is recorded on a suitable substrate.
EXAMPLES
Example I
[0091] Materials
[0092] Millipore Direct-Q 3 deionized water (18.2 M.OMEGA.m) was
used in all preparations. PAH (nominal molecular weight 120-200
kDa) and PVAm (nominal molecular weight 25 kDa) were purchased from
Polysciences, Inc. (Warrington, Pa.). PPi and TPP (both sodium
salts) were obtained from Sigma-Aldrich (St. Louis, Mo.).
Hydrochloric acid (HCl) and sodium hydroxide (NaOH) were purchased
from Fisher Scientific (Fair Lawn, N.J.) and VWR (West Chester,
Pa.), respectively. The glass, PMMA, and Teflon.RTM. plates used
for adhesion tests were cut from Fisherfinest premium microscope
slides (Fisher Scientific), and OPTIX 0.093''-thick clear acrylic
sheet, and a 0.25''-thick sheet of polytetrafluoroethylene,
respectively. All materials were used as received.
[0093] Phase Studies
[0094] PAH/PPi and PAH/TPP mixtures were prepared by mixing PAH and
multivalent counterion stock solutions, whose pH was adjusted to
7.0 using NaOH and HCl. The parent PAH solutions ranged between
0.016 wt % and 0.16 wt % (1.7-17.1 mM) in concentration, and were
mixed with different volumes (40-400 .mu.L) of PPi and TPP
solutions, where the parent PPi solution concentrations varied
between 0.16 and 1.6 wt % (5.4-54.1 mM), and the parent TPP
solution concentrations varied between 0.16 wt % and 1.6 wt %
(4.4-43.5 mM). The cuvettes were then shaken for approximately 2
seconds and allowed to equilibrate for one month. During
equilibration, DLS (Zetasizer Nano ZS; Malvern, UK) was used to
detect the formation and coagulation of colloidal dispersions. A
sudden increase in the light scattering intensity indicated the
formation of colloidal dispersions. Similarly, a drastic increase
in the colloid hydrodynamic diameter and polydispersity (and
visible macroscopic precipitation) indicated the coagulation of
colloidal complexes into adhesive gels. These DLS measurements were
performed using non-invasive back-scattering (NIBS) detection at
the 173.degree. scattering angle.
[0095] Zeta Potential Measurements
[0096] The evoluation in the apparent .zeta.-potential with the
addition of PPi and TPP to PAH mixtures was tracked via
electrophoretic light scattering using the Zetasizer Nano ZS
instrument (where the .zeta.-potentials were estimated from the
electrophoretic mobilities via the Smoluchowski equation). Here,
160 .mu.L aliquots of either 0.37 wt % PPi or 0.40 wt % TPP were
sequentially added to 10 mL of 0.04 wt % PAH solution while
stirring the mixtures at 800 rpm with cylindrical magnetic stir
bars (10 mm.times.5 mm). The .zeta.-potential was measured after
each addition, following 10 minutes of equilibration. Each
titration was repeated thrice.
[0097] Rheology Measurements
[0098] Dynamic rheology was performed at room temperature using a
Rheometric Scientific RDA III (Piscataway, N.J.) strain-controlled
rheometer equipped with 25 mm parallel plates. The gel samples were
prepared by slowly adding 26 mL of 3.9 wt % PPi solution or 15 mL
of 5.7 wt % TPP solution to 1000 mL of 0.1 wt % PAH. The receiving
PAH solutions were stirred at 300 rpm with cylindrical magnetic
stir bars (5 cm.times.1 cm). The ionically-crosslinked complexes
were allowed to coagulate for 3 days, whereupon the gels were
scraped from the bottom of the beaker. The gels were then loaded
into the rheometer, compressed to a 0.5 mm gap thickness, and
allowed to relax between the plates until the normal force was
below 100 g. The excess gel was removed using a spatula and water
was applied to the exposed, outer edges of the gel to prevent
drying. After performing strain amplitude sweeps to determine the
linear viscoelasticity region, frequency sweeps were performed in
triplicate at angular velocities raging 0.1-500 rad/s at a 1.0%
strain amplitude. Additionally, the self-healing properties of the
gels were tested by first breaking the gels using 0.1-200% strain
sweeps (oscillation frequency=1 rad/s), and then tracking the
recovery in G' at a 0.4% strain amplitude, at which the gel network
was stable, at the same oscillation frequency. All rheological
measurements were performed in triplicate.
[0099] Adhesion Tests
[0100] PAH/PPi and PAH/TPP gel samples for adhesion tests were
prepared by slowly adding either 66 mL of 4.2 wt % PPi solution or
43 mL of 5.6 wt % TPP solution to 1000 mL of 0.3 wt % PAH (while
magnetically stirring at 300 rpm). The complexes were then allowed
to coagulate for 3 days, whereupon the adhesive gels were scraped
from the bottom of the beaker and used to adhere two 2.5
cm.times.2.5 cm substrate surfaces (glass, PMMA, and Teflon.RTM.).
All substrate surfaces were super-glued onto custom-made Plexiglass
brackets (using Loctite.RTM. Glass Glue for the glass substrate and
Gorilla.RTM. Super Glue for the PMMA and Teflon.RTM. substrates),
which allowed them to be clamped into the stress-strain analyzer.
To adhere the plates, a 0.5-1.0 g piece of gel (a more precise
application was difficult because the gel adhered to the spatula)
was placed between the two plates and compressed for 3 hours using
24 kPa of pressure, which uniformly spread the gel over the entire
adhesion area (in a 0.33-0.43 mm thick layer). After trimming the
excess gel that was squeezed out, the adhered plates remained
submerged in deionized water for 15-30 minutes, until a tensile
bond test was performed using an Instron 4400R Universal Testing
Machine (UTM; Norwood, Mass.). The adhered plates were then clamped
into the grips of the UTM and separated at a cross-head speed of
0.85 mm/s while measuring both the force and displacement.
[0101] Results
[0102] To characterize the properties of the gels, the phase
behavior, rheology, and adhesion properties of the PAH/PPi and
PAH/TPP mixtures were examined. When PAH was mixed with PPi or TPP
at pH 7.0, its phase behavior (recorded after 1 month of
equilibration) depended on the PAH multivalent ion:PAH monomer
ratio. At PPi:PAH molar ratios below 0.13:1, the PAH/PPi mixtures
formed clear solutions ("S Region" in FIG. 1A). Above that point,
PPi crosslinked the PAH and formed colloidal complexes ("D Region"
in FIG. 1A). The DLS analysis of these dispersions revealed the
stably-dispersed colloidal complexes have narrow size distributions
(PDI 0.1-0.2) and z-average hydrodynamic diameters ranging between
roughly 100 and 250 nm. (See FIGS. 12A-12D.) These diameters
increased with the PAH concentration and remained stable with time.
(FIGS. 12A-12B.) The complexes had fairly narrow size
distributions, with polydispersity indices (PDIs) ranging between
roughly 0.1 and 0.2. (FIGS. 12C-12D.)
[0103] The stably-dispersed complexes maintained highly positive
.zeta.-potentials (+50 mV to +70 mV; FIG. 2.) As the PPi:PAH molar
ratio exceeded 0.25:1, however, a dramatic reduction in the
.zeta.-potentials occurred, to +23 mV at the 0.30:1 PPi:PAH ratio
and then +15 mV at the 0.35:1 PPi:PAH ratio, whereupon the
.zeta.-potentials remained below +11 mV. At this point, the
dispersions became destabilized and coagulated into macroscopic,
sticky gel-like coacervates. (See FIG. 1C and "G+S Region" in FIG.
1A.) The macroscopic gel-like coacervates occupied a small fraction
of the sample volume, and were in equilibrium with dilute
supernatant solutions. These gels were highly adhesive and bound to
dissimilar substrates, such as glass, metal, human skin, and
Teflon.RTM. (as shown in FIG. 1D, where a Teflon.RTM.-coated
magnetic stir bar was adhered to a fingertip with the gel, and in
FIG. 1E, where the same gel adhered to the metallic rheometer
plates).
[0104] Similar to the PAH/PPi complexes, the PAH mixtures with
pentavalent TPP underwent a progression from clear solutions to
colloidal dispersions and adhesive macroscopic gel-like coacervates
with an increasing TPP:PAH ratio (see FIG. 1B). Their complexation,
however, exhibited two qualitative differences. First, unlike the
PAH/PPi system, where stable colloidal complexes began forming only
above the 0.11:1 PPi:PAH molar ratio, the onset of stable complex
formation in the PAH/TPP system was sensitive to the PAH
concentration; at higher PAH monomer concentrations (i.e., above
6.+-.2 mM), stable colloidal complexes only formed above the 0.09:1
TPP:PAH molar ratio (FIG. 1B), while at lower concentrations,
colloidal complexes form at TPP:PAH ratios as low as 0.02:1.
Second, although at higher TPP concentrations (above the 0.19:1
TPP:PAH molar ratio) the PAH/TPP complexes coagulated into
macroscopic gel-like coacervates (see the "G+S" region in FIG. 1B),
no macroscopic gel-like coacervates formed during the month-long
experiment where the TPP:PAH molar ratio exceeded 0.25:1 (FIG. 1B).
Instead, the PAH/TPP mixtures formed colloidal dispersions with
negative .zeta.-potentials, ranging between -10 mV and -20 mV (FIG.
2B). Over the month-long equilibration time, these negatively
charged complexes slowly grew in size (see FIG. 13), indicating
that the dispersions had lower colloidal stabilities than those
formed at low ion:monomer molar ratios, and were only kinetically
stable.
[0105] At the mixture compositions where macroscopic gels formed,
the rates at which the PAH/PPi and PAH/TPP dispersions coagulated
into gels were sensitive to the PAH concentration, ion:monomer
molar ratio and stirring speed. At low PAH concentrations (e.g.,
0.016 wt %), the coagulation was slow and occurred over multiple
days. Conversely, at higher PAH concentrations, such as those used
to prepare samples for the rheology and adhesion tests (i.e.,
0.10-0.30 wt %), most of the gel phase formed within minutes. This
rapid gel formation, however, was followed by a second, slower
stage, where the remaining colloidal complexes (whose concentration
was now quite low) required 2-3 days to fully coagulate. Similarly,
the coagulation rates were accelerated by stirring and the use of
ion:monomer ratios where the .zeta.-potentials were near zero.
[0106] Once formed, both the PAH/PPi and PAH/TPP gel-like adhesives
contained only 25-30 wt % water. This water content indicated very
dense network structures and (within the limits of the "S+G" region
in FIGS. 1A-1B) was insensitive to the PAH and ionic crosslinker
concentrations. Thus, the gel-like coacervate phase composition
remained roughly constant with the overall polymer and crosslinker
concentration, and, because virtually all of the PAH was in the
gel-like coacervate phase, the amount of adhesive formed scaled
linearly with the concentration of the parent PAH solution.
Furthermore, the final adhesive gel properties were insensitive to
the mixing procedure used in their preparation. This insensitivity
to the mixing method and ability to form the adhesive gels rapidly
(i.e., at the higher PAH concentrations) indicate that the adhesive
gel preparation process can easily be scaled up.
[0107] Gel formation was not analyzed in terms of the ion:monomer
charge ratios due to the uncertainty of the ionization states of
PPi and TPP. While the pK.sub.a's of PPi (pK.sub.a,2=2.3,
pK.sub.a,3=6.6, and pK.sub.a,4=9.3) and TPP (pK.sub.a,3=2.8,
pK.sub.a,4=6.5, and pK.sub.a,5=9.2), and effective pK.sub.a of PAH,
are known in solution, the acid-base equilibria invariably shift
towards the ionized states when the PPi and TPP bind to the PAH.
FIGS. 1A-1B indicate that the onset of gel formation occurred at
near-stoichiometric charge ratios. Without wishing to be bound by
theory, it is believed that PAH, PPi, and TPP are fully ionized at
pH 7. However, as seen from FIGS. 10A-10B, the gel-like complexes
can form over a broader range of charge ratios depending on the
ionic strength and possibly pH.
[0108] The gel-like properties of the macroscopic complexes
(specifically, those formed using 0.33:1 PPi:PAH and 0.20:1 TPP:PAH
molar ratios) were confirmed by dynamic rheology. Both the PAH/PPi
and PAH/TPP complexes (FIGS. 2A-2B) had almost identical and
frequency-independent storage moduli (G') that were consistently
higher than the loss moduli (G'') for the entire frequency (w)
range tested, thus confirming gel-like properties. The complexes
had storage moduli and loss moduli that were quite high
(G'.sub..infin..about.4.times.10.sup.5 Pa; see FIGS. 3A-3B)
compared to the majority of other physical (non-covalently
crosslinked) gels, whose G' values are typically on the order of
10.sup.3-10.sup.4 Pa. These high G'-values reflect the very dense
ionic crosslinking, mediated by the high linear charge density of
PAH and strong binding of PPi and TPP. These high values exceeded
those obtained through the ionic crosslinking of PAH with
weaker-binding citrate and phosphate ions. Likewise, these
G'-values significantly exceeded those obtained from Ca.sup.2- and
Mg.sup.2+ crosslinked complex coacervates used in the biomimetic
underwater adhesion strategies (whose G'-values are typically below
10.sup.4 Pa).
[0109] As seen in FIGS. 3A-3B, the G' and G'' approach one another
at lower oscillation frequencies, indicating that the G'/G''
crossover would eventually occur at lower .omega. values. This
indicates that, like other physical gels, these complexes undergo
plastic deformation when stress is applied over long timescales
(i.e., reflecting the reversibility of the ionic crosslinks) This
reversibility allows the PAH/PPi and PAH/TPP networks to self-heal
when torn, a property that was confirmed by dynamic rheology using
a two-step procedure. Briefly, the gels were first broken by
applying a 200% strain through a strain amplitude sweep, thereby
breaking their network and causing their G' values to drop
precipitously (FIGS. 4A-4B). The healing of the gel was then
monitored over time by continuing to oscilate the gels at low
(0.4%) strain amplitude, which was in the linear viscoelasticity
region and did not disrupt the ionic network. As seen in FIGS.
4A-4B, all of the PAH/PPi and PAH/TPP samples recovered their
original G' values within 10-30 minutes, although there was some
variation in the recovery kinetics between the individual tests.
The PAH/TPP networks healed slightly faster than the PAH/PPi
networks.
[0110] In addition to being self-healing, the gels were moldable,
ductile, and adhesive (see FIGS. 1D-1F), even under water, which
allows the ionically crosslinked polyamines to serve to
pressure-sensitive underwater adhesives. Tensile-load tests were
performed to quantify the ability of PAH/PPi and PAH/TPP gels to
bond various dissimilar substrates. The adhesive gels were
compressed between two flat plates made of either: (1) hydrophilic
glass, (2) moderately hydrophobic poly(methyl methacrylate) (PMMA),
or (3) very hydrophobic Teflon.RTM.. The adhesive application was
performed under deionized water and a compressive pressure of 24
kPa for 3 hours. To prevent drying, the adhered plates were not
removed from water until the adhesion strength was tested. The
plates were then pulled apart at a rate of 0.85 mm/s while
recording the force and displacement.
[0111] The adhesion strength achieved with the gels was insensitive
to the substrate type and hydrophibicity, as seen in FIG. 5A. The
PAH/PPi gels had very similar average tensile bond strengths
regardless of the substrate being used (361 kPa for glass, 374 kPa
for PMMA, and 366 kPa for Teflon.RTM.; see FIG. 5A). Likewise, the
PAH/TPP gels provided only slightly higher tensile-bond strengths
when adhered to glass (435 kPa) and PMMA (417 kPa) than when
adhered to Teflon.RTM. (341 kPa). The tensile-bond strengths of
these gels are roughly comparable to, if not better than, those of
mussel and barnacle adhesion.
[0112] Despite the similarities in adhesion strengths, the modes of
failure were substrate-dependent. When the PMMA and Teflon.RTM.
substrates were used, the bonds failed at the gel/substrate
interface. Conversely, when the glass substrate was used, the
failure was cohesive. Therefore, without wishing to be bound by
theory, although the gels yield stronger interfacial adhesion
strengths when bonded to glass (relative to the PMMA and
Teflon.RTM. substrates), the tensile bond strengths that the gels
provide increase modestly because of their limited cohesive
strengths.
[0113] Additionally, while the reversibility of ionically
crosslinked networks limits their ability to withstand sustained
stress, the ability of the PAH/PPi and PAH/TPP gels to self-heal
(shown in FIGS. 4A-4B) also allows them to partially regenerate
their bonds if the bonds fail. This reversibility was quantified by
separating glass plates adhered with the gels, then recompressing
the plates again (using the same underwater procedure as in their
initial adhesion), and then remeasuring their bond strength. As
shown in FIG. 5B, after the initial gel failure and recompression,
the gels recovered 30-40% of their original bond strength in the
third adhesion/debonding cycle. This moderate reduction in tensile
bond strength upon recompression reflects the introduction of
defects into the network when the adhesive gels are reattached.
[0114] The adhesive gels were easily dissolved on demand by
changing the pH because the ionization states of PAH, PPi, and TPP
are pH-dependent. The adhesive gels were easily removed from
laboratory equipment by raising the pH to 12, which quickly
deprotonated the PAH amine groups and dissolved the ionic networks.
Likewise, by protonating the PPi and TPP ions, the adhesive gels
can be dissolved at low pH conditions. The effect of changing pH on
the adhesive gels is shown in FIGS. 11A-11B. Complex formation was
sensitive to pH, and occurred most readily near pH 7-8, due to the
high ion and PAH charge. As seen in FIGS. 11A-11B, the complexes
did not form at a pH greater than 8.5. This stimulus sensitivity is
useful in situations where underwater adhesion needs to be
reversed, and further distinguishes these ionotropic adhesive gels
from covalently crosslinked gels. Furthermore, the PAH/PPi and
PAH/TPP complexes did not dissolve in high ionic strength
solutions, such as those containing 500 mM NaCl, although the ionic
strength did affect their rheology. FIGS. 10A-10B show that the
colloidal stability can be tuned with NaCl, which has little impact
on the complex formation phase boundary but causes the dispersions
to coagulate.
[0115] Payload Uptake and Release
[0116] Isothermal titration calorimetry (ITC) was used to
characterize the binding of model payloads (Fast Green FCF and
Rhodamine B dyes) to the PAH/PPi or PAH/TPP complexes (MicroCal
VP-ITC, GE Healthcare, U.S.A.). Colloidal PAH/PPi complexes were
formed by adding 29.0 .mu.l of 3.93 wt % PPi to 10.0 ml of 0.016 wt
% PAH, and PAH/TPP complexes were formed by adding 19.0 .mu.l of
5.84 wt % TPP to 10.0 ml of 0.016 wt % PAH. The Fast Green FCF and
Rhodamine B solutions were then prepared at concentrations of 0.43
and 0.80 wt %, respectively. All solutions were adjusted to pH 7
using HCl and NaOH. ITC experiments were run by placing the PAH/PPi
or PAH/TPP complex solutions in the sample cell, and loading the
dye solution into the injector. The dye solutions were then
titrated using twenty 15-.mu.L injections, with 20 min
equilibration intervals between each injection. The contents of the
sample cell were stirred at 307 rpm with the impeller-shaped
injector tip. The instrument software was used to integrate the raw
ITC data obtained from these titrations. The heat of dilution,
which was obtained by titrating the dye solutions into DI water,
was then subtracted from the integrated data to obtain the final
thermograms. To ensure reproducibility, each ITC test was repeated
twice.
[0117] The dye-loaded coacervates were prepared by adding either
0.93 ml of 7.5 wt % PPi solution or 0.80 ml of 7.5 wt % TPP
solution to 0.75 ml of 10.0 wt % PAH in a microcentrifuge tube
(inner diameter: 1.0 cm) with all solutions containing one of the
dyes (Fast Green FCF or Rhodamine B) at a concentration of 0.5,
1.0, or 4.0 mg/ml. All solutions were adjusted to pH 7 using HCl
and NaOH prior to mixing. The mixtures were than centrifuged at
15,000 rpm for 90 min using an Ependorf centrifuge. This resulted
in a gel-like plug at the bottom of the microcentrifuge tube
approximately 7-8 mm thick with an aqueous supernatant above it.
The loading capacity (LC) and loading efficiency (LE) for each
loading procedure was calculated by subtracting the mass of dye in
the supernatant from the total mass of dye originally in the
solutions in the microcentrifuge tubes. The total dye mass in the
supernatant was determined by measuring the supernatant mass in
each tube and determining the supernatant dye concentration using
UV-Vis spectroscopy (Varian Cary 50 Spectrophotometer) and the
absorbance at a wavelength of 614 nm (.epsilon.=9.57.times.103
m2/mol) and 555 nm (.epsilon.=9.82.times.103 m2/mol) for Fast Green
FCF and Rhodamine B, respectively. The LC and LE were then
calculated using the following equations:
LC = V t C i - V s C f V c .times. 100 % Equation 1 LE = V s C f V
t C i .times. 100 % Equation 2 ##EQU00001##
[0118] where Ci is the dye concentration in the parent PAH and
PPi/TPP solutions, Cf is the final dye concentration in the
supernatant, Vt is the solution total volume of solution initially
added to the microcentrifuge tube, Vs is the supernatant volume
recovered after centrifugation, and Vc is the total volume of
coacervate after centrifugation.
[0119] Similarly, the LEs of the dye-loaded complexes was measured
at a constant initial dye concentration of 1 mg/ml while the PAH
concentration was varied. The dye-loaded complex samples were
prepared as outlined above; however, the PAH concentration was
varied (between 2.5 and 10.0 wt %). The concentrations of the
counterion solutions were also varied (between 1.9 and 7.5 wt %) to
keep the counterion:PAH molar ratio constant (0.33 PPi:PAH and 0.20
TPP:PAH). The LC- and LE-values were then determined using
Equations 1 and 2.
[0120] The adhesive PAH/PPi and PAH/TPP complexes were prepared
using the procedure described above, with and without dye. The
initial mass of the complexes was measured by removing the
supernatant from the microcentrifuge tube, measuring the mass of
the microcentrifuge tube containing the adhesive complex, and then
subtracting the mass of the microcentrifuge tube. The complexes
were stored in 1 ml of 1.times.PBS with the PBS solution replaced
daily, and weighed weekly using the same procedure used to
determine their initial mass.
[0121] To characterize the release rates from PAH/PPi and PAH/TPP
complexes, dye-loaded complexes were prepared using the procedure
outlined above at an original Fast Green FCF or Rhodamine B
concentration of 1.0 or 4.0 mg/ml (release measurements on
complexes loaded with 0.5 mg/ml Fast Green FCF or Rhodamine B were
not run). The supernatant was removed from the microcentrifuge
tube, and the inside of the tube was rinsed with DI water to ensure
all excess supernatant was removed. The release medium, 1 ml of
1.times.PBS, was then added to the microcentrifuge tube. During the
release evaluations, the contents of the microcentrifuge tube was
mixed at 400 rpm in an Ependorf Thermomixer at 37.degree. C. The
old PBS solution was replaced with fresh PBS daily. UV-Vis
spectroscopy was used to determine the amount of dye released
within this one day interval. The concentration of dye in the PBS
was determined using the absorbance of the solution as described
above. These release studies were run until the amount of dye
released in one day became so low that a discernable peak in the
UV-Vis spectrum could not be obtained. The weight of the adhesives
was also tracked during the release process using the procedure
described above. Each release experiment was repeated thrice to
ensure reproducibility. An additional release study was run (using
the same complex/dye compositions) with tap water at room
temperature as the release medium.
[0122] The long-term sustained release properties of the gels were
examined using the small molecule dyes Fast Green FCF and Rhodamine
B. As shown in FIGS. 7A-7B, only 1-2% of the payload was released
in the first week, thus demonstrating the capability of the gels to
take up and controllably release a payload over a long period of
time.
[0123] The degradation of the adhesive PAH/PPi and PAH/TPP adhesive
coacervates in 1.times. phosphate buffered saline (PBS) at
37.degree. C. (conditions mimicking those seen in the human body)
was tracked for multiple months (see FIG. 24). The masses of the
adhesive networks increased sharply to roughly 110% of their
initial values after the first week (likely due to swelling).
However, after this initial increase, the mass of PAH/PPi and
PAH/TPP coacervates stayed roughly constant, exhibiting almost no
change over 200 days. (FIG. 24.) This stable coacervate weight
indicates that these coacervates degrade very slowly under
physiological conditions.
[0124] Similarly, to further demonstrate the stability of these
adhesive coacervates, the dynamic rheology of the PAH/PPi and
PAH/TPP complexes was probed before and after being stored in
1.times. PBS (i.e., at physiological pH and ionic strength) for 2
weeks. As mentioned above, increasing the ionic strength causes a
reduction in the network stiffness and relaxation time. Testing the
rheology of the adhesives after 2 weeks of storage in PBS revealed
only minor changes in the dynamic rheology of these complexes, with
a reduction in the G'-value only occurring at low co (indicating a
reduction in relaxation time) and the G.sub..infin.'-values of
these complexes remaining roughly constant (see FIGS. 25A-25B).
This means that these adhesive networks retain their high crosslink
density even at elevated ionic strengths similar to those seen in
the human body. Notably, the rheology of the adhesives after 2
weeks of storage in PBS was very similar to that measured after 3
days of storage. This indicates that, after an immediate, ionic
strength-induced reduction in relaxation time, the rheology of the
adhesive gels remained fairly stable.
[0125] The cationic nature of the PAH and anionic nature of the PPi
and TPP presents a situation in which a binding interaction could
exist between the charged payload molecules and the ionically
crosslinked network of the adhesive. Fast Green FCF and Rhodamine B
are anionic and cationic, respectively. ITC was used to probe the
binding of these dye molecules to the ionically crosslinked
networks. In ITC the heat required to maintain a sample cell (which
contains a binding substrate solution) at a constant temperature
upon the addition of a binding ligand solution is measured, with
endothermic or exothermic peaks in the ITC raw data indicating
binding. The Fast Green FCF or Rhodamine B solutions were titrated
into a PAH/PPi or PAH/TPP dispersion (prepared at a near
stoichiometric PAH:crosslinker charge ratio). The thermograms
obtained are shown in FIGS. 26A-26D, and indicate the binding
enthalpy per mole of dye injected as a function of the dye:PAH
molar ratio inside the sample cell. The plots obtained from the
Fast Green FCF titrations are characteristic of a system with
strong cooperative binding, with the exothermic binding signals
(and consequently the binding strength) increasing with the dye:PAH
molar ratio up to a ratio near 0.2:1. However, the plots from the
Rhodamine B titrations exhibited much weaker exothermic signals,
which continuously decreased as the dye:PAH molar ratio increased,
meaning that Rhodamine B binds only very weakly and
non-cooperatively with the network
[0126] The preparation of the dye loaded PAH/PPi and PAH/TPP
coacervates was simple. The PAH was mixed with one of the
multivalent anions using PAH and multivalent anion solutions
containing either Fast Green FCF or Rhodamine B. This caused dye
molecules to become entrapped in the PAH/PPi and PAH/TPP networks
during crosslinking The loading capacities (LCs) and loading
efficiencies (LEs) were dependent on the dye type and the initial
dye concentration in the parent PAH, PPi, and TPP solutions (see
Table 1).
TABLE-US-00001 TABLE 1 Loading efficiencies and capacities at
different initial dye concentrations. PAH/PPi PAH/TPP Loading
Loading Loading Loading Efficiency Capacity Efficiency Capacity (%)
(mg/ml) (%) (mg/ml) Fast Green (0.5 mg/ml) 56.48 .+-. 3.04 3.16
.+-. 0.17 64.01 .+-. 1.45 3.31 .+-. 0.07 Fast Green (1 mg/ml) 63.03
.+-. 1.37 7.04 .+-. 0.15 72.94 .+-. 3.09 7.54 .+-. 0.32 Fast Green
(4 mg/ml) 68.54 .+-. 2.09 30.62 .+-. 0.93 74.74 .+-. 3.47 30.89
.+-. 1.43 Rhodamine B (0.5 mg/ml) 9.83 .+-. 2.79 0.55 .+-. 0.16
8.71 .+-. 2.10 0.45 .+-. 0.11 Rhodamine B (1 mg/ml) 6.38 .+-. 0.79
0.71 .+-. 0.09 6.27 .+-. 1.04 0.65 .+-. 0.11 Rhodamine B (4 mg/ml)
7.76 .+-. 2.77 3.46 .+-. 1.24 6.62 .+-. 1.41 2.74 .+-. 0.58
[0127] The LCs and LEs of Fast Green FCF were much higher than
those of Rhodamine B. Without wishing to be bound by theory, it is
believed this stemmed from the strong binding of Fast Green FCF to
the PAH/PPi and PAH/TPP networks. Similarly, the relatively low LC
and LE of Rhodamine B likely reflected its very weak binding to the
PAH/PPi and PAH/TPP networks. Moreover, the Fast Green FCF
LE-values increased with the dye concentration, while the Rhodamine
B LE-values remained roughly constant. This increase in the Fast
Green FCF LEs at higher dye concentrations was consistent with the
cooperative binding between Fast Green FCF and the coacervates
revealed by ITC (see FIGS. 26A-26B).
[0128] The LEs for each dye type were also measured at a constant
initial dye concentration (1 mg/ml) while varying the PAH
concentration used during the encapsulation procedure (see Table
2). The LEs of the Rhodamine B-loaded adhesives increased linearly
with PAH concentration (see FIG. 27). As the PAH concentration
decreased, a lesser adhesive volume formed. Thus, less of the
Rhodamine B could be enmeshed by the networks resulting in lower
LEs. In contrast, the LEs of the Fast Green FCF-loaded adhesives
remained fairly constant with the PAH concentration (Table 2). This
was because a decrease in PAH concentration (as Fast Green FCF
concentration remained constant) increased the dye:PAH molar ratio,
which due to the cooperative nature of the Fast Green FCF/network
binding, increased the fractional coverage of the PAH binding
sites. Accordingly, the LE remained roughly constant even at a
lower PAH concentration. Furthermore, in the case of the Fast Green
FCF-loaded adhesives, the gel-like plugs at the bottoms of the
microcentrifuge tubes did not form when 2.5 wt % PAH was used
during the encapsulation procedure. Instead, the PAH/ionic
crosslinker/dye complexes formed a flaky precipitate, which evenly
coated the insides of the centrifuge tubes even after
centrifugation. Without wishing to be bound by theory, it is
believed this was because at this composition the Fast Green
FCF:PAH ratio became so high that the Fast Green FCF bound to the
PAH began to displace the PPi and TTP anions, which drastically
changed the properties of the complexes.
TABLE-US-00002 TABLE 2 Loading efficiencies at different PAH
concentrations used during complex formation. PAH/PPi PAH/TPP
Loading Loading Efficiency Efficiency (%) (%) Fast Green (2.5% PAH)
N/A N/A Fast Green (5% PAH) 61.69 .+-. 1.13 72.40 .+-. 0.53 Fast
Green (10% PAH) 63.03 .+-. 1.37 72.94 .+-. 3.09 Rhodamine B (2.5%
PAH) 1.49 .+-. 0.30 1.59 .+-. 0.65 Rhodamine B (5% PAH) 3.76 .+-.
0.45 3.78 .+-. 0.32 Rhodamine B (10% PAH) 6.38 .+-. 0.79 6.27 .+-.
1.04
[0129] To characterize the controlled released properties of
adhesive PAH/PPi and PAH/TPP coacervates, 1 ml of 1.times.PBS was
added to microcentrifuge tubes containing approximately 0.2 ml of
the dye-loaded adhesives, whereupon the samples were continuously
mixed at 37.degree. C. The released dye concentration in the PBS
was determined using UV-Vis spectroscopy, with the PBS being
replaced daily. This procedure was repeated until discernable
UV-Vis peaks could no longer be obtained from the PBS samples in
order to obtain release profiles.
[0130] The complexes loaded with Fast Green FCF were capable of
releasing a very small fraction (<5%) of their payload over a
time span of up to 164 days, after which the release became too
slow to obtain a discernable UV-Vis peak. The adhesives prepared at
a Fast Green FCF concentration of 4.0 mg/ml released a lower
percentage of dye and over longer timescales than those prepared at
1.0 mg/ml dye concentration (see FIGS. 28A-28B). As mentioned
previously, Fast Green FCF binds cooperatively to the ionic
network. Thus, when the initial Fast Green FCF concentration is
increased from 1.0 mg/ml to 4.0 mg/ml, its stronger binding to the
network prolongs its release. Yet, due to their higher LC-values
(Table 1), the adhesives prepared at a Fast Green FCF concentration
of 4.0 mg/ml released a higher total mass of dye than those
prepared at the 1.0 mg/ml dye concentration (see FIGS. 28C-28D).
This increase in the total rate of release indicates that the
release rates can be easily tuned by varying the payload
concentration used during the encapsulation procedure. Furthermore,
the greater mass of dye being released allowed the dye release from
the samples prepared at a Fast Green FCF concentration of 4.0 mg/ml
to be tracked longer than those prepared at 1.0 mg/ml dye
concentration. Because less dye was released from the adhesives
prepared at a Fast Green FCF concentration of 1.0 mg/ml, it was
difficult to obtain a discernable UV-Vis peak beyond 42 days for
the PAH/PPi complexes and 80 days for the PAH/TPP complexes.
Moreover, the mass of these dye-loaded coacervates was tracked
during each release experiment (see FIGS. 29A-29B). After some
initial swelling the mass of PAH/PPi and PAH/TPP coacervates
decreased only very slightly over the entire release experiment (up
to 164 days). This indicates that these Fast Green FCF-loaded
coacervates degrade very slowly under physiological conditions.
[0131] The stability of the Fast Green FCF-loaded PAH/PPi and
PAH/TPP coacervates (the same coacervates used during the dye
release experiments) was determined by tracking the mass of these
coacervates during their release in 1.times.PBS at 37.degree. C.
(see FIGS. 29A-29B). Measurements were taken once a week throughout
the entire release experiment. Noticeable swelling occurred within
the first 1-2 weeks of release. After this initial swelling, the
mass of PAH/PPi and PAH/TPP coacervates decreased only very
slightly over the entire release experiment (up to 164 days),
meaning that these Fast Green FCF-loaded coacervates degrade very
slowly under physiological conditions. Similarly, the mass of the
Rhodamine B-loaded PAH/PPi and PAH/TPP coacervates was also tracked
during their release (see FIGS. 30A-30B). Again, after the initial
swelling, the mass of the coacervates remained roughly constant
over the entire release experiment (up to 42 days), indicating that
these Rhodamine B-loaded coacervates are largely stable under
physiological conditions.
[0132] The adhesives loaded with Rhodamine B were also capable of
releasing a very small fraction of their payload over multiple week
timescales (see FIGS. 31A-31D). Again, the time over which the
experiments were performed were limited to the early portions of
the release profiles, where a discernable UV-Vis peak could be
obtained (even though further release still occurred). Because
Rhodamine B had significantly lower LEs than Fast Green FCF (see
Table 1), there was significantly less Rhodamine B loaded into the
coacervates compared to the Fast Green FCF. Consequently, a lower
total mass of Rhodamine B was released (see FIGS. 31A-31D), which
made it difficult to obtain discernable UV-Vis peaks over long
timescales. Furthermore, unlike the adhesives loaded with Fast
Green FCF, the adhesives loaded with Rhodamine B dye released
approximately the same percentage of dye over time regardless of
the initial dye concentration (see FIGS. 31A-31B). This was because
the binding of the Rhodamine B to the network was very weak.
Therefore, the rate of release was likely largely determined by the
crosslink density and the concentration gradient within the
network. Under these conditions, the mass of dye released over time
should scale linearly with the LC. This is why the coacervates
loaded with Rhodamine B dye released approximately the same
percentage of dye over time regardless of the initial dye
concentration. Again, after tracking the mass of these dye-loaded
coacervates (see FIGS. 30A-30B), it can be concluded that the
Rhodamine B-loaded coacervates are quite stable under physiological
conditions.
[0133] While the slow release of Fast Green FCF molecules may in
part be attributed to their binding to the ionic networks, the slow
release of non-binding Rhodamine B indicates the permeability of
PAH/PPi and PAH/TPP networks as the reason for this slow release.
Without wishing to be bound by theory, it is believed that this
property stems from their high crosslink densities, and indicates
that PAH/PPi and PAH/TPP complexes can be used for the
multiple-month delivery of small molecules, even those small
molecules that do not bind to the ionic network.
[0134] Furthermore, the long-term release of small molecules
achieved with these complexes is not limited only to a PBS medium.
Release experiments were also performed using tap water at room
temperature as the release medium. These experiments yielded
similar slow release rates. (See FIGS. 32-33.) A release study was
run using the same procedure described above, however, the release
medium was changed from 1.times.PBS to tap water at room
temperature. The results for the Fast Green FCF and Rhodamine B
loaded hydrogels are plotted in FIGS. 32A-32D and FIGS. 33A-33D,
respectively.
[0135] Additionally, only a small percentage of the loaded dye was
released even after multiple months (see FIGS. 28, 31).
Qualitatively, the color of the PAH/PPi and PAH/TPP complexes (blue
in the case of Fast Green FCF and pink in the case of Rhodamine B)
faded much more near the surface than throughout the bulk of the
gel-like plugs. This means that in the timescales studied in these
experiments, diffusion of dye molecules was likely only occurring
near the exposed surface of the adhesives.
[0136] Release experiments were also run on dye loads adhesive
samples with a thickness of only 1-2 mm (rather than 7-8 mm), and a
much higher percent of the loaded dye was released (up to 15%). To
determine the effect of PAH/PPi and PAH/TPP complex thickness on
their release profiles, the coacervates were prepared using the
procedure outlined above; however, only 0.047 ml of a 7.5 wt % PPi
or 0.040 ml of a 7.5 wt % TPP were added to 0.038 ml of a 10.0 wt %
PAH. All solutions contained 4.0 mg/ml of either Fast Green FCF or
Rhodamine B. This procedure yielded 1-2 mm thick dye-loaded
gel-like plugs at the bottoms of the microcentrifuge tubes. The
release profiles were then obtained using the procedure outlined
above. (See FIGS. 34A-34B.) These thinner gel-like plugs released a
much higher percentage of the dye (.about.5-15%) than the thicker
plugs described above.
[0137] Because the release rates of the dyes from the network is at
least partially determined by the crosslink density of the
networks, increasing the pH of the parent solutions used to form
the adhesive coacervates increases release rates. Therefore,
altering the pH at which the complexes are formed can be utilized
as a mechanism for triggering payload release.
Example II
[0138] Effect of pH on PAH/PPi and PAH/TPP Complex Formation
[0139] The compositions for adhesive formation were determined by
mixing 0.04 wt % PAH solutions with different volumes of either
0.37 wt % PPi or 0.40 wt % on the ion:PAH monomer ratios where the
dispersions coagulated to form the gel-like macroscopic adhesives
("G+S" regions in FIGS. 14A-14B). When the parent solutions were at
pH-levels ranging between 2.0 and 8.0, colloidal dispersions formed
at lower ion:PAH monomer ratios as the pH of their parent solutions
were increased (see "S" to "D" region transitions in FIGS.
14A-14B). The parent solution pH had a similar impact on the
ion:PAH monomer ratios where the dispersions coagulated to form the
gel-like macroscopic adhesives ("G+S" regions in FIGS. 14A-14B).
The same downward shift also occurred for the ion:PAH monomer
ratios where the PPi and TPP-rich complexes ceased to undergo
macroscopic phase separation (see "G+S" to "D" transitions at high
ion:monomer ratios; FIGS. 14A-14B). As the pH of the parent
solutions was raised to 8.8, however, the colloidal and macroscopic
complexes ceased to form (FIGS. 14A-14B).
[0140] The pH dependence of each of these transitions reflected the
acid-base equilibria of PAH, PPi, and TPP. The ionization of PPi
and TPP increased at higher pH-levels, thereby reducing the ionic
cross-linker requirements for the formation of colloidal and
macroscopic complexes. Without wishing to be bound by theory, it is
believed that this reduction in PPi and TPP requirement for each of
the state transitions stemmed from: (1) the increased ionic
cross-linker:PAH charge ratios and (2) a possible amplification in
the polymer/crosslinker binding strength (where both occur due to
an increase in PPi and TPP valence). As the pH of the parent
solutions was raised above the effective pK.sub.a of PAH, however
(i.e., above pH 8.5), the linear charge density of PAH became much
lower and the complexes ceased to form. This inability to form
colloidal and macroscopic complexes at higher pH-levels indicates
that the high linear charge density of PAH is important for its
ionic cross-linking
[0141] Furthermore, PAH/PPi and PAH/TPP complexation can cause
significant changes in the mixture pH. This stems from shifts in
the acid-base equilibria of PAH, PPi and TPP, and is highlighted in
the state diagrams in FIG. 14C and FIG. 14D, where the ordinates
now indicate the final pH after mixing (recorded after 1 day of
equilibration) instead of the parent solution pH. When the PAH was
initially not fully protonated (e.g., at a parent solution pH of
7.0 or 8.0), the complexation of its ionized amine groups with PPi
or TPP favored its further protonation. This led to a depletion of
protons and increased the pH. Conversely, when PPi and TPP were
initially not fully deprotonated (which was the case for every pH
level examined), their association with PAH drove their further
deprotonation, which lowered the ambient pH.
[0142] The respective magnitudes of these competing effects on pH
depended on the initial PAH, PPi and TPP protonation states. At
pH-levels above 7.0, there were significant numbers of uncharged
PAH amine groups, while the PPi and TPP were mostly ionized. Thus,
the proton uptake by the PAH outweighed their release by the PPi
and TPP, and the pH upon PAH/PPi and PAH/TPP mixing increased by up
to two full units (FIG. 14C and FIG. 14D). Notably, FIGS. 14C-14D
show that upon mixing, the complexes can form at a pH of up to
about 10. When the parent solution pH was lower, however, an
opposite pH drift sometimes occurred. This was most evident when
the parent solution pH was 4.5, where nearly all PAH amine groups
were protonated, but the PPi and TPP ions each bore two protons
that could be released upon their binding to PAH. Accordingly,
ionic cross-linking under these conditions led to a reduction in pH
(cf. FIGS. 14A-14D).
[0143] The resulting pH drifts occurred regardless of whether
insoluble complexes formed (as seen from the vertical shift of many
of the data points in the "S" regions; cf. FIGS. 14A, 14C and FIGS.
14B, 14D). This confirmed that PPi and TPP bound to the PAH even in
the absence of ionotropic gelation. Once all of the PAH was
ionically cross-linked, however, the pH remained nearly constant,
irrespective of whether the complexes remained dispersed or were
coagulated into macroscopic, gel-like adhesives. This constant pH
reflected the fact that little further PPi and TPP binding to PAH
occurred under these conditions. The binding-induced pH changes in
FIGS. 14A-14D were also consistent with those occurring upon the
complexation between weak polyelectrolytes (including PAH), where
similar changes in the effective pK.sub.a values and solution pH
occur.
[0144] Further insight into the pH effects on the PAH/counterion
aggregation states was gained by probing the evolutions in their
.zeta.-potentials upon the titration of PPi and TPP into PAH, using
parent solutions at pH 6.0, 7.0, and 8.0 (see FIGS. 15A-15B). Here,
colloidal complexes formed from the very first PPi and TPP
additions but dissolved upon equilibration if the ion:PAH monomer
ratios were below the "S/D" phase boundaries in FIGS. 14A-14B. At
each pH, the .zeta.-potentials stayed roughly constant (at values
ranging between +40 and +70 mV) at lower ion:PAH monomer ratios but
diminished sharply (to values below +30 mV) once a critical ion:PAH
monomer ratio was reached. This drop in .zeta.-potential
corresponded to the point where nearly all of the PAH molecules
were ionically crosslinked, and instead of forming new colloidal
complexes, the PPi and TPP began to bind to the surfaces of
existing ones. Moreover, this sudden drop in .zeta.-potential
corresponded to the point where the colloidal complexes began
coagulating into macroscopic adhesives, likely because of: (1) the
reduction in electrostatic repulsion between the dispersed colloids
and (2) bridging flocculation mediated by the surface-bound PPi or
TPP.
[0145] As the pH increased, the multivalent ion:PAH monomer ratios
at which the .zeta.-potential reduction occurred decreased, again
reflecting the increased ionization of PPi and TPP, and decreased
ionization of PAH. Interestingly, this effect was most pronounced
when the parent solution pH was raised to 8.0 (i.e., approached the
effective pK.sub.a of PAH). This was likely because the charge of
PAH near its effective pK.sub.a was much more sensitive to pH than
that of PPi and TPP which--due to their widely spaced, multiple
pK.sub.a-values--exhibited a much slower fractional change in
charge as the pH was varied. As the multivalent ion:PAH monomer
ratio was increased further, the .zeta.-potential continued to
diminish and, in some cases (i.e., in all PAH/TPP mixtures and in
PAH/PPi mixtures that started at pH 8.0), became negative. When the
.zeta.-potential became sufficiently negative (between -10 and -20
mV; see FIGS. 15A-15B), the colloidal dispersions that formed at
high multivalent ion:PAH monomer ratios persisted throughout the
month-long experiment (cf. FIGS. 14A-14B). While the aggregation
states and .zeta.-potential trends for the PAH/TPP system were all
qualitatively consistent with those reported previously for
mixtures prepared from parent solutions at pH 7.0, the stable
anionic dispersions that formed in PPi-rich PAH/PPi mixtures from
parent solutions at pH 8.0 were not seen at lower pH levels. This
likely reflected the higher valence of PPi (and therefore its
stronger surface adsorption) at the higher pH values.
[0146] As the multivalent ion:PAH monomer ratio was increased
further, the .zeta.-potential continued to diminish and, in some
cases (i.e., in all PAH/TPP mixtures and in PAH/PPi mixtures that
started at pH 8.0), became negative. When the .zeta.-potential
became sufficiently negative (between -10 and -20 mV; see FIGS.
15A-15B), the colloidal dispersions that formed at high multivalent
ion:PAH monomer ratios persisted throughout the month-long
experiment (cf. FIGS. 14A-14B). While the aggregation states and
.zeta.-potential trends for the PAH/TPP system were all
qualitatively consistent with those reported previously for
mixtures prepared from parent solutions at pH 7.0, the stable
anionic dispersions that formed in PPi-rich PAH/PPi mixtures from
parent solutions at pH 8.0 were not seen at lower pH levels.
Without wishing to be bound by theory, it is believed this is
reflected the higher valence of PPi (and therefore its stronger
surface adsorption) at the higher pH values.
[0147] Effects of pH on Water Content
[0148] In addition to affecting the compositions at which the
gel-like adhesives formed, the pH also affected their
physicochemical properties. The first property examined was their
water content. This was achieved by measuring their mass change
upon drying as described above. All adhesive complexes were
solute-rich (containing less than 40 wt % water), which reflected
their high ionic cross-link densities, and had properties that were
insensitive to the multivalent ion:PAH monomer ratios used in their
preparation. Yet, the adhesives prepared from solutions at the
acidic pH of 6.0 had the lowest water contents, while those
prepared from solutions at pH 8.0 had the highest water contents
(see Table 3). The amplified water content of adhesives prepared at
pH 8.0 (where the final pH-values for PAH/PPi and PAH/TPP mixtures
were 10.1 and 10.3, respectively) likely reflected the partial
deprotonation of PAH amine groups, which diminished the cross-link
density within the ionic networks and increased their swelling.
Moreover, the water content might also reflect variations in the
multivalent ion:PAH binding stoichiometry, where at lower pH-values
more ions are needed to neutralize the PAH amine groups (i.e.,
because the PPi and TPP valence decreases and PAH ionization
increases). The PPi and TPP content within these complexes is
therefore likely higher at lower pH values. This explains the
reduction in water content at a parent solution pH of 6.0 (where
the final PAH/PPi and PAH/TPP mixture pH values were 6.6-6.7) and
is consistent with the state diagrams, where more PPi and TPP was
needed to form the cross-linked complexes as the pH was
diminished.
TABLE-US-00003 TABLE 3 Final pH Values and Average Water Contents
of Adhesive PAH/PPi and PAH/TPP Complexes Prepared using PAH, PPi,
and TPP Solutions at pH 6.0, 7.0, and 8.0 (.+-.Standard
Deviation).sup.a. initial final pH water content (wt %) pH PAH/PPi
PAH/TPP PAH/PPi PAH/TPP 6.0 6.6 .+-. 0.1 6.7 .+-. 0.1 24.5 .+-. 0.4
25.0 .+-. 1.1 7.0 8.4 .+-. 0.1 9.1 .+-. 0.2 30.2 .+-. 0.1 26.0 .+-.
0.1 8.0 10.1 .+-. 0.2 10.3 .+-. 0.2 37.7 .+-. 0.4 37.3 .+-. 0.8
.sup.aThe final pH values are the readings obtained from the
supernatant phases.
[0149] Effects of pH on Rheology
[0150] The pH also had a strong impact on the rheology of PAH/PPi
and PAH/TPP adhesives (which was probed as described above). When
prepared from solutions at pH 7.0, both materials had storage
moduli (G') that exceeded the loss moduli (G'') for the entire
frequency range tested (thus indicating gel-like properties) and
remarkably high G.sub..infin.'-values of 4.times.10.sup.5 Pa (see
FIGS. 16A-16B). Yet, as the preparation pH was decreased to 6.0,
there was a reduction in the G'-values at lower frequencies for
both adhesive types. Indeed, the PAH/PPi complex exhibited
crossover in its G'/G'' spectra at we .about.0.1 rad/s, signifying
the adhesive complexes to be more fluid-like (FIG. 16A). This
decrease in G' likely resulted from the more significant
protonation of PPi and TPP ions, which reduced the PAH/PPi and
PAH/TPP binding strength and led to faster network relaxation.
Moreover, as the preparation pH was increased to 8.0 (i.e., to near
the effective pK.sub.a of PAH), there was a drastic decrease in the
G'-value with a G'/G'' crossover occurring near 10 rad/s for the
PAH/PPi complex and near 0.3 rad/s for the PAH/TPP complex. Indeed,
in contrast to the complexes prepared at pH 6.0 and 7.0, the
G.sub..infin.' was not reached for complexes prepared at pH 8.0
over the entire frequency spectrum tested, reflecting their much
faster relaxation times. These faster relaxation dynamics likely
reflect the deprotonation of PAH amine groups (which weakens the
ionic crosslinking) and again indicates that a high polymer linear
charge density is essential to preparing gel-like PPi and
TPP-crosslinked adhesives.
[0151] While FIGS. 16A-16B clearly show that PAH/PPi and PAH/TPP
complex rheology is acutely sensitive to the pH of the parent PAH,
PPi, and TPP solutions, the high moduli of these adhesive
coacervates (once they are formed under optimized solutions) appear
to persist over a significant range of pH-values. This was revealed
by the drastically different final pH-values of the adhesives
prepared from parent solutions at pH 7.0 and those prepared from
solutions at pH 6.0, where the G' values were not much smaller
(despite the dissimilar final pH; see Table 3) Similarly, for the
adhesives prepared from solutions at pH 8.0 (where the G' value was
drastically diminished), the final supernatant pH exceeded 10
(Table 3). This high variability in final pH-values indicates that,
despite their sensitivity to their preparation pH, these adhesive
coacervates are robust enough to withstand minor pH fluctuations in
their environment. This was confirmed by placing the adhesives
prepared from parent solutions at pH 7.0 (whose final pH was 8.4
for the PAH/PPi complexes and 9.1 for PAH/TPP complexes) into water
at pH 6.5 and equilibrating them at that pH for 3 days. Although
the pH to which the adhesives were exposed was lowered by 1.9-2.6
units, the rheological properties of the adhesives remained
essentially unchanged. Interestingly, the relaxation times in these
samples were longer than in those that were prepared from parent
solutions at pH 6.0 (whose final pH was 6.6-6.7; see Table 3). This
difference in relaxation indicates that protonation in PAH/PPi and
PAH/TPP complexes might be more sensitive to small changes in their
preparation pH than to minor pH fluctuations after they form. When
the pH of the same complexes was raised significantly to 10.2 (to
match the final pH of complexes prepared at pH 8.0), however, the
PAH amine groups were deprotonated, and the rheology of the
adhesive complexes became nearly identical to that of those
prepared from PAH, PPi, and TPP solutions at pH 8. Similarly, the
adhesives became more fluid-like when equilibrated at pH 5.0 and
6.0. This indicates that preformed PAH/PPi and PAH/TPP adhesives
can withstand small fluctuations in pH but become more fluid-like
(and ultimately dissolve) when exposed to high- and low-pH
environments.
[0152] Effects of pH on Adhesion Strength
[0153] The pH effects on the rheology of PAH/PPi and PAH/TPP
complexes also had a strong impact on their underwater adhesion
properties. These were probed via tensile adhesion strength
measurements, which were performed using glass and Teflon as model
hydrophilic and hydrophobic substrates. The short-term tensile
adhesion strength was measured by separating two bonded glass or
Teflon plates at a rate of 0.85 mm/s. The PAH/PPi and PAH/TPP
complexes prepared from solutions at pH 7.0 had the highest average
bond strength (see FIGS. 17A-17B). When adhered to glass, the
average adhesion strength was 361 kPa for the PAH/PPi complex and
435 kPa for the PAH/TPP complex, while when adhered to Teflon the
average adhesion strength was 366 kPa for the PAH/PPi complex and
341 kPa for the PAH/TPP complex. When the parent solution pH was
reduced to 6.0, there was a slight (10-20%) reduction in average
adhesion strength. Conversely, when the parent solution pH was
raised to 8.0, a drastic (over 80%) reduction in adhesion strength
occurred, where all tensile bond strengths diminished to around 60
kPa (FIGS. 17A-17B).
[0154] While for the glass substrate the adhesion failure was
always cohesive, the failure mode for the adhesion to Teflon was
pH-dependent. The bonds failed adhesively (at the
adhesive-substrate interfaces) when the adhesives were prepared at
pH 6.0 and 7.0, but underwent cohesive failure when the adhesives
were prepared at pH 8.0. These trends in short-term adhesion
strength (and shift from adhesive to cohesive failure in the
Teflon-bonded complexes) indicate that the pH sensitivity of the
adhesion achieved using the PAH/PPi and PAH/TPP complexes primarily
stems from changes in their cohesive strength. They also confirm
that, in addition to triggering their dissolution (e.g., by raising
the ambient solution pH to 12), changes in pH can strongly affect
their underwater adhesion strength.
[0155] Furthermore, while the PAH/PPi and PAH/TPP adhesives
exhibited significant adhesion strengths over short times, the
reversibility of their ionic cross-links made them susceptible to
plastic deformation (and ultimate failure) when subjected to
sustained stress. To quantify this phenomenon, the bonds that these
adhesive complexes formed between two glass plates were subjected
to a sustained 17.8-kPa tensile stress, whereupon the times
required for each bond to fail were recorded. Consistent with the
short-term adhesion tests, the bond longevity was highly sensitive
to the pH of the parent PAH, PPi and TPP solutions (see FIG. 18).
The PAH/PPi and PAH/TPP samples prepared from solutions at pH 7.0
supported the stress the longest, followed closely by those
prepared at pH 6.0, where each bond on average lasted over an hour.
Conversely, bonds formed by the complexes prepared at pH 8.0 lasted
for only around 30 s. This indicates that these underwater
adhesives yield the most durable bonds when prepared from solutions
at near-neutral pH (where both PAH and cross-linking ions are
highly ionized), again reflecting the variations in their cohesive
strength.
[0156] Effects of Ionic Strength on Complex Formation
[0157] Because their various potential uses may subject these
underwater adhesives to different ionic strengths, the effect of
NaCl concentration on PAH/PPi and PAH/TPP complexes was also
investigated. Here, the aggregation states of PAH/PPi and PAH/TPP
mixtures prepared from parent PAH, PPi and TPP solutions at pH 7.0
were monitored at various ion:monomer molar ratios and NaCl
concentrations. When no NaCl was added, colloidal dispersions
formed above a PPi:PAH molar ratio of 0.11:1 and a TPP:PAH molar
ratio of 0.01:1 and coagulated above a PPi:PAH molar ratio of
0.26:1 and a TPP:PAH molar ratio of 0.19:1 (see FIGS. 19A-19B).
Above a TPP:PAH molar ratio of 0.25:1, however, the coagulation was
slowed to the point that the complexes remained dispersed
throughout the month-long experiment, while the PAH/PPi mixtures
continued to coagulate even at a 0.50:1 PPi:PAH ratio. The
multivalent ion:PAH monomer ratio at which PAH/PPi colloidal
dispersions began forming was completely insensitive to the NaCl
concentration (see "S/D" phase boundary in FIG. 19A). Similarly,
the onset of dispersion formation in PAH/TPP mixtures shifted from
0.01:1 to 0.09:1 when 5 mM NaCl was added but remained fixed at
0.09:1 as the NaCl concentration was raised from 5 to 500 mM (FIG.
19B). The 0.09:1 TPP:PAH molar ratio was also the molar ratio at
which colloidal complexes formed under NaCl-free conditions when
higher PAH concentrations (>6.+-.2 mM) were used, which
indicates that the previously reported sensitivity of the "S/D"
phase transition to the PAH concentration was an ionic strength
effect. The insensitivity of the phase boundary to further NaCl
addition, however, indicates that PAH/PPi and PAH/TPP complex
formation is (aside from PAH/TPP mixtures at very low ionic
strengths) insensitive to monovalent salt.
[0158] Despite the insensitivity of PAH/PPi and PAH/TPP
complexation to the ionic strength, NaCl had a significant impact
on the PPi:PAH and TPP:PAH molar ratios where these complexes
coagulated into macroscopic adhesives, which decreased as the NaCl
concentration increased (see "D" to "G+S" transitions in FIGS.
19A-19B). Indeed, at NaCl concentrations above 400 mM, the mixtures
changed directly from solutions to macroscopic adhesives as the
multivalent ion:PAH monomer ratios were increased, with no stable
dispersions forming in between. The NaCl effect on the coagulation
of PAH/TPP dispersions that formed at higher TPP:PAH molar ratios
was even more drastic, with colloidally stable dispersions ceasing
to form even at 5 mM NaCl (FIG. 19B).
[0159] To further analyze the NaCl effects on the aggregation
states, .zeta.-potential measurements were performed. Unlike the
.zeta.-potential curves at varying pH-levels, which kept their
general shapes but shifted in the ion:monomer molar ratios where
the precipitous .zeta.-potential drop occurred (cf. FIGS. 15A-15B),
NaCl addition drastically changed the shape of the .zeta.-potential
curves (see FIGS. 20A-20B). Without added NaCl, the colloidal
complexes that formed at PPi:PAH molar ratios below 0.26:1 and
TPP:PAH molar ratios below 0.19:1 had consistently-high
.zeta.-potentials (+50 to +70 mV) and were very stable. At the 150
and 500 mM NaCl concentrations, however, the apparent potentials
were much lower and (especially in 150 mM NaCl) increased at the
ion:monomer ratios where the complexes began to form (see FIGS.
19A-19B). For PAH/PPi mixtures, this difference in curve shape was
attributed to variations in the ionic cross-linking kinetics (which
become much slower at higher NaCl concentrations). Without added
NaCl, the colloidal complexes initially formed even when the
overall sample compositions were in the "S" regions in the state
diagrams (due to the elevated local PPi concentrations during their
titration into the PAH solution). Thus, .zeta.-potentials
reflective of colloidal complexes were detected from the beginning
of the titrations (although the unstable complexes at low PPi:PAH
monomer ratios dissolved after 1 day). Conversely, when NaCl was
added to the mixtures, the colloid formation was much slower. Thus,
the PPi was uniformly mixed into the PAH solution before the
colloidal complexes could assemble, and no colloidal complexes
formed during the titrations until the compositions reached the "D"
region in FIG. 19A (where the peaks in the .zeta.-potential curves
were detected; see FIG. 20A). While the same kinetic arguments
apply to the PAH/TPP mixtures, the compositions tested in the
.zeta.-potential titrations under the NaCl-free condition were all
in the "D" region in FIG. 19B (cf. FIG. 19B and FIG. 20B). Thus,
the altered shape of the PAH/TPP .zeta.-potential curves also
reflected a salt-triggered-shift in the "S/D" phase boundary.
Furthermore, because the .zeta.-potentials and the electrostatic
repulsion between the complexes were lower at higher salt
concentrations, the PAH/PPi and PAH/TPP complexes were more prone
to coagulation at lower multivalent ion:PAH monomer ratios. This
was especially true in 500 mM NaCl, where the .zeta.-potentials
never rose above +30 mV (FIGS. 20A-20B), and due to their weakened
electrostatic repulsion, the colloidal PAH/PPi and PAH/TPP
complexes rapidly coagulated within 1 h of their formation.
[0160] At higher ion:PAH monomer ratios, where the
.zeta.-potentials began to decrease with the ionic cross-linker
concentration, the .zeta.-potential reduction became less drastic
at higher ionic strengths. Indeed, at high ion:monomer ratios the
.zeta.-potentials increased with NaCl concentration (and no charge
inversion occurred in the PAH/TPP system in the presence of added
NaCl). This was consistent with previous work on chitosan/TPP
mixtures (where NaCl made the .zeta.-potentials of chitosan/TPP
microgels less sensitive to further TPP addition) and on
multivalent ion adsorption to polyelectrolyte brushes, and likely
reflected the competitive binding of monovalent ions to
cross-link-forming ionic groups. Interestingly, when charge
inversion occurred, the colloidal complexes remained dispersed for
one month even though the .zeta.-potentials (which ranged between
-10 and -20 mV) were quite low. This indicates that the coagulation
rates in PAH/PPi and PAH/TPP mixtures depend on both the colloidal
collision frequency--which increases with NaCl concentration due to
reduced electrostatic repulsion--and the probability of ionic
bridging (which likely diminishes upon charge inversion due to a
lack of free amine groups).
[0161] Effects of Ionic Strength on Water Content
[0162] As indicated by the .zeta.-potential measurements, the
strength of PAH/PPi and PAH/TPP binding decreased with the NaCl
concentration. Because of this impact on ionic cross-linking
strength, the salt effect on the ionic network swelling was
opposite to that in covalently cross-linked polyelectrolyte
networks. Instead of collapsing upon the addition of NaCl, PAH/PPi
and PAH/TPP networks increased their water content (albeit
modestly) with increasing NaCl concentration (see Table 4). This
effect indicated a reduction in crosslink density caused by the
weaker PPi and TPP binding and was more significant for the PAH/PPi
adhesives than PAH/TPP adhesives (presumably because TPP forms
stronger cross-links than PPi).
TABLE-US-00004 TABLE 4 Final pH Values and Average Water Contents
of Adhesive PAH/PPi and PAH/TPP Complexes Prepared at Different
NaCl Concentrations and a Parent PAH, PPi, and TPP Solution pH of
7.0 (.+-.Standard Deviation).sup.a. [NaCl] final pH water content
(wt %) (mM) PAH/PPi PAH/TPP PAH/PPi PAH/TPP 0 8.4 .+-. 0.2 9.1 .+-.
0.2 30.2 .+-. 0.1 26.0 .+-. 0.1 150 6.7 .+-. 0.1 6.4 .+-. 0.1 31.3
.+-. 0.6 28.0 .+-. 0.6 300 6.7 .+-. 0.1 6.4 .+-. 0.1 36.9 .+-. 0.6
28.9 .+-. 0.1 .sup.aThe final pH values are the readings obtained
from the supernatants.
[0163] Notably, the ionic strength also affected the pH drift
during PAH/PPi and PAH/TPP complexation (Table 4). Although the
parent solution pH was 7.0 in each case, complexation at 0 mM NaCl
increased the pH to 8.4-9.1, while complexation in 150 and 300 mM
NaCl decreased the pH to 6.4-6.7. This difference reflected the
ionic strength effects on the polyelectrolyte pK.sub.a, where
higher ionic strengths favored higher solution-phase PAH ionization
and therefore diminished the PAH effect on the pH drift. Despite
these variations in pH, however, the increase in water content
caused by the added salt was opposite to that expected with a pH
reduction (cf. Table 3) and confirmed that the swelling was a salt
(rather than a pH) effect.
[0164] Effects of Ionic Strength on Rheology
[0165] The weaker ionic cross-linking at higher NaCl concentrations
also affected the rheology of PAH/PPi and PAH/TPP complexes.
Despite the modest salt-induced changes in the water contents of
these adhesives, their G.sub..infin.'-values (and therefore their
network densities) appeared to be insensitive to salt (see FIGS.
21A-21B). Yet, as the NaCl concentration was increased from 0 to
150 mM, there was a decrease in the G'-values at lower frequencies
for both adhesive types, with G'/G'' crossover occurring at
.omega.c.about.0.1 rad/s and corresponding to a roughly 10 s
relaxation time. This decrease in relaxation time indicated that
both adhesives became more fluid-like and was qualitatively
consistent with the salt effects reported for polyelectrolyte
complexes (i.e., complexes between oppositely charged polymer
species).
[0166] As the NaCl concentration was increased further (to 300 mM),
there was an additional decrease in the G'-values at lower
frequencies for the PAH/PPi complex (with the crossover occurring
at an even higher frequency; .omega.c.about.0.5 rad/s). Conversely,
the rheology of the PAH/TPP complex exhibited little change as the
NaCl concentration was raised from 150 to 300 mM, thus indicating
the rheological properties of PAH/PPi adhesives to be more
sensitive to NaCl concentration than those of PAH/TPP adhesives.
This was consistent with the weaker impact of NaCl on the PAH/TPP
water content and again likely reflected the stronger PAH/TPP
binding. These salt-induced changes in relaxation dynamics were
roughly the same regardless of whether the NaCl was added before or
after the complexes formed. Interestingly, adhesive gels stored in
a supernatant at 150 mM NaCl concentration after originally
prepared with no added NaCl had very similar rheology to those
originally prepared in 150 mM NaCl.
[0167] Effects of Ionic Strength on Adhesion Strength
[0168] The ionic strength also affected the underwater adhesion
strength mediated by the PAH/PPi and PAH/TPP complexes. The
adhesive complexes prepared without added NaCl yielded the highest
tensile bond strengths, which became weaker at higher ionic
strengths (see FIGS. 22A-22B). As indicated by the rheology data,
this salt effect was stronger for the PAH/PPi complexes than for
the PAH/TPP complexes (especially in 300 mM NaCl, where the
adhesion strength of the PAH/TPP complex was twice that of the
PAH/PPi complex). Moreover, while the reduction in adhesion
strength in 300 mM NaCl was 2-4-fold, the reduction in average
adhesion strength in 150 mM NaCl (which roughly corresponds to
near-physiological ionic strength) was only modest (roughly 20-30%;
with short term bond strengths still ranging between 250 and 321
kPa). At each salt concentration the mode of bond failure was
cohesive for the glass substrate and adhesive for the Teflon
substrate. This indicates that elevated ionic strengths diminish
both the cohesive strength of the ionic networks and the strengths
of their interfacial interactions.
[0169] The addition of NaCl had an even stronger effect on adhesion
longevity. When the adhesives were prepared in 150 mM NaCl, the
average duration of their adhesion to glass (under a sustained
17.8-kPa tensile stress) diminished greatly, from roughly 3900 to
1000 s for the PAH/PPi complexes and from roughly 4600 to 2300 s
for the PAH/TPP complexes. This reduction in bond longevity was
even more drastic when the adhesives were formed in 300 mM NaCl,
where the bonds created by the PAH/PPi and PAH/TPP complexes lasted
only 202 and 230 s, respectively. This 5-10-fold further reduction
in bond longevity was surprising in the case of the PAH/TPP
complexes, whose rheology in 150 and 300 mM NaCl was quite similar
(see FIG. 23), and may have reflected rheological differences at
low w-values outside the experimental frequency range. In summary,
the reduction in PAH/PPi and PAH/TPP adhesion longevity at higher
salt concentrations is even more drastic than that of their
short-term adhesion strength and indicates that these complexes
form their strongest and most durable bonds at lower ionic
strengths.
[0170] Injectability
[0171] The sensitivity of PAH/PPi and PAH/TPP complexes to pH and
ionic strength weakens their adhesion when they are prepared at (or
exposed to) high/low pH and elevated salt concentrations. While
their pH sensitivity can be partially addressed by using PAH
analogues with higher effective pK.sub.a-values (to maintain their
high charge in alkaline solutions), their salt sensitivity
indicates that the adhesive properties of these ionotropic
complexes is especially useful at relatively low (roughly 0-150 mM)
ionic strengths. This ionic strength range is sufficient for using
these underwater adhesives in biomedical and pharmaceutical
applications. The sensitivity of PAH/PPi and PAH/TPP mixtures to pH
and ionic strength can also be exploited to expand their
functionality. For instance, these adhesive complexes can be
dissolved on demand by changing the pH. Another attractive feature
of this salt and pH sensitivity is that it can be leveraged to
develop injectable adhesives (for minimally invasive application)
composed of low-viscosity dispersions that coagulate into gel-like
macroscopic adhesives upon injection into their target sites. This
effect has previously been achieved with biomimetic polymers and
can be achieved with the readily available PAH/PPi and PAH/TPP
mixtures. To demonstrate this, a TPP/PAH mixture was prepared at a
TPP:PAH molar ratio of 0.40:1, where low-viscosity colloidal
dispersions form in the absence of added salt (see FIG. 19B). The
dispersion was then injected through a 21-gauge needle into
1.times. phosphate buffered saline (PBS) solution, which contained
a Teflon-coated magnetic stir bar (see FIGS. 9A-9C). Upon its
injection into PBS (where the ionic strength was near 150 mM), the
dispersion spontaneously coagulated into a gel-like layer.
Time-series photographs of this process are shown in FIGS. 9A-9D.
As seen in FIG. 9D, the magnetic stir bar adhered to the test tube
glass with a thin layer of gel 5 minutes after injection. This
gel-like layer formed within 5 min of injection and adhered the
Teflon-coated stir bar (5 mm.times.3 mm) to the bottom of the glass
test tube (as shown by the tube inversion test in FIG. 9D). Thus,
the salt sensitivity of PAH/PPi and PAH/TPP complexes (and likely
also their pH sensitivity) can be harnessed to design injectable
underwater adhesive formulations.
[0172] The short-term tensile bond strengths of PAH/PPi and PAH/TPP
adhesives prepared at near-optimal conditions (i.e., of around 400
kPa; see FIGS. 17, 22) are similar to those reported for the
natural underwater adhesives produced by barnacles and sandcastle
worms. The reversibility and pH/salt sensitivity of their ionic
cross-links, however, makes the bonds formed by PAH/PPi and PAH/TPP
adhesives less permanent and robust than those achieved using
covalent/biomimetic (e.g., catechol-based) chemistries. Despite
this, their stimulus sensitivity and lack of potentially harmful
covalent cross-linking render PAH/PPi and PAH/TPP adhesives
especially useful in situations where: (1) temporary or reversible
adhesion is desired and (2) harmful side reactions must be avoided.
Furthermore, their simple, scalable and inexpensive preparation
from readily-available ingredients make these underwater adhesives
advantageous for larger-scale use, where the application of highly
specialized biological (or biomimetic) polymers might not be
feasible.
[0173] Materials and Methods
[0174] All experiments were conducted using Millipore Direct-Q 3
deionized water (18.2 M.omega.m). PAH (nominal molecular weight
120-200 kDa) was obtained from Polysciences, Inc. (Warrington, Pa.)
and Alpha Aesar (Ward Hill, Mass.). PPi and TPP (both sodium salts)
were purchased from Sigma-Aldrich (St. Louis, Mo.). HCl and NaCl
were obtained from Fisher Scientific (Fair Lawn, N.J.) and VWR
(West Chester, Pa.), respectively. PBS was purchased in powder form
from Fisher Scientific (Fair Lawn, N.J.). The glass and Teflon
plates used for the adhesion tests were cut from Fisherfinest
premium microscope slides (Fisher Scientific) and 0.25-in.-thick
sheets of polytetrafluoroethylene (PTFE), respectively. All
materials were used as received.
[0175] To construct the pH dependent state diagrams, 0.04 wt % (4.4
mM) PAH stock solutions were mixed with either 0.37 wt % (13.5 mM)
PPi or 0.40 wt % (11.1 mM) TPP stock solutions at matching pH
levels (which were varied between 2.0 and 12.0 using HCl and NaOH).
Specifically, 2.5 mL aliquots of the PAH solution were injected
with different volumes (40-400 .mu.L) of either PPi or TPP
solution. The cuvettes were then shaken by hand and allowed to
equilibrate for 1 month. During equilibration, the formation and
coagulation of colloidal dispersions was detected by dynamic light
scattering (DLS), using a Zetasizer Nano ZS dynamic and
electrophoretic light scattering instrument (Malvern, UK) as
described previously. Here, a sudden increase in the light
scattering intensity indicated the formation of colloidal
dispersions, while a drastic increase in hydrodynamic diameter and
polydispersity index (PDI), along with visible macroscopic
precipitation, revealed the coagulation of colloidal complexes into
gel-like adhesives. The ionic strength-dependent state diagrams
were then constructed using the same procedure; however, the PAH,
PPi, and TPP stock solutions were now prepared at variable NaCl
concentrations (ranging between 0 and 500 mM) and a constant pH of
7.0.
[0176] The changes in the apparent .zeta.-potentials upon the
addition of PPi and TPP to PAH were characterized by
electrophoretic light scattering, again using the Zetasizer Nano ZS
instrument (where the .zeta.-potentials were estimated from
electrophoretic mobility measurements via the Smoluchowski
equation). Here, 160 .mu.L aliquots of either 0.37 wt % PPi or 0.40
wt % TPP at pH 6.0, 7.0, or 8.0 were sequentially added to 10 mL of
0.04 wt % PAH solution at a matching pH, while stirring the
mixtures at 800 rpm with cylindrical magnetic stir bars (10
mm.times.5 mm). The .zeta.-potentials were then measured after each
addition (following 10 min of equilibration). The evolutions in the
apparent potentials with the addition of PPi and TPP to PAH
mixtures at different NaCl concentrations were measured using the
same procedure; however, the PPi, TPP and PAH solutions were now
prepared at a fixed pH (of 7.0) and at variable NaCl concentrations
(i.e., 0, 150 or 500 mM). Each measurement was performed using
three replicate samples.
[0177] To quantify the pH and ionic strength effects on the water
content within the adhesives, their samples were prepared at
various pH and ionic strength levels, whereupon their wet and dry
weights were measured. The samples at variable pH-levels were
prepared by slowly adding either 3.9 wt % PPi solution or 5.7 wt %
TPP solution at pH 6.0, 7.0 or 8.0 to 1000 mL of 0.1 wt % PAH at a
matching pH. Since the amount of PPi and TPP required for
macroscopic phase separation was pH-dependent, the volume of PPi
and TPP solution added to the PAH solution varied with the pH. At
pH 6.0 either 30 mL of PPi or 17 mL of TPP solution was added, at
pH 7.0 either 26 mL of PPi or 14 mL of TPP solution was added, and
at pH 8.0 either 22 mL of PPi or 11 mL of TPP solution was added.
Furthermore, to ensure that the water content within the adhesive
complexes was insensitive to the PPi, TPP and PAH compositions,
additional complexes were prepared by adding either 30 mL of PPi
solution or 17 mL of TPP solution to the PAH solution at pH 7.0
(which revealed no change in water content). Samples at variable
NaCl concentrations were prepared in a similar fashion, by slowly
adding 26 mL of 3.9 wt % PPi solution or 14 mL of 5.7 wt % TPP
solution at pH 7.0 and 0, 150 or 300 mM NaCl to 1000 mL of 0.1 wt %
PAH at a matching pH and NaCl concentration. During the PPi and TPP
additions, the receiving PAH solutions were stirred at 300 rpm with
cylindrical magnetic stir bars (5 cm.times.1 cm). The ionically
cross-linked complexes were allowed to coagulate for 3 days,
whereupon they were scraped from the bottoms of the beakers. The
wet weight of each adhesive sample (0.175-0.190 g) was then
recorded after carefully removing all excess supernatant from the
sample surface with a KimWipe. The samples were dried for 72 h in
an oven at 37.degree. C., whereupon the dry mass of the adhesive
was recorded. All measurements were performed in triplicate.
[0178] The dynamic rheology of the adhesives was characterized at
room temperature using a Rheometric Scientific RDA III (Piscataway,
N.J.) strain-controlled rheometer equipped with 25-mm parallel
plates. The samples were prepared using the procedure described
above. The gel-like complexes were then loaded into the rheometer,
compressed to a 0.5-mm gap thickness and allowed to relax between
the plates until the normal force was below 100 g. The excess
sample was then removed using a spatula, and water was applied to
its exposed outer edges to prevent drying. After performing strain
amplitude sweeps to determine the linear viscoelasticity region,
frequency sweeps were performed at 0.1-500 rad/s angular velocities
and a 1.0% strain amplitude for all of the samples except for those
prepared from parent solutions at pH 8.0. When prepared at this
higher pH, the adhesives were much softer and required a 10.0%
strain amplitude (which for these samples was still within the
linear viscoelasticity region) to achieve sufficient torque. Three
replicate samples were analyzed under each condition.
[0179] The adhesive complexes were prepared at variable pH-levels
by slowly adding 4.2 wt % (158 mM) PPi or 5.6 wt % (152 mM) TPP
solution at pH 6.0, 7.0, or 8.0 to 1000 mL of 0.3 wt % (32 mM) PAH
at the same pH. The volume of PPi and TPP solution added to the PAH
again depended on the pH used (with either 76 mL of PPi or 50 mL of
TPP solution added at pH 6.0, either 66 mL of PPi or 42 mL of TPP
solution added at pH 7.0, and either 56 mL of PPi or 33 mL of TPP
solution added at pH 8.0). This variance in PPi and TPP solution
volume was again necessary to ensure that adhesive formation
occurred and had no discernible impact on adhesive properties (as
confirmed by probing the rheology and adhesion strengths of
complexes prepared from solutions at pH 7.0 and PPi:PAH or TPP:PAH
molar ratios ranging between 0.33-0.38:1 and 0.20-0.24:1,
respectively; data not shown). The adhesive samples were similarly
prepared at variable NaCl concentrations by slowly adding 66 mL of
4.2 wt % PPi solution or 42 mL of 5.6 wt % TPP solution at pH 7.0
and 0, 150 or 300 mM NaCl to 1000 mL of 0.3 wt % PAH solution at a
matching pH and NaCl concentration. Each receiving PAH solution was
stirred at 300 rpm as indicated above.
[0180] The gel-like complexes were then equilibrated for 3 days
before being collected from the bottoms of the beakers and being
used to adhere two 2.5 cm.times.2.5 cm substrate surfaces (either
glass or Teflon). Each substrate was superglued onto custom-made
Plexiglas brackets (using Loctite Glass Glue for the glass and
Gorilla Super Glue for the Teflon), which enabled its placement
into the stress-strain analyzer. To adhere the two plates, 0.5-1.0
g of the adhesive complex (a more precise application was
complicated by its adhesion to the spatula) was pressed between the
plates to a final thickness of 0.33-0.43 mm. For the samples
prepared at pH 6.0 and 7.0 and NaCl concentrations of 0 and 150 mM,
this was achieved by pressing the two plates together for 3 h under
deionized water and 24 kPa of pressure. Conversely, for the samples
prepared at pH 8.0 and 300 mM NaCl, this was achieved by hand
pressing the two plates together under deionized water to the
specified (0.33-0.43 mm) thickness. This alternative procedure was
used because these adhesive complexes were much softer and applying
24 kPa of pressure for 3 h caused most of the adhesive to be
squeezed out from between the two plates (resulting in much thinner
adhesive layers). After removing the excess adhesive from the sides
of the plates, the adhered plates remained submerged in deionized
water (for 15-30 min) until the tensile bond strength test was
performed using an Instron 4400R Universal Testing Machine (UTM;
Norwood, Mass.). The adhered plates were then clamped into the
grips of the UTM and immediately separated at a crosshead speed of
0.85 mm/s while measuring both the force and displacement. Each
measurement was repeated six times.
[0181] The samples for the adhesion longevity tests were prepared
and compressed between two glass plates using the procedure
described above. One plate was then held stationary while a 1.1-kg
weight was suspended from the second plate (thereby applying a
17.8-kPa tensile stress to the bond). The time required for the
adhesion to fail at each pH-value and NaCl concentration was then
measured while reapplying deionized water to the exposed edges of
the adhesives every 5 min to prevent them from drying. To ensure
reproducibility, each measurement was replicated thrice.
[0182] To demonstrating preparation of stimulus-responsive
injectable adhesives, 1.40 mL of 5.7 wt % TPP solution was added to
10.0 mL of 0.5 wt % PAH solution (both at pH 7.0 and 0 mM NaCl), so
that the TPP:PAH molar ratio was 0.40:1. One mL of the dispersion
was then injected through a 21-gauge syringe needle into 1 mL of
1.times.PBS containing a Teflon-coated magnetic stir bar (5
mm.times.3 mm). After the dispersion was added to the PBS solution,
the mixture remained undisturbed (without stirring) for 5 min,
whereupon the test tube was inverted to determine whether the
Teflon-coated stir bar was adhered to the glass test tube.
[0183] Certain embodiments of the compositions and methods
disclosed herein are defined in the above examples. It should be
understood that these examples, while indicating particular
embodiments of the invention, are given by way of illustration
only. From the above discussion and these examples one skilled in
the art can ascertain the essential characteristics of this
disclosure, and without departing from the spirit and scope
thereof, can make various changes and modifications to adapt the
compositions and methods described herein to various usages and
conditions. Various changes may be made and equivalents may be
substituted for elements thereof without departing from the
essential scope of the disclosure. In addition, many modifications
may be made to adapt a particular situation or material to the
teachings of the disclosure without departing from the essential
scope thereof.
* * * * *