SELF-ASSEMBLING PEPTIDE COMPOSITIONS
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit under 35 U.S.C. §119(e) of U.S. provisional patent
application serial no. 61/950,529, filed March 10, 2014, which application is hereby incorporated
by reference in its entirety.
SEQUENCE LISTING
[0002] This application makes reference to a sequence listing submitted in electronic form as
an ascii .txt file named "2004837-0046_Sequences.txt". The .txt file was generated on March 9,
2015 and is 1 kb in size.
BACKGROUND
[0003] Peptide agents with the ability to self-assemble into gel structures have a wide variety
of uses in therapeutic and research contexts. One such peptide agent, for example, a synthetic,
16-amino acid polypeptide with a repeating sequence of arginine, alanine, and aspartic acid (i.e.,
RADARADARADARADA [SEQ ID NO:l], also known as "RADA16"), is commercially
available under the trade names PuraStat®, PuraMatrix®, and PuraMatrix GMP® from 3-D
Matrix Medical Technology, and has demonstrated utility in a wide range of laboratory and
clinical applications, including cell culture, drug delivery, accelerated cartilage and bone growth,
and regeneration of CNS, soft tissue, and cardiac muscle, and furthermore as a matrix, scaffold,
or tether that can be associated with one or more detectable agents, biologically active agents,
cells, and/or cellular components.
SUMMARY
[0004] The present disclosure provides, among other things, certain peptide compositions
(and particularly certain compositions of self-assembling peptide agents), and technologies
relating thereto. In some embodiments, such compositions may be or comprise solutions. In
some embodiments, such compositions may be or comprise gels. In some embodiments, such
compositions may be or comprise solid (e.g., dried/lyophilized) peptides.
[0005] For example, the present disclosure demonstrates that particular peptide compositions
(i.e., peptide compositions having specific concentration, ionic strength, pH, viscosity and/or
other characteristics) have useful and/or surprising attributes (e.g., gelation or self-assembly
kinetics [e.g., rate of gelation and/or rate and reversibility of peptide self-assembly], stiffness
[e.g., as assessed via storage modulus], and/or other mechanical properties). In some
embodiments, the present disclosure demonstrates particular utility of certain such compositions
in specific contexts (e.g., in certain in vivo or in vitro applications).
[0006] Among other things, the present disclosure provides guidelines that permit selection,
design, and/or formulation of particular peptide compositions useful in certain contexts or
applications.
[0007] The present disclosure establishes the extent to which certain cations and anions
interact with self-assembling peptide agents, and furthermore how such interactions can alter
certain material (e.g., rheological) properties (e.g., increase mechanical stiffness and/or viscosity)
of peptide compositions. Still further, the present disclosure establishes how such interactions
can influence, among other things, gelation kinetics, restoration of gelled state (e.g., timing
and/or extent of gelation and/or restoration of gel properties) after exposure to deformation (e.g.,
mechanical perturbation or other disruption).
[0008] Studies described herein have identified the source of various problems with certain
existing self-assembling peptide technologies, and furthermore define particularly useful and/or
necessary attributes and/or characteristics specific to particular applications of peptide
composition technologies.
[0009] In some embodiments, peptides included in provided compositions are selfassembling
peptides. In some embodiments, peptides included in provided compositions are
amphiphilic peptides. In some embodiments, peptides included in provided compositions have
an amino acid sequence characterized by at least one stretch (e.g., of at least 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20 etc amino acids) of alternating hydrophilic and hydrophobic
amino acids. In some embodiments, peptides included in provided compositions have an amino
acid sequence that includes one or more repeats of Arg-Ala-Asp-Ala (RADA). In some
embodiments, peptides included in provided compositions have an amino acid sequence that
comprises or consists of repeated units of the sequence Lys-Leu-Asp-Leu (KLDL). In some
embodiments, peptides included in provided compositions have an amino acid sequence that
comprises or consists of repeated units of the sequence Ile-Glu-Ile-Lys (IEIK). In some
embodiments, the peptides may be IEIK13, KLD12, or RADA16. In some embodiments,
compositions of these peptides may have enhanced properties relative to appropriate reference
compositions that have different (e.g., lower) pH level, and/or ionic strength.
[0010] Peptide compositions at a milder pH level may have stiffer rheological properties
rendering them suitable for a broader range of applications. Environmental pH change to over
4.0 may also beneficially impact gelation kinetics from peptide compositions. In some
embodiments, the increased pH may be physiological pH which may occur when the peptide
compositions are placed into the body.
[0011] In accordance with one or more aspects, rheological properties of certain peptide
compositions, including but not limited to IEIK13, KLD12, and RADA16, may be enhanced by
maintaining increased ionic strength. In some embodiments, the ionic strength may be lower
than critical ionic strength. In some embodiments, peptides compositions may be dissolved in
water with salts instead of pure water. In some embodiments, the ionic strengths may be lower
than their critical ionic strengths.
[0012] In some embodiment, increased ionic strength may beneficially impact stiffness
and/or gelation kinetics to peptide compositions rendering them suitable for a broader range of
applications. In some embodiments, increased ionic strength may be physiological ionic
strength, which may occur when peptide compositions are placed into the body.
[0013] In accordance with one or more aspects, properties of certain peptide compositions,
including but not limited to IEIK13, KLD12, and RADA16, may be enhanced by maintaining
their pH level at about 3.5 or less and, at the same time, their salt concentrations at less than their
critical ionic strength levels (i.e. no precipitation).
[0014] In accordance with one or more aspects, self-assembling peptides, for example,
IEIK13, KLD12, and RADA16, may be characterized in terms of properties including
appearance, pH level, ionic strength level, gelation kinetics, rheological properties, and cell
viability to optimize peptide formulations for various applications. IEIK13 and KLD12 may be
characterized as similar peptide compositions to RADA16 in terms of basic gelation properties
and other characteristics.
[0015] In some embodiments, a peptide may have a length within the range of about 6 to
about 20 amino acids and an amino acid sequence of alternating hydrophobic amino acid and
hydrophilic amino acids.
[0016] In some embodiments, a peptide composition may be solution, gel, or any
combination thereof.
[0017] In some embodiments, a peptide composition may be at a concentration of at least
0.05%. In some embodiments, a peptide composition may be present at a concentration of less
than 3%
[0018] In some embodiments, a peptide composition may have a pH within the range of
about 2.5 to about 4.0, or within the range of about 3.0 to about 4.0. In some embodiments, pH
of a peptide composition can be achieved with a solution selected from the group consisting of
sodium hydroxide or, potassium hydroxide, calcium hydroxide, sodium carbonate, sodium
acetate, sodium sulfide, DMEM (Dulbecco's modified Eagle's medium), and PBS(Phosphate-
Buffered Saline).
[0019] In some embodiments, an ionic strength of a peptide composition may be about
0.0001 M to about 1.5 M. In some embodiments, an ionic strength of a peptide composition may
be adjusted by mixing common salts, for example, NaCl, KC1, MgCl2, CaCl2, CaS0 4, DPBS
(Dulbecco's Phosphate-Buffered Saline, 10X). In some embodiments, ionic strengths of peptide
compositions may be adjusted by mixing common salts, wherein one or more common salts are
composed of one or more salt forming cations and one or more salt forming anions, wherein the
salt forming cations are selected from the group consisting of ammonium, calcium, iron,
magnesium, potassium, pyridinium, quaternary ammonium, and sodium, wherein the salt
forming anions are selected from the group consisting of acetate, carbonate, chloride, citrate,
cyanide, floride, nitrate, nitrite, and phosphate.
[0020] In some embodiments, a peptide composition may have a viscosity with the range of
about 1 to about 10000 Pa-S. In some embodiments, a peptide composition may have a storage
modulus with the range of about 50 to about 2500 Pa.
[0021] In some embodiments, a method of selecting a peptide composition for applications
to a particular in vivo site may comprise steps of determining one or more parameters selected
from the group consisting of storage modulus, viscosity, gelation time, restoration time and/or
extent, etc for the peptide composition, comparing the determined parameters to specifications
for various applications, choosing the peptide composition in light of the comparison; and
administering the chosen peptide composition to the site.
[0022] In some particular embodiments, the present disclosure provides liquid peptide
compositions that may, for example, comprise a peptide having a length within the range of
about 6 to about 20 amino acids and an amino acid sequence of alternating hydrophobic amino
acid and hydrophilic amino acids, and may be characterized in that (i) it has a viscosity within
the range of about 1 Pa-s to about 500,000 Pa-s at room temperature; (ii) ithas a storage modulus
at 1 rad/sec of frequency and 1 Pa of oscillation stress within the range of about 1 to about 5000
Pa; and/or (iii) it forms a gel within a time period about 0 to about 30s when exposed
to/maintained under pH within the range of about 2.5 to about 4.0 or and/or ionic strength within
the range of about 0.0001 M to about 1.5 M. In some embodiments, such a composition is an
aqueous composition.
[0023] Also in some particular embodiments, the present disclosure provides methods of
designing, selecting, and or producing a peptide composition that is particularly appropriate for
use in a certain specific context. In some such embodiments, the certain specific context is or
comprises application to a particular in vivo site. In some embodiments, such provided methods
may comprise, for example: (i) determining one or more parameters selected from the group
consisting of storage modulus, viscosity, gelation time, shear-thinning property, peptide nanofiber
re-assembly time that is , and/or one or more other parameters as described herein that is
appropriate for application to the particular in vivo site; and (ii) designing, selecting, and/or
producing a peptide composition characterized by such parameters, in accordance with guidance
provided herein.
[0024] Alternatively or additionally, in some particular embodiments, the present disclosure
provides methods of selecting particular peptide compositions, for example for administration to
certain in vivo sites; exemplary such methods may comprise steps of (i) determining one or more
parameters selected from the group consisting of storage modulus, viscosity, gelation time,
shear-thinning property, peptide nano-fiber re-assembly time that is , and/or one or more other
parameters as described herein for a peptide composition; (ii) comparing the determined one or
more parameters to a set of characteristics determined to be appropriate for application to the
particular in vivo site; (iii) choosing the peptide composition in light of the comparison; and (iv)
administering the chosen peptide composition to the site.
BRIEF DESCRIPTION OF THE DRAWING
[0025] Objects and features of the invention can be better understood with reference to the
drawings described below, and the claims.
[0026] Figure 1 shows exemplary gel formations of peptide compositions in PBS buffer
solutions. RADA16, IEIK13, and KLD12 were plated at varying concentrations of 0.5%, 1.0%,
1.5%, 2.0% and 2.5%. RADA16, IEIK13, and KLD were gelled at all concentrations.
[0027] Figures 2A and 2B show exemplary rheological properties of RADA16. Figure 2A
depicts a stress sweep test performed at 1 Pa and 10 rad/s. Figure 2B shows measured storage
modulus as a function of RADA16 concentration. Storage modulus of RADA16 compositions
may have a linear relationship with their concentration.
[0028] Figures 3A and 3B show exemplary rheological properties of IEIK13. Figure 3A
depicts a stress sweep test performed at 1 Pa and 10 rad/s. Figure 3B shows measured storage
moduli as a function of IEIK concentration. Storage moduli of IEIK13 compositions may have a
linear relationship with their concentrations.
[0029] Figures 4A and 4B show exemplary rheological properties of KLD12. Figure 4A
depicts a stress sweep test performed at 1 Pa and 10 rad/s. Figure 4B shows measured storage
moduli as a function of KLD12 concentration. Storage moduli of KLD12 compositions may
have a linear relationship with their concentrations.
[0030] Figures 5A and 5B are bar graphs showing the effect of DMEM (Dulbecco's
modified Eagle's medium) treatment on 1% peptide compositions. Figure 5A depicts storage
modulus data (performed at 1 Pa and 10 rad/s) before or after the DMEM treatment on peptide
compositions. Figure 5B shows fold increases of storage modulus after the DMEM treatment.
[0031] Figure 6A is a picture of RADA16 and DMEM mixture (1:1 volume ratio). The
mixture was runny and cloudy. Figure 6B shows the mixture after centrifugation. RADA16 (i.e.
the translucent accumulation at the bottom of the centrifuge tube) was precipitated from the
mixture.
[0032] Figure 7 illustrates nanostructures of RADA16, KLD12, and IEIK13 compositions at
pH 2-3, and at physiological pH (DMEM). While the DMEM treatment may form stiff
compositions, mixing DMEM may precipitate peptides.
[0033] Figure 8 shows exemplary stress sweep tests at 10 rad/s of 1% KLD12 compositions
at pH = 2.0 and 3.4. Figure 9 shows exemplary stress sweep tests at 10 rad/s of 1% IEIK13
compositions at pH = 2.1 and 3.7. Figure 10 shows exemplary stress sweep tests at 10 rad/s of
1% RADA16 compositions at pH = 2.5 and 3.4. Storage moduli of peptide compositions were
increased with pH increase.
[0034] Figures 11A and 1IB show exemplary frequency sweep tests of RADA16 at 1 Pa.
Figure 11A is measurements of 1% RADA16 at pH 2.5 and 3.4. Figure 1IB is measurements of
2.5% RADA16 at pH 2.5 and 3.4.
[0035] Figure 12 is pictures of 2.5% RADA16 at pH 3.2, 3.4, 3.6 and 4.0. The composition
was clear when pH level was about 3.5, and slightly cloudy at pH = 3.6. The composition was
precipitated at pH = 4.0.
[0036] Figure 13 illustrates nanostructures and/or reassembly of RADA16, KLD12, and
IEIK13 at different pH levels, with or without shear stress. Dominant interactions may be
determined by pH and shear stress.
[0037] Figures 14-16 show cell viabilities (mMSCs) of RADA16, IEIK and KLD,
respectively, at selected concentrations. * is denoted that the cell viability is significantly lower
than the cell viability at next left column (p-value < 0.05). # is denoted that the cell viability is
significantly higher than the cell viability at next left column (p-value < 0.05).
[0038] Figure 17A illustrates a structure of IEIK13. Figure 17B shows SEM images of
IEIK1 3 before and after the DMEM treatment. IEIK1 3 fibers after the DMEM treatment may be
thicker than the fibers before the DMEM treatment.
[0039] Figure 18 is a bar graph of storage modulus, showing the effect of pH on the
rheological properties of 2.5% RADA16. The storage moduli were measured at 1 rad/s.
[0040] Figure 19 is a bar graph of storage modulus, showing the effect of pH on the
rheological properties of 1.5% IEIK13. The storage moduli were measured at 1 rad/s.
[0041] Figure 20A shows exemplary flow viscosity tests of 1% IEIK13 at pH = 2.1, 3.0, 3.3
and 3.5. Figure 20B is a bar graph of exemplary viscosity measurements with the shear rate of
0.003 1/sec.
[0042] Figures 2 1A, 21B, 21C and 2 ID show storage modulus measurements as a function
of time after applying high shear stress to 1% IEIK13 at pH = 2.1, 3.0, 3.3, and 3.5, respectively.
The horizontal lines indicate the original storage moduli of 1% IEIK13.
[0043] Figures 22A and 22B are bar graphs of storage modulus as a function of RADA16
concentration at pH 2.2 and 3.4. Figure 22A is measurements before the DMEM treatment.
Figure 22B is measurements after the DMEM treatment.
[0044] Figures 23A and 23B are bar graphs of storage modulus as a function of IEIK
concentration at pH 2.3 and 3.4. Figure 23A is measurements before the DMEM treatment.
Figure 23B is measurements after the DMEM treatment.
[0045] Figure 24 and 25 show storage modulus measurement as a function of time. Figure
24 includes 2.5% RADA16 data at pH 2.2, 2.6, 2.8, 3.1 and 3.4. Figure 25 includes 1.5%
IEIK13 data at pH 2.3, 2.6, 2.9 and 3.2. Time sweep tests were performed at 1 rad/sec and at 1
Pa with 20 mm plates and 500 mih gap distance. During time sweep tests, DMEM was added
into the chamber surrounding the measuring plates to soak the peptides at time = 0.
[0046] Figure 26 illustrates nanostructures and/or reassembly of peptides at low salt
conditions or high salt conditions (i.e. over critical ionic strength). The application methods of
salt solutions (treatment or mixing) may change the nanostructures. While treating peptides with
a salt solution may form stiff gel, mixing a salt solution to peptides may cause phase separation.
[0047] Figures 27-29 show exemplary frequency sweep tests from 1 rad/s to 10 rad/s at 1 Pa.
Figure 27 is measurements of 1% KLD12 with or without NaCl solution (0.2M ionic strength).
Figure 28 is measurements of 1% IEIK13 with or without NaCl solution (0.02M ionic strength).
Figure 29 is measurements of 1% RADA16 with or without NaCl solution (0.7 M ionic
strength).
[0048] Figure 30 is a bar graph of storage modulus at 1 rad/s. 1.0%> RADA16 was exposed
to NaCl of which ionic strengths varied from 0 to 1.0 M.
[0049] Figure 31 is a bar graph of storage modulus at 1 rad/s. 1.0%> IEIK13 was exposed to
NaCl of which ionic strengths varied from 0 to 0.04 M.
[0050] Figure 32A shows flow viscosity tests of 1% IEIK13 at NaCl ionic strengths of 0,
0.01 and 0.02 M. Figure 32B is a bar graph of viscosity with the shear rate of 0.003 1/sec.
[0051] Figures 33A, 33B and 33C show storage modulus measurements as a function of time
after applying high shear stress to 1% IEIK13 at NaCl ionic strength of 0, 0.01, and 0.02,
respectively. The horizontal lines indicate the original storage moduli of each 1% IEIK13
compositions.
[0052] Figure 34 shows exemplary storage moduli of 1% RADA16 at 1Pa as a function of
NaCl ionic strength before or after the DMEM treatment.
[0053] Figure 35 shows exemplary storage moduli of 1% IEIK13 at lPa as a function of
NaCl ionic strength before or after the DMEM treatment.
[0054] Figure 36 shows exemplary storage moduli of 1% RADA16 with selected salts
(NaCl, KC1, MgCl 2, and CaCl2) . * denotes that G' is significantly higher than control (no salt)
(P < 0.05). # denotes that G' is significantly lower than 1% RADA16 with NaCl ionic strength
of0.15M (P < 0.05).
[0055] Figure 37 shows exemplary storage moduli of 1% RADA16 with selected salts
(NaCl, KC1, MgCl 2, and CaCl2) after the DMEM treatment. * denotes that G' is significantly
higher than control (no salt, after DMEM treatment) (P < 0.05).
[0056] Figure 38 shows exemplary storage modulus measurements as a function of time for
1.5% IEIK13, 1.5% KLD12, and 2.5% RADA16 after the saline buffer treatment. Time sweep
tests were performed at 1 rad/sec and at 1 Pa with 20 mm plates and 500 m gap distance.
During time sweep tests, the saline buffer was added into the chamber surrounding the
measuring plates to soak the peptides at time = 0.
[0057] Figure 39 illustrates nanostructures and/or reassembly of RADA16, KLD12, and
IEIK13, at certain ionic strengths. High shear stress may change the nanostructures and/or
reassembly.
[0058] Figure 40 shows storage moduli of 2.5% RADA16 at 1 rad/sec. NaCl addition and
pH elevation increased storage moduli of 2.5% RADA16.
[0059] Figures 41A, 41B, 41C and 41D illustrate steps utilized in preparing peptide
compositions with different salts and/or salt concentrations as described in Examples 4 and 7. Those
of ordinary skill will appreciate that a similar strategy can be utilized, for example, to analyze peptide
compositions with different pHs, peptide concentrations, etc. In Figure 4 1A, the peptide powder was
placed with a glass vial. In Figure 41B, the peptide powder was dissolved first in deionized water at
a selected fraction of the final volume; vortexing and/or sonication was utilized as desired to achieve
or ensure complete solubilization. In Figure 41C, a concentrated salt solution was added on top, in
an amount and concentration dependent on the volume of deionized water used. In Figure 4ID, the
solution ws mixed, for example by vortexing.
[0060] Figure 42A, 42B, 42C, 42D, 42E, 42F and 42G are upright and inverted pictures of 0.5%
RADA16 and 0, 0.005, 0.05, 0.125, 0.250, 0.500, and 1M CaCh mixture, respectively. Figure 42E
shows an optimal and fully functional gel. Figure 42F shows a semi-functional gel. Figure 42G
shows a non-functional gel.
[0061] Figure 43 shows storage modulus measurements of 0.5% RADA16 mixed with NaCl,
KC1, and CaCh at concentrations of 0.125, 0.250, and 0.500 M.
[0062] Figure 44 shows storage modulus measurements of 2.5% RADA16 and 2.5% RADA16
with 0.125 M CaCh .
[0063] Figure 4 A and 45B show shows storage modulus measurements of RADA16 with
0.125, 0.250, and 0.500 M after mechanical perturbation. The * denotes that the control sample and
the perturbed sample are significantly different.
[0064] Figure 46 is storage modulus measures of 2.5% RADA16 treated with CaCl 2 and
CaS0 4 as a function of time.
[0065] Figure 47A is exemplary rheological data of IEIK13 and IEIK13 with Indigo
Carmine. Figure 47B shows stiffness of IEIK13 and IEIK13 with Indigo Carmine. Figure 47C
shows stiffness of RADA16 and RADA16 with Ringer's Solution. Figure 47D is a picture of
RADA16 with Ringer's Solution inverted. Figure 46E is pictures of IEIK13 and IEIK13 with
Indigo Carmine inverted. Figure 47F is a picture of IEIK13 with Indigo Carmine and placed
within a syringe.
[0066] Figure 48 is a graph of 2.5% RADA1 6 and 2.5% RADA1 6 with NaCl, showing an
increase in burst pressure of a lung.
DEFINITIONS
[0067] The term "agent" as used herein may refer to a compound or entity of any chemical
class including, for example, polypeptides, nucleic acids, saccharides, lipids, small molecules,
metals, or combinations thereof. In some embodiments, an agent is or comprises a natural
product in that it is found in and/or is obtained from nature. In some embodiments, an agent is
or comprises one or more entities that is man-made in that it is designed, engineered, and/or
produced through action of the hand of man and/or is not found in nature. In some
embodiments, an agent may be utilized in isolated or pure form; in some embodiments, an agent
may be utilized in crude form. In some embodiments, potential agents are provided as
collections or libraries, for example that may be screened to identify or characterize active agents
within them. Some particular embodiments of agents that may be utilized in accordance with the
present invention include small molecules, antibodies, antibody fragments, aptamers, nucleic
acids (e.g., siRNAs, shRNAs, DNA/RNA hybrids, antisense oligonucleotides, ribozymes),
peptides, peptide mimetics, etc. In some embodiments, an agent is or comprises a polymer. In
some embodiments, an agent is not a polymer and/or is substantially free of any polymer. In
some embodiments, an agent contains at least one polymeric moiety. In some embodiments, an
agent lacks or is substantially free of any polymeric moiety.
[0068] As used herein, the term "amino acid," in its broadest sense, refers to any compound
and/or substance that can be incorporated into a polypeptide chain, e.g., through formation of one
or more peptide bonds. In some embodiments, an amino acid has the general structure H2NC(
H)(R)-COOH. In some embodiments, an amino acid is a naturally-occurring amino acid. In
some embodiments, an amino acid is a synthetic amino acid; in some embodiments, an amino
acid is a D-amino acid; in some embodiments, an amino acid is an L-amino acid. "Standard
amino acid" refers to any of the twenty standard L-amino acids commonly found in naturally
occurring peptides. "Nonstandard amino acid" refers to any amino acid, other than the standard
amino acids, regardless of whether it is prepared synthetically or obtained from a natural source.
In some embodiments, an amino acid, including a carboxy- and/or amino-terminal amino acid in
a polypeptide, can contain a structural modification as compared with the general structure
above. For example, in some embodiments, an amino acid may be modified by methylation,
amidation, acetylation, and/or substitution as compared with the general structure. In some
embodiments, such modification may, for example, alter the circulating half life of a polypeptide
containing the modified amino acid as compared with one containing an otherwise identical
unmodified amino acid. In some embodiments, such modification does not significantly alter a
relevant activity of a polypeptide containing the modified amino acid, as compared with one
containing an otherwise identical unmodified amino acid. As will be clear from context, in some
embodiments, the term "amino acid" is used to refer to a free amino acid; in some embodiments
it is used to refer to an amino acid residue of a polypeptide.
[0069] As used herein, the term "approximately" or "about," as applied to one or more
values of interest, refers to a value that is similar to a stated reference value. In certain
embodiments, the term "approximately" or "about" refers to a range of values that fall within
25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%,
3% , 2%>, 1% , or less in either direction (greater than or less than) of the stated reference value
unless otherwise stated or otherwise evident from the context (except where such number would
exceed 100% of a possible value).
[0070] Two events or entities are "associated" with one another, as that term is used herein,
if the presence, level and/or form of one is correlated with that of the other. For example, a
particular entity (e.g., polypeptide, genetic signature, metabolite, etc) is considered to be
associated with a particular disease, disorder, or condition, if its presence, level and/or form
correlates with incidence of and/or susceptibility to the disease, disorder, or condition (e.g.,
across a relevant population). In some embodiments, two or more entities are physically
"associated" with one another if they interact, directly or indirectly, so that they are and/or
remain in physical proximity with one another. In some embodiments, two or more entities that
are physically associated with one another are covalently linked to one another; in some
embodiments, two or more entities that are physically associated with one another are not
covalently linked to one another but are non-covalently associated, for example by means of
hydrogen bonds, van der Waals interaction, hydrophobic interactions, magnetism, and
combinations thereof.
[0071] The term "comparable" is used herein to describe two (or more) sets of conditions,
circumstances, individuals, or populations that are sufficiently similar to one another to permit
comparison of results obtained or phenomena observed. In some embodiments, comparable sets
of conditions, circumstances, individuals, or populations are characterized by a plurality of
substantially identical features and one or a small number of varied features. Those of ordinary
skill in the art will appreciate that sets of circumstances, individuals, or populations are
comparable to one another when characterized by a sufficient number and type of substantially
identical features to warrant a reasonable conclusion that differences in results obtained or
phenomena observed under or with different sets of circumstances, individuals, or populations
are caused by or indicative of the variation in those features that are varied. Those skilled in the
art will appreciate that relative language used herein (e.g., enhanced, activated, reduced,
inhibited, etc) will typically refer to comparisons made under comparable conditions.)
[0072] Certain methodologies described herein include a step of "determining". Those of
ordinary skill in the art, reading the present specification, will appreciate that such "determining"
can utilize or be accomplished through use of any of a variety of techniques available to those
skilled in the art, including for example specific techniques explicitly referred to herein. In some
embodiments, determining involves manipulation of a physical sample. In some embodiments,
determining involves consideration and/or manipulation of data or information, for example
utilizing a computer or other processing unit adapted to perform a relevant analysis. In some
embodiments, determining involves receiving relevant information and/or materials from a
source. In some embodiments, determining involves comparing one or more features of a
sample or entity to a comparable reference.
[0073] The term "gel" as used herein refers to viscoelastic materials whose rheological
properties distinguish them from solutions, solids, etc. In some embodiments, a composition is
considered to be a gel if its storage modulus ( ) is larger than its modulus (G"). In some
embodiments, a composition is considered to be a gel if there are chemical or physical crosslinked
networks in solution, which is distinguished from entangled molecules in viscous solution.
[0074] The term "in vitro " as used herein refers to events that occur in an artificial
environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within a multi
cellular organism.
[0075] The term "in vivo " as used herein refers to events that occur within a multi-cellular
organism, such as a human and a non-human animal. In the context of cell-based systems, the
term may be used to refer to events that occur within a living cell (as opposed to, for example, in
vitro systems).
[0076] The term "peptide" as used herein refers to a polypeptide that is typically relatively
short, for example having a length of less than about 100 amino acids, less than about 50 amino
acids, less than 20 amino acids, or less than 10 amino acids.
[0077] The term "polypeptide" as used herein refers to any polymeric chain of amino acids.
In some embodiments, a polypeptide has an amino acid sequence that occurs in nature. In some
embodiments, a polypeptide has an amino acid sequence that does not occur in nature. In some
embodiments, a polypeptide has an amino acid sequence that is engineered in that it is designed
and/or produced through action of the hand of man. In some embodiments, a polypeptide may
comprise or consist of natural amino acids, non-natural amino acids, or both. In some
embodiments, a polypeptide may comprise or consist of only natural amino acids or only nonnatural
amino acids. In some embodiments, a polypeptide may comprise D-amino acids, Lamino
acids, or both. In some embodiments, a polypeptide may comprise only D-amino acids.
In some embodiments, a polypeptide may comprise only L-amino acids. In some embodiments,
a polypeptide may include one or more pendant groups or other modifications, e.g., modifying or
attached to one or more amino acid side chains, at the polypeptide's N-terminus, at the
polypeptide's C-terminus, or any combination thereof. In some embodiments, such pendant
groups or modifications may be selected from the group consisting of acetylation, amidation,
lipidation, methylation, pegylation, etc., including combinations thereof. In some embodiments,
a polypeptide may be cyclic, and/or may comprise a cyclic portion. In some embodiments, a
polypeptide is not cyclic and/or does not comprise any cyclic portion. In some embodiments, a
polypeptide is linear. In some embodiments, a polypeptide may be or comprise a stapled
polypeptide. In some embodiments, the term "polypeptide" may be appended to a name of a
reference polypeptide, activity, or structure; in such instances it is used herein to refer to
polypeptides that share the relevant activity or structure and thus can be considered to be
members of the same class or family of polypeptides. For each such class, the present
specification provides and/or those skilled in the art will be aware of exemplary polypeptides
within the class whose amino acid sequences and/or functions are known; in some embodiments,
such exemplary polypeptides are reference polypeptides for the polypeptide class or family. In
some embodiments, a member of a polypeptide class or family shows significant sequence
homology or identity with, shares a common sequence motif (e.g., a characteristic sequence
element) with, and/or shares a common activity (in some embodiments at a comparable level or
within a designated range) with a reference polypeptide of the class; in some embodiments with
all polypeptides within the class). For example, in some embodiments, a member polypeptide
shows an overall degree of sequence homology or identity with a reference polypeptide that is at
least about 30-40%, and is often greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99% or more and/or includes at least one region (e.g., a
conserved region that may in some embodiments be or comprise a characteristic sequence
element) that shows very high sequence identity, often greater than 90%> or even 95%, 96%,
97%, 98%o, or 99%. Such a conserved region usually encompasses at least 3-4 and often up to 20
or more amino acids; in some embodiments, a conserved region encompasses at least one stretch
of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids. In some
embodiments, a useful polypeptide may comprise or consist of a fragment of a parent
polypeptide. In some embodiments, a useful polypeptide as may comprise or consist of a
plurality of fragments, each of which is found in the same parent polypeptide in a different
spatial arrangement relative to one another than is found in the polypeptide of interest (e.g.,
fragments that are directly linked in the parent may be spatially separated in the polypeptide of
interest or vice versa, and/or fragments may be present in a different order in the polypeptide of
interest than in the parent), so that the polypeptide of interest is a derivative of its parent
polypeptide.
[0078] The term "reference" as used herein describes a standard or control relative to which
a comparison is performed. For example, in some embodiments, an agent, animal, individual,
population, sample, sequence or value of interest is compared with a reference or control agent,
animal, individual, population, sample, sequence or value. In some embodiments, a reference or
control is tested and/or determined substantially simultaneously with the testing or determination
of interest. In some embodiments, a reference or control is a historical reference or control,
optionally embodied in a tangible medium. Typically, as would be understood by those skilled
in the art, a reference or control is determined or characterized under comparable conditions or
circumstances to those under assessment. Those skilled in the art will appreciate when sufficient
similarities are present to justify reliance on and/or comparison to a particular possible reference
or control.
[0079] The term "self-assembling" is used herein in reference to certain polypeptides that,
under appropriate conditions, can spontaneously self-associate into structures so that, for
example, solutions (e.g., aqueous solutions) containing them develop gel character. In some
embodiments, interactions between and among individual self-assembling polypeptides within a
composition are reversible, such that the composition may reversibly transition between a gel
state and a solution state. In some embodiments, self-assembly (and/or dis-assembly) is
responsive to one or more environmental triggers (e.g., change in one or more of pH,
temperature, ionic strength, osmolarity, osmolality, applied pressure, applied shear stress, etc).
In some embodiments, compositions of self-assembling polypeptides are characterized by
detectable beta-sheet structure when the polypeptides are in an assembled state.
DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS
[0080] In accordance with one or more embodiments, the present invention provides
preparations of certain peptides that may offer enhanced utility and improved performance as
compared with other preparations of the same peptides. In some embodiments, disclosed
preparations may offer different or unique properties that, for example may address previously
unmet requirements associated with various research and/or clinical applications. In some
embodiments, certain desirable features of provided peptide preparations are provided by
elevating pH level of the preparation as compared with a standard or reference preparation of the
peptide and/or by adding one or more salts to the preparation, as compared with the type and/or
amount of salt in a standard or reference preparation. In some embodiments, provided
preparations are characterized by more stable hydrogel formation, and/or other attributes relative
to a standard or reference preparation, as described herein.
Peptides
[0081] In accordance with one or more embodiments, peptide compositions may include an
amphiphilic polypeptide having about 6 to about 200 amino acid residues. In certain
embodiments, the may have a length of at least about 7 amino acids. In certain embodiments, the
polypeptides may have a length of between about 7 to about 17 amino acids. In certain
embodiments, the polypeptides may have a length of at least 8 amino acids, at least about 1
amino acids, or at least about 16 amino acids.
[0082] In some embodiments, as is understood in the art, an amphiphilic polypeptide is one
whose sequence includes both hydrophilic amino acids and hydrophobic amino acids. In some
embodiments, such hydrophilic amino acids and hydrophobic amino acids may be alternately
bonded, so that the peptide has an amino acid sequence of alternating hydrophilic and
hydrophobic amino acids. In some embodiments, a polypeptide for use in accordance with the
present disclosure has an amino acid sequence that comprises or consists of repeated units of the
sequence Arg-Ala-Asp-Ala (RADA). In some embodiments, a polypeptide for use in accordance
with the present disclosure has an amino acid sequence that comprises or consists of repeated
units of the sequence Lys-Leu-Asp-Leu (KLDL). In some embodiments, a polypeptide for use
in accordance with the present disclosure has an amino acid sequence that comprises or consists
of repeated units of the sequence Ile-Glu-Ile-Lys (IEIK).
[0083] In some embodiments, a peptide for use in accordance with the present disclosure,
may generally be self-assembling, and/or may exhibit a beta-sheet structure in aqueous solution
under certain conditions.
[0084] In some embodiments, a peptide for use in accordance with the present disclosure has
an amino acid sequence: Arg-Ala-Asp-Ala-Arg-Ala-Asp-Ala-Arg- Ala-Asp-Ala- Arg-Ala-Asp-
Ala (i.e., RADA16, aka [RADA]4; SEQ ID NO:l). In some embodiments, a peptide for use in
accordance with the present disclosure has an amino acid sequence: Lys-Leu-Asp-Leu-Lys-Leu-
Asp-Leu-Lys-Leu- Asp-Leu (i.e., KLDL12, aka [KLDL])3 aka KLD12; SEQ ID NO: 2). a
peptide for use in accordance with the present disclosure has an amino acid sequence: Ile-Glu-
Ile-Lys-Ile-Glu-Ile-Lys-Ile-Glu-Ile-Lys-Ile (i.e., IEIK13, aka (IEIK)3I; SEQ ID NO:3)
[0085] Those skilled in the art, reading the present specification, will appreciate that any of a
variety of other peptides may alternatively be employed in the practice of the present invention.
In some embodiments, for example, one or more peptides as described in Published US Patent
Application US2009/01 11734 Al, Published US Patent Application US2008/0032934 Al,
Published US Patent Application US2014/0038909 Al, Issued US Patent US7,846891 B2,
Issued US Patent US7,7 13923 B2, Issued US Patent US 5670483 B2, the relevant contents of
which are incorporated herein by reference.
[0086] In some embodiments, a peptide for use in accordance with the present invention
have an amino acid sequence that comprises or consists of a sequence represented by one of the
following formulae: .
((XY)1-(ZY) m)
Formula (a)
(YX)1_(YZ) m)
Formula (b)
((ZY)1-(XY) m)
Formula (c)
((YZ)1-(YX) m)
Formula (d),
wherein X represents an acidic amino acid, Y represents a hydrophobic amino acid and Z
represents a basic amino acid, and 1, m and n are all integers (n(l+m)<200), ( 1 KLD12 > RADA16, so a
composition of IEIK13 showed greater rheological strength than did a composition of KLD12,
which in turn showed greater rheological strength than did a composition of RADA l 6 when
peptide concentration was the same in each case.
[0096] In some embodiments, peptide concentration in a peptide composition for use in
accordance with the present is at least 0.05%>, at least 0.25%>, at least 0.5%>, at least 0.75%>, at
least 1.0% or more. In some embodiments, peptide concentration in a peptide composition for
use in accordance with the present is less than 5%, less than 4.5%, less than 4%, less than 3.5%,
less than 3%, or less. In some embodiments, peptide concentration in a peptide composition for
use in accordance with the present invention is within a range between about 0.5% and about 3%.
In some embodiments, peptide concentration in a peptide composition for use in accordance with
the present invention is within a range between about 0.5% and about 2.5%. In some
embodiments, peptide concentration in a peptide composition for use in accordance with the
present invention is within a range between about 1% and about 3%. In some embodiments,
peptide concentration in a peptide composition for use in accordance with the present invention
is within a range between about 1% and about 2.5%. In some embodiments, peptide
concentration in a peptide composition for use in accordance with the present invention is about
0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, or more. In some particular
embodiments, where the peptide is RADA16, peptide concentration in peptide compositions of
the present invention is within a range of about 0.05% to about 10%.
[0097] In some particular embodiments, where the peptide is KLD12, peptide concentration
in peptide compositions of the present invention is within a range of about 0.05% to about 10%.
[0098] In some particular embodiments, where the peptide is IEIK13, peptide concentration
in peptide compositions of the present invention is within a range of about 0.05% to about 10%.
pH
[0099] The present disclosure demonstrates, among other things, that pH may impact
properties of peptide compositions. As described herein, optimizing pH of peptide compositions
may improve mechanical strengths, so that peptide compositions can be used for various clinical
applications. Example 3 in this disclosure illustrates details of certain specific embodiments.
[00100] In accordance with one or more embodiments, provided peptide compositions may
have a pH above (e.g., significantly above) the pi of the relevant peptide and/or of that obtained
when the peptide is solubilized in water. In some embodiments, properties of peptide
compositions may be controlled with pH. For example, in some embodiments, at pH within the
range of about 2.5 to about 4.0, stiffness and/or viscosity of peptide compositions may be
increased relative to that of an appropriate reference composition (e.g., of the same peptide at the
same concentration in water).
[00101] In some embodiments, peptide compositions may comprise peptide and a solvent,
typically an aqueous solvent, and pH may be adjusted via a pH-adjusting agent such as a base or
acid. In some embodiments, peptide compositions comprise peptide and a buffer.
[00102] In some embodiments, a pH-adjusted peptide composition may comprise one or more
of sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate, sodium
acetate, sodium sulfide, DMEM and/or PBS.
[00103] In some embodiments, an automated titration device may be implemented for pH
adjustment.
[00104] In some embodiments, provided compositions have and/or are maintained at a pH
above (e.g., materially above) the pi for the relevant peptide. In some embodiments, provided
compositions have and/or are maintained at a pH above (e.g., materially above) that of a water
solution of the same peptide at the same concentration. In some embodiments, provided
compositions have and/or are maintained at a pH below that at which the composition is or
becomes cloudy.
[00105] In some embodiments, provided compositions are characterized by a pH at or above
about 2.5-4.0; in some embodiments, provided compositions are characterized by a pH closer to
physiological pH. In some embodiments, provided compositions have a pH within the range of
about 3.0-4.0. In some embodiments, provided compositions have a pH at or above about 2.5,
2.6, 2.7, 2.8, 2.9, 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5 or higher. In some
embodiments, provided compositions have a pH at or below about 4.3, about 4.2, about 4.1,
about 4.0, about 39., about 3.7, about 3.6, about 3.5, about 3.4, or lower.
[00106] In some embodiments, elevated-pH compositions (i.e., compositions with a pH at or
above about 2.5) as described herein are characterized by greater rheological stiffness and/or
improved gelation properties as compared with an appropriate reference composition (e.g. a
comparable composition of the same peptide at the same concentration and optionally with the
same salts but at a different pH). In some embodiments, elevated pH compositions are useful in
a wider range of applications than are corresponding reference compositions of lower pH.
[00107] The present disclosure specifically demonstrates that, in some embodiments, at
elevated pH 3.5 or less, stiffness of IEIK13 compositions may be increased significantly, while
those of RADA16 and KLD12 compositions may not. Without wishing to be bound by any
particular theory, the present disclosure proposes that different behaviors of the peptide
compositions at pH 3.5 or less are likely related to the pKa of aspartic acid (D) (pKa = 3.71) in
RADA16 and KLD12 and glutamic acid (E) (pKa = 4.15) in IEIK13.
[00108] When pH is higher than pKa of aspartic acid (D) and glutamic acid (E), acidic groups
in peptide chains are mostly negatively-charged. Negatively charged groups induce intra- or
inter-molecular attractive charge-charge interactions with positively-charged groups in the
peptide chains (i.e. arginine (R) in RADA16 and lysine (K) in IEIK13 and KLD12) to form
larger aggregates, which are a translucent or opaque (i.e. it is above its cloud point) and provide
possible phase separation, rather than form nano-fibers (i.e. clear viscous compositions).
[00109] When pH is lower than pKa of aspartic acid (D) and glutamic acid (E) but close to the
pKa, the more populated negatively-charged groups may induce stronger attractive chargecharge
interactions with positive groups. The compositions may maintain the nano-fiber
formation, so that the stiffness increases.
[00110] Certain particular exemplary peptide compositions having a pH of about 3.5 are
presented in Table 2. Such compositions, which are considered "elevated pH compositions"
herein, may provide improved performance (e.g., relative to otherwise comparable compositions
of lower pH, such as relevant reference compositions as described herein, including in the
Examples) in various applications. Mechanical strength and versatility of peptide compositions
may be enhanced with elevated pH.
Table 2 Representative formulations for selected peptide compositions at around pH 3.5
[00111] In accordance with one or more embodiments, pH may impact gelation kinetics (e.g.,
response time to begin gelation). Effects of pH on the gelation kinetics may be evaluated to
identify optimized pH for peptide compositions as described herein.
[00112] In some embodiment, peptide compositions may gel faster at higher pH. For
example, as described herein IEIK13 compositions without pH adjustment show immediate
storage modulus increase, while RADA16 compositions without pH adjustment (pH 2.2) do not
show storage modulus increase for first 13 seconds. With pH adjustment, both IEIK13 and
RADA16 show immediate storage modulus increase due to fast gelation, as shown in Figures 24
and 25.
Ionic Strength
[00113] The present disclosure demonstrates that ionic strength may change rheo logical
properties of peptide compositions. Increasing the ionic strength of peptide compositions may
generally improve mechanical properties for various clinical applications. Effects of ionic
strength on the properties of peptide compositions may be evaluated, e.g., as described herein, to
identify optimized ionic strengths for peptide compositions as described herein.
[00114] In some embodiments, at ionic strength within the range of about 0.0001 M to about
1.5 M, stiffness, viscosity, and/or gelation kinetics of peptide compositions may be increased. In
some embodiments, peptide compositions may be controlled with ionic strength.
[00115] In accordance with one or more embodiments, ionic strengths of peptide
compositions may be adjusted with one or more common salts including but not limited to NaCl,
KC1, MgCl2, CaCl2 and CaS0 4. Common salts are composed of cations and anions. In some
embodiments, cations may be selected from the group comprising of ammonium, calcium, iron,
magnesium, potassium, pyridinium, quaternary ammonium, and sodium. In some embodiments,
anions may be selected from the group comprising of acetate, carbonate, chloride, citrate,
cyanide, floride, nitrate, and phosphate.
[00116] In accordance with one or more embodiments, when ionic strength approaches an
optimal level (e.g., highest stiffness), addition of one or more salt or salt solutions may be
carefully regulated. In some embodiments, pure water may be added if ionic strength is higher
than desired. Addition of one or more salt or salt solutions may be regulated to adjust its
osmolality to be hypotonic, isotonic or hypertonic depending on its applications.
[00117] In some embodiments, to adjust ionic strengths of certain peptide compositions by
way of example, certain salt buffer solutions, for example, NaCl, KC1, MgCl2, CaCl2 and DPBS
(Dulbecco's Phosphate -Buffered Saline, 10X) may be added.
[00118] In some embodiments, provided compositions include one or more salts, the identity
and/or concentration of which maintain the composition at a critical ionic strength below that at
which material precipitation of the peptide is observed. In some embodiments, material
precipitation is considered to have occurred when a liquid composition is cloudy (e.g., as
assessed by visual inspection). Thus, in some embodiments, provided compositions are not
cloudy, and have lower ionic strengths than otherwise comparable compositions (e.g., of the
same peptide at the same concentration) that are cloudy.
[00119] In some embodiments, provided compositions are characterized by an elevated ionic
strength relative to that of an appropriate reference composition (e.g., a composition of the same
peptide at the same concentration and pH but with different salt or different concentration of the
same salt). In some embodiments, provided compositions are characterized by ionic strength
close to or at physiological strength. In some embodiments, compositions as described herein
are characterized by greater rheological stiffness and/or improved gelation properties as
compared with an appropriate reference composition of different ionic strength. In some
embodiments, provided compositions are suitable for use in a broader range of applications that
are corresponding reference compositions of different (e.g., lower) ionic strength.
[00120] In accordance with certain particular embodiments, peptide compositions comprising
IEIK13, KLD12, or RADA16 in a salt solution are provided, which compositions have an ionic
strength different from that a reference composition of the relevant peptide dissolved in water
and show one or more improved material (e.g., rheological) properties relative to that reference
composition. In some embodiments, the provided compositions are stiffer than the relevant
reference compositions. In some embodiments, the provided compositions have elevated ionic
strengths relative to the reference compositions, but still have an ionic strength below their
critical salt points.
Table 10. Visual observation of solubility of certain self-assembling peptide compositions with
selected salts
[00121] Without wishing to be bound by any particular theory, the present disclosure proposes
that properties of peptide compositions with increased ionic strength may be related to solubility
of peptides. Solubility of self-assembling peptides at pH level about 2 to 4 is mostly high
enough to make clear and homogeneous peptide compositions. Increased ionic strength around
peptide chains decreases solubility of peptides. When solubility of peptides is low so that
compositions become cloudy, this status may be called as a critical point. When increased ionic
strength is lower than its critical point but close to it, peptides may induce stronger hydrophobic
interactions increasing stiffness. When peptide solubility is decreased below its critical point
(i.e. high ionic strength), peptide compositions may be translucent or opaque (i.e. it is above its
cloud point), and may be precipitated (i.e. phase separation). Peptides may not form nano-fibers
that make clear and viscous solutions. Random hydrophobic interactions may be dominant over
hydrophobic interactions that create self-assembled nano fibers at high ionic strength due to
salting out effect. Random intra- and/or inter-molecular aggregates may cause phase separations.
[00122] In accordance with one or more embodiments, critical ionic strengths may vary
depending on salt and peptide identities. The relationship between solubility and salt
concentration can be expressed by following Cohen equation:
log S = B - KI
where S is a solubility of a peptide, B is a peptide-specific constant, K is a salt-specific constant,
and I is an ionic strength of salts. B is related to pH, and temperature. K is related to pH.
[00123] In some embodiments, solubility of peptides may be governed by salting out
constant K and ionic strength I, when temperature and pH are constant (i.e. B is constant). The
higher K and I result in the lower peptide solubility. At constant pH and temperature, K is
decided by ion identities in salts. Overall, the order of constant K among the four salts is NaCl >
KC1 > MgCl2 = CaCl2.
[00124] In accordance with one or more embodiments, solubility of peptides may be
determined by amino acid sequence (e.g., by compositions of hydrophilic and hydrophobic
amino acid residues in the peptide). Peptides with relatively high hydrophobic amino acid
contents (e.g. IEIK13) typically have low solubility in aqueous solvents. Such peptides often are
characterized by strong hydrophobic interactions between self-assembled peptide chains,
resulting in high stiffness. As demonstrated herein, compositions of such peptides may show
dramatic stiffness increases with addition of a small amount of salt. By contrast, peptides with
relatively low hydrophobic amino acid contents (e.g. RADA16) have high solubility in aqueous
solvents. These peptides typically have weak hydrophobic interactions between self-assembled
peptides, resulting in low stiffness. Stiffness of compositions of such peptides does not increase
significantly even with addition of a large amount of salt. Consistent with this model, the present
disclosure demonstrates an order of critical ionic strength (e.g. when the composition becomes
cloudy) among three particular exemplified peptides that parallels relative hydrophobicity:
RADA16 (0.9-1.2 M) > KLD12 (0.3-0.4 M) > IEIK13 (0.03-0.04 M).
[00125] In accordance with one or more embodiments, ionic strength of a peptide composition
may impact its gelation kinetics. In some embodiment, elevated ionic strengths may accelerate
gelation of peptide compositions. The required ionic strengths for gelation may depend on salt
and/or peptide identities. For example, when RADA16, KLD12, and IEIK13 peptides were
exposed to saline buffer (i.e. 0.15 M NaCl, comparable to the isotonic body fluid), only gelation
of IEIK13 was initiated. RADA16 and KLD12 showed no or negligible gelation. These
findings may reflect decreased solubility of peptides with elevated ionic strength. IEIK13 is
more sensitive to ionic strength than RADA16 and KLD12 as described above.
[00126] In some embodiments, ionic strength of a peptide composition may impact its
recovery characteristics, for example after mixing and/or agitation processes break down
initially-formed assemblies (e.g., nano fibers) that result from (typically hydrophobic) peptidepeptide
interactions.
CombinedpH and Salt Effects
[00127] The present disclosure demonstrates that simultaneous adjustment of pH and ionic
strength (e.g., via exposure to physiological conditions) can alter rheological properties of
peptide compositions. For example, as described herein, increased pH level and ionic strength
due to inclusion of cell culture medium in provided peptide compositions can impact various
properties (e.g., rheological properties) of such compositions.
[00128] In some embodiments, stiffness, viscosity and/or gelation kinetics of peptide
compositions may be increased under physiological conditions. In some embodiments,
properties of peptide compositions may be controlled with the combination of pH and ionic
strength.
[00129] Without wishing to be bound by any particular theory, the present disclosure proposes
that there are two main intermolecular interactions that relate to stiffness of peptide
compositions: hydrophobic interactions and charge-charge interactions.
[00130] First, hydrophobic interactions and repulsive electrostatic interactions are the main
driving force for forming viscous solutions through b-sheet nanofiber formation at low pH.
These interactions are predicted to be significant at low pH, where a majority of aspartic acid and
glutamic acids are protonated without negative charges and a majority of arginine and lysine are
positively charged. The peptide molecules are self-assembled to form nano-fibers due to
hydrophobic interactions, while the surfaces of the nano-fibers are hydrated due to repulsive
electrostatic interactions between the peptide molecules.
[00131] In some embodiments, stiffness of peptide compositions around pH levels of about 2
to about 3 should be mainly related to their hydrophobicity. IEIK13 has seven isoleucine groups
(strong hydrophobic group), KLD12 has six leucine groups (strong hydrophobic group), and
RADA16 has eight alanine groups (weak hydrophobic group). IEIK13 has higher storage
modulus than KLD12 and RADA16 at the same pH and concentration.
[00132] As shown in Figure 17B, when IEIK13 molecules in an aqueous solution were treated
with simulated body fluid, for example, DMEM, their fibrous structure became thicker. The
thicker fibrous structure may occur due to increased hydrophobic interactions at physiological
pH and osmolality between neighboring nano fibers.
[00133] Hydrophobic interactions may induce nanofiber formation in an aqueous environment,
creating a viscous composition. After application of high shear stress (i.e. reduced viscosity and
stiffness), peptides may also reform nanofibers to recover their properties. Thus, the peptides
show thixotropic property at pH 2-3. Peptide compositions slowly recover their original
properties once the applied shear stress is removed.
[00134] Second, attractive charge-charge interactions may occur simultaneously with existing
hydrophobic interactions at physiological conditions. When the pH around peptide molecules
changes from acidic to neutral, existing hydrophobic interactions may not break down.
Negatively-charged groups and positively-charged groups induce additional attractive chargecharge
intermolecular interactions, so that peptide compositions may be stiffer as demonstrated
in Figure 7.
[00135] However, when peptide assemblies at a physiological condition are exposed to high
shear stress, the peptide assemblies break down to peptide aggregates. This is an irreversible
process, as illustrated in Figure 7.
[00136] For example, when 0.5 mL of DMEM was mixed with 0.5 mL of 2% RADA16 by
pipetting several times, RADA16 did not form clear and viscous peptide assemblies (i.e. cloudy
and runny emulsions). When the mixture was centrifuged at 2500 rpm for 5 min, phase
separation of cloudy RADA16 precipitated from the mixture. In this case, the peptide assemblies
(i.e. initially were formed via hydrophobic interactions) were likely broken down during the
mixing process. Attractive charge-charge interactions were dominant over hydrophobic
interactions, which induce formation of random intra- and inter-molecular aggregates. The phase
separation is illustrated in Figure 7.
[00137] In accordance with one or more embodiments, IEIK13, KLD12, or RADA16 may be
dissolved in salt buffer (e.g. NaCl), and their pH may be elevated with alkali salt buffer (e.g.
NaOH). Their salt ionic strengths may be under their critical salt points. Their pH may be about
2.5 to about 4.0. The compositions may have increased stiffness and viscosity relative to an
appropriate reference composition of the same peptide at the same concentration.
[00138] In some embodiments, physiological conditions (e.g.,. elevated pH and salt ionic
strength) may accelerate gelation of peptide compositions. Accelerated gelation of IEIK13 under
a physiological condition may be related to two driving forces i.e. increased pH and ionic
strength. Accelerate gelation of RADA16 under a physiological condition may have only one
driving force i.e. increased pH. In some embodiments, accelerated gelation of peptide
compositions with body fluid may generally improve its function and responding time for
various clinical applications.
Cell compatibility
[00139] In accordance with one or more embodiments, provided peptide compositions are
generally associated with high cell viability.
[00140] In some embodiments, KLD12 and IEIK13 may have similar or higher cell viabilities
compared to RADA16. The order of overall cell viability among these peptide compositions was
KLD12 > IEIK13 > RADA16. In some embodiments, peptide compositions may have cell
viabilities higher than 80% when their concentrations are about or less than 0.75%. In some
embodiments, cell viabilities may be decreased when peptide concentrations are higher than
0.75%.
Form
[00141] In some embodiments, peptide compositions in accordance with the present invention
are in the form of a dry powder, a solution, a gel (e.g., a hydrogel), or any combination thereof.
[00142] In some embodiments, a dry powder composition comprises peptide in an appropriate
amount to result in a solution of desired concentration upon addition of a selected volume of
solvent (e.g., aqueous solvent, optionally including one or more salts and/or one or more pHadjusting
agents). In some embodiments, a dry powder composition comprise peptide and salt of
appropriate types and relative amounts to result in a solution of desired peptide concentration and
ionic strength as described herein upon addition of a selected volume of solvent (e.g., aqueous
solvent, optionally including one or more additional salts and/or one or more pH-adjusting
agents). In some embodiments, a dry powder composition comprise peptide and pH adjusting
agent of appropriate types and relative amounts to result in a solution of desired peptide
concentration and pH as described herein upon addition of a selected volume of solvent (e.g.,
aqueous solvent, optionally including one or more salts and/or one or more additional pHadjusting
agents). In some embodiments, a dry powder composition comprises peptide, salt, and
pH adjusting agent of appropriate types and relative amounts to result in a solution of desired
peptide concentration, pH, and/or ionic strength as described herein upon addition of a selected
volume of solvent (e.g., aqueous solvent, optionally including one or more additional salts and/or
one or more additional pH-adjusting agents).
[00143] In some embodiments, a provided composition is housed in a container (e.g., a
syringe, vial, well, etc). In some embodiments, the container is a graduated container in that it
includes volume indications. In some embodiments, the container is adapted for connection to a
delivery device such as a cannula or syringe. In some embodiments, the container is sealed in a
manner (e.g., a penetrable covering) that permits addition and/or removal of flowable (e.g.,
liquid) material without removal of the seal.
Applications
[00144] In some embodiments, the present disclosure provides a system for selecting peptide
compositions for use in particular applications. Effects of peptide identity, peptide
concentration, pH, salt identity and/or salt concentration, as described herein, can impact
characterstics, and therefore utility of particular peptide compositions for certain applications.
[00145] To give but a few examples, in general, peptide compositions with higher stiffness are
more suited to applications characterized by hemostasis, tissue plugs, anti-adhesion, or certain
tissue regeneration. Peptide compositions with more rapid gelation times may be particularly
suited to certain tissue plug applications such as, for example, hemostasis, tissue plug, antiadhesion,
or drug delivery, vascular plug, for which gelation times below about 1 minute to
about 1 hour are typically required or preferred. Peptide compositions with more rapid recovery
times may be particularly suited to hemostasis, tissue plug, or vascular plug.
[00146] As noted herein, self-assembling peptide compositions have provide to be extremely
useful in a variety of in vivo and in vitro contexts, including for example as cell scaffolds,
barriers to liquid movement, hemostats, void fillers, and more. Different such compositions, as
described herein, may be more useful in different contexts.
[00147] For example, contexts that involve administration of a peptide composition during
surgery (e.g., as a hemostat) may benefit from gelation kinetics that permit the composition to
remain substantially liquid for a period of time appropriate for administration to the surgical site,
followed by rapid gelation to form a stable, preferably clear and relatively stiff gel through which
surgical manipulations can readily proceed.
[00148] To give specific examples, as described herein, in some embodiments, IEIK13
compositions may be particularly useful in a variety of biomedical applications, for example that
require certain stiffness and fast gelation. The present disclosure demonstrates that certain
IEIK13 compositions are characterized by relatively high stiffness and/or rapid recovery rates
after application of high shear stress (e.g. the fastest self-assembly). Also, the present disclosure
demonstrates that certain IEIK13 compositions may show particularly useful (e.g., high) stiffness
when contacted with a physiological medium.
[00149] The present disclosure also demonstrates that certain KLD12 compositions may be
particularly useful, for example, when easy injection is required together with high final
stiffness. In some embodiments, self-assembled nano fibers of KLD12 may be disassembled
with high shear stress, and then slowly assemble back.
[00150] The present disclosure also demonstrates that certain KLD12 compositions may be
particularly useful when high cell viability is required. In some embodiments, concentrations of
KLD12 may be increased to have a required stiffness for a certain application.
EXEMPLIFICATION
Example 1: Optical clarity of certain reference peptide compositions
[00151] The present Example illustrates optical clarity and phase stability (i.e., absence of
phase separation) of certain reference peptide compositions in which the indicated peptide was
dissolved in water. In some embodiments, optical clarity (and/or phase stability) of provided
peptide compositions is assessed relative to that of such reference compositions. In some
embodiments, provided compositions of a particular peptide at a particular concentration show
optical clarity and/or phase stability at least as good as that of a reference composition of the
same peptide at the same composition dissolved in water.
[00152] As can be seen with reference to Table 1, various reference peptide compositions
were prepared that showed optical clarity (and also phase stability) across a range of peptide
concentrations. In particular, peptide compositions at concentrations of 0.05%, 0.1%, 0.5%>, 1%,
1.5%, 2%, 2.5% or more showed a clear optical character and absence of phase separation.
Table 1 Appearance of peptides
Example 2 : Rheological properties of peptide compositions as a function of concentration
[00153] The present Example describes effects of peptide concentration on rheological
properties of certain peptide compositions. In some embodiments, rheological properties may
have a linear relationship with peptide concentration.
[00154] In some embodiments, peptide compositions with a specific desired stiffness may be
formulated to have a particular peptide concentration determined using a mathematical model
(i.e., the modulus trend-line equation). A formulation chart of peptide compositions may relate
their concentrations in deionized water to their specific storage moduli, for example as is
presented below in Table 3A. From such a chart, one skilled in the art may formulate a peptide
composition with desired rheological properties by selecting a particular peptide and an
appropriate peptide concentration so that the formulated composition has a desired stiffness..
[00155] For example, as described herein, in general, the order of rheological strength of
peptide compositions containing RADA16, KLD12, or IEIK13 peptides is demonstrated to be
IEIK13 > KLD12 > RADA16.
Table 3A Formulation chart of peptide compositions by varying their concentration with
deionized water to obtain specifically desired storage moduli, calculated with the linear trendline
equations presented above.
800 5.1 2.5 1.1
900 2.7 1.2
1000 2.9 1.3
1050 3.0 1.4
1200 3.3 1.5
1400 3.7 1.7
1600 4.1 1.9
1800 4.5 2.1
2000 5.0 2.3
2200 2.5
[00156] The present Example further describes measurement of various rheological properties
of certain peptide compositions at selected concentrations using a rheometer (AR500, TA
Instruments) with 40 mm plates. A peptide composition (700 m ) was placed on the rheometer
plate and excess composition was gently removed by Kimwipes. Measurements were performed
after 2 minutes of relaxation time at 37°C. Storage modulus, loss modulus, and viscosity (h')
were measured at 37°C with the plates placed at a measuring geometry gap of 300 mih, and stress
sweep tests were performed at 0.1 Pa ~ 1000 Pa of oscillation stress with angular frequency at 10
rad/s.
[00157] Results are shown in Figures 2-4 for RADA16, IEIK13, and KLD12. As shown in
Figures 2-4, peptide compositions showed near plateau moduli when oscillation stress was less
than about 10 to 200 Pa. They all had a dramatic modulus decrease at a certain yield oscillation
stress. At least within the tested concentration range of about 1% to about 2.5%, peptide
compositions showed a linear increase of storage modulus with increased concentration (R2 of
linear trend-lines are between 0.971-0.992). Certain peptide compositions demonstrated a shear
thinning property over a critical stress level.
[00158] The determined rheological results for various peptide compositions tested in the
present Example are listed in Table 3. As can be seen, the storage modulus of 1.5% KLD12 was
found to be similar to that of 2.5% RADA16. The storage modulus of 1% IEIK13 was found to
be similar to that of 2.5% KLD12 and higher than that of 2.5% RADA16. Overall, the order of
rheological strength among the compositions tested here was IEIK13 > KLD12 > RADA16.
Table 3 Rheological properties of peptide compositions at selected concentrations
*: at 1 Pa of oscillation stress
# : Maximum viscosity data was adapted in viscosity plots at the range of measured stress.
Example 3 : Rheological properties of peptide compositions as a function of pH
[00159] The present Example describes effects of pH on rheological properties of certain
peptide compositions. In some embodiments, pH may be a control parameter that impacts
stiffness, viscosity, and/or recovery time of peptide compositions.
[00160] Table 2, below, presents pH concentrations observed for reference compositions in
which the indicated peptide is solubilized in water at the indicated concentration.
Table 2 pH values of reference peptide compositions (in water) at selected concentrations.
1.0% 2.0
0.5% 2.2
[00161] In this Example, pH levels of peptide compositions was adjusted, for example, by
addition of 0.1 N NaOH to 2 mL of a 2.5% peptide composition. pH and appearance of the
adjusted compositions were observed. An acidic salt was added if the pH level was higher than
the desired level.
[00162] Results are shown in Table 5. Notably, a pH increase (i.e., up to about 3.5 or less) did
not change the clear color of RADA16, IEIK13, and KLD12 compositions, while their apparent
stiffness was increased. With certain compositions, when pH levels of peptide compositions
were higher than 3.5 (RADA16 and KLD12) or 3.7 (IEIK13), the peptide compositions began
phase separation (i.e. becoming cloudy). In some embodiments, provided peptide compositions
have a pH within the range of about 3.0 to about 3.7 (particularly for IEIK3), or about 3.0 to
about 3.5 (particularly for RADA16 and/or KLD12).
Table 5 Visual observation of certain e tide com ositions at selected H levels
KLD12 0 2.5 2.1 Clear, thick gel
50 2.38 2.4 Clear, thick gel
100 2.27 2.6 Clear, thick gel
150 2.17 2.9 Clear, thick and stiffer gel
200 2.08 3.3 Clear, thick and stiffer gel
225 2.04 3.6 Clear, thick and stiffer gel
250 2.0 4.0 Slightly cloudy, brittle gel
300 1.92 4.7 Cloudy, brittle gel
350 1.85 5.2 Cloudy, phase-separated
7.0 Cloudy, phase-separated
[00163] Rheological properties of certain peptide compositions were observed before and
after adjusting their pH levels to 3.4 (RADA16 and KLD12) or 3.7 (IEIK13). Rheological
properties of peptides were evaluated using a rheometer (AR500, TA Instruments) with 40 mm
plates. Specifically, a peptide composition (700 m ) was placed on the rheometer plate and
excess composition was gently removed by Kimwipes. Measurements were performed after 2
minutes of relaxation time at 37°C. Stress sweep test results are shown in Figure 8-1 1.
RADA16, KLD12 and IEIK13 compositions at elevated pH were stiffer than those at 2.5
(RADA16), 2.0 (KLD12), and 2.1 (IEIK13).
[00164] Storage modulus of certain peptide compositions at selected pH levels were evaluated
using a rheometer (DHR-1, TA Instruments) with 20 mm plates. Storage modulus of RADA16
and IEIK13 compositions were increased with pH increase up to 3.4. Determined storage moduli
for tested peptide compositions are shown in Figure 18 for RADA16 and Figure 19 for IEIK13,
respectively.
[00165] Viscosities of 1% IEIK13 compositions at selected pH levels were evaluated using a
rheometer (DHR-1, TA Instruments) with 20 mm plates. Viscosities of IEIK13 compositions
increased with pH increase up to 3.5. IEIK13 compositions showed a typical shear thinning
property. The results are shown in Figures 20A and 20B.
[00166] Recovery times of rheological properties were evaluated at selected pH after applying
high shear stress to 1% IEIK13 compositions. Using DHR-1 rheomether (TA Instruments),
storage modulus changes of 1% IEIK13 were measured with at 1 rad/s at 1 Pa after applying
1000 1/sec of shear rate to samples for 1 min. IEIK13 compositions at selected pH showed a
typical thixotropic behavior, which means their rheological properties were slowly recovered.
Without wishing to be bound by any particular theory, we propose that rheological property
recovery times represent time required for reassembly of peptide molecules to form selfassociations
(e.g., nano fibers) again in the compositions. Complete reassembling time of 1%
IEIK13 control composition (pH 2.3) was up to 12 hours or less, while those of pH-elevated
IEIK13 compositions were 6—10 min. Representative results are shown in Figure 21A-21D for
IEIK13.
Example 4 : Rheo logical properties of peptide compositions as a function of ionic strength
[00167] The present Example describes effects of ionic strength on rheo logical properties of
certain peptide compositions. In some embodiments, ionic strength may be a control parameter
of stiffness, viscosity, and/or recovery time of peptide compositions.
[00168] Visual observations of RADA16, KLD12, and IEIK13 compositions with selected
salts (e.g. KC1, MgCl 2, CaCl 2) are summarized in Tables 7-9. Peptide compositions at certain
ionic strengths were clear and showed higher stiffness than those at lower ionic strength. For
RADA16 (Table 7), ionic strength within the range of approximately 0.85 -1.15 M (depending
on salt identities) did not noticeably change the opacity of RADA16 compositions. For KLD 12
(Table 8), ionic strength within the range of approximately 0.25-0.35 M (depending on salt
identities) did not noticeably change the opacity of KLD 12 compositions. For IEIK13 (Table 9),
ionic strength within the range of approximately 0.025-0.035 M (depending on salt identities)
did not change the opacity of IEIK13 compositions. Apparent stiffness of RADA16, KLD12 and
IEIK13 compositions was increased with increased ionic strength.
[00169] Without wishing to be bound by any particular theory, we propose that increased
rheological properties may relate to the salting out constant, K, of each salt. The constant K of
NaCl for RADA16 may be higher than the other salts. Rheological properties of the RADA16
compositions with NaCl were slightly higher than those with KC1 and CaCl 2.
Table 7 Visual observation of RADA16 compositions with selected salts at room
temperature
(3 M-as a 52.6 1.43 0.15 0.15 Clear, thick, stiffer gel
stock 111.1 1.35 0.3 0.3 Clear, thick, stiffer gel
solution) 176.5 1.27 0.45 0.45 Clear, thick, stiffer gel
250 1.2 0.6 0.6 Clear, thick, stiffer gel
333.3 1.13 0.75 0.75 Clear, thick, stiffer gel
363.6 1.10 0.8 0.8 Clear, thick, stiffer gel
395.3 1.08 0.85 0.85 Clear, thick, stiffer gel
428.6 1.05 0.9 0.9 Slightly cloudy, brittle gel
463.4 1.03 0.95 0.95 Cloudy, phase-separated
500 1.0 1.0 1.0 Cloudy, phase-separated
KC1 0 1.5 0 0 Clear, thick gel
(3 M-as a 52.6 1.43 0.15 0.15 Clear, thick, stiffer gel
stock 111.1 1.35 0.3 0.3 Clear, thick, stiffer gel
solution) 176.5 1.27 0.45 0.45 Clear, thick, stiffer gel
250 1.2 0.6 0.6 Clear, thick, stiffer gel
333.3 1.13 0.75 0.75 Clear, thick, stiffer gel
428.6 1.05 0.9 0.9 Clear, thick, stiffer gel
463.4 1.03 0.95 0.95 Clear, thick, stiffer gel
500 1.0 1.0 1.0 Clear, thick, stiffer gel
538.5 0.98 1.05 1.05 Slightly cloudy, thick, stiffer gel
578.9 0.95 1.1 1.1 Slightly cloudy, brittle gel
621.6 0.93 1.15 1.15 Cloudy, phase-separated
MgCl 0 1.5 0 0 Clear, thick gel
(3 M-as a 16.9 1.48 0.05 0.15 Clear, thick, stiffer gel
stock 34.5 1.45 0.1 0.3 Clear, thick, stiffer gel
solution) 52.6 1.43 0.15 0.45 Clear, thick, stiffer gel
71.4 1.4 0.2 0.6 Clear, thick, stiffer gel
90.9 1.38 0.25 0.75 Clear, thick, stiffer gel
111.1 1.35 0.3 0.9 Clear, thick, stiffer gel
132.1 1.32 0.35 1.05 Clear, thick, stiffer gel
146.5 1.31 0.383 1.15 Clear, thick, stiffer gel
153.8 1.3 0.4 1.2 Slightly cloudy, thick, stiffer gel
161.3 1.29 0.417 1.25 Slightly cloudy, brittle gel
168.8 1.28 0.433 1.3 Cloudy, phase-separated
CaCl 0 1.5 0 0 Clear, thick gel
(3 M-as a 16.9 1.48 0.05 0.15 Clear, thick, stiffer gel
stock 34.5 1.45 0.1 0.3 Clear, thick, stiffer gel
solution) 52.6 1.43 0.15 0.45 Clear, thick, stiffer gel
71.4 1.4 0.2 0.6 Clear, thick, stiffer gel
90.9 1.38 0.25 0.75 Clear, thick, stiffer gel
111.1 1.35 0.3 0.9 Clear, thick, stiffer gel
132.1 1.32 0.35 1.05 Clear, thick, stiffer gel
146.5 1.31 0.383 1.15 Clear, thick, stiffer gel
153.8 1.3 0.4 1.2 Slightly cloudy, thick, stiffer gel
161.3 1.29 0.417 1.25 Slightly cloudy, brittle gel
168.8 1.28 0.433 1.3 Cloudy, phase-separated
DPBS 0 1.5 0 0 Clear, thick gel
(pH 3.2) 111.1 1.35 0.15 0.15 Clear, thick, stiffer gel
(10X - 250 1.2 0.3 0.3 Clear, thick, stiffer gel
1.5 M -as 428.6 1.05 0.45 0.45 Clear, thick, stiffer gel
a stock 666.7 0.9 0.6 0.6 Clear, thick, stiffer gel
solution) 1000 0.75 0.75 0.75 Clear, thick, stiffer gel
1500 0.6 0.9 0.9 Clear, thick, stiffer gel
1725 0.55 0.95 0.95 Slightly cloudy, brittle gel
2000 0.5 1.0 1.0 Cloudy, phase-separated
Table 8 Visual observation of KLD12 compositions with selected salts at room
tem erature
CaCl2 0 1.5 0 0 Clear, thick gel
(3M-as a 16.9 1.48 0.05 0.15 Clear, thick, stiffer gel
stock 22.7 1.47 0.067 0.2 Clear, thick, stiffer gel
solution) 28.6 1.46 0.083 0.25 Clear, thick, stiffer gel
34.5 1.45 0.1 0.3 Clear, thick, stiffer gel
40.2 1.44 0.1 17 0.35 Clear, thick, stiffer gel
46.5 1.43 0.133 0.4 Slightly cloudy, thick, stiffer gel
52.6 1.43 0.15 0.45 Slightly cloudy, brittle gel
58.8 1.42 0.167 0.5 Cloudy, phase-separated
Table 9 Visual observation of IEIK13 compositions with selected salts at room
tem erature
100.9 1.36 0.0183 0.055 Cloudy, phase-separated
CaCl2 0 1.5 0 0 Clear, thick gel
(0.2 M-as 25.6 1.46 0.005 0.015 Clear, thick, stiffer gel
a stock 34.5 1.45 0.0067 0.02 Clear, thick, stiffer gel
solution) 43.5 1.44 0.0083 0.025 Clear, thick, stiffer gel
52.6 1.43 0.01 0.03 Clear, thick, stiffer gel
61.9 1.41 0.01 17 0.035 Clear, thick, stiffer gel
71.4 1.40 0.0133 0.04 Slightly cloudy, thick, stiffer gel
81.1 1.39 0.015 0.045 Slightly cloudy, thick, stiffer gel
91.1 1.37 0.0167 0.05 Slightly cloudy, brittle gel
100.9 1.36 0.0183 0.055 Cloudy, phase-separated
[00170] Critical ion strengths were determined from the visual observations recorded in
Tables 7-9. When the ionic strengths of peptide compositions were higher than 0.9 M
(RADA16), 0.3 M (KLD12) or 0.03 M (IEIK13), the peptide compositions began phase
separation. 0.9 M, 0.3 M and 0.03 M may represent critical ion strengths for RADA16, KDL12
and IEIK13, respectively.
[00171] Figures 27-29 show rheological properties measured with a rheometer (DHR-1, TA
Instruments) with 20 mm plates when ion strengths are slightly lower than the critical ion
strengths. Ion strengths of RADA16, KDL12 and IEIK13 were 0.7 M, 0.2 M and 0.02M,
respectively for the measurements. Rheological properties of RADA16, KLD12 and IEIK13
compositions were higher after adjusting their ionic strength levels with NaCl to 0.7 M
(RADA16), 0.2 M (KLD12) or 0.02 M (IEIK13).
[00172] Rheological properties of peptide compositions at selected ionic strengths were
measured using a rheometer (DHR-1, TA Instruments) with 20 mm plates. Rheological
properties of 1% RADA16 compositions were increased with ionic strength adjustment up to 0.7
M, while decreased with ionic strength higher than 0.7 M. Rheological properties of 1% IEIK13
compositions were increased with ionic strength adjustment up to 0.03 M, while decreased with
ionic strength higher than 0.03 M. Results well matched with visual inspections of peptide
compositions at selected salt ionic strengths. Results are shown in Figure 30 for RADA16 and in
Figure 3 1 for IEIK13.
[00173] Viscosities of peptide compositions at selected ionic strength levels were evaluated.
Viscosities of IEIK13 compositions were increased with the increased ionic strength. 1%
IEIK13 compositions showed a typical shear thinning property. Viscosities of 1% IEIK13
compositions were evaluated using a rheometer (DHR-1, TA Instruments) with 20 mm plates.
The results are shown in Figures 32A and 32B for IEIK13.
[00174] Recovery times of rheological properties were evaluated after applying high shear
stress to 1% IEIK13 compositions at selected ionic strengths. Using a DHR-1 rheomether (TA
Instruments), storage modulus changes of 1% IEIK13 were measured with time sweep tests at 1
rad/s at 1 Pa after applying 1000 1/sec of shear rate to samples for 1 min. IEIK13 compositions
at selected ionic strengths showed a typical thixotropic behavior, recovering their rheological
properties slowly. Recovery times of rheological properties are based on reassembly of peptide
molecules to form nano fibers again. Complete reassembling time of 1% IEIK13 control
compositions without salt addition was up to 12 hours or less, while those of IEIK13
compositions with NaCl 0.01M and 0.02M were less than 1 min ~ 3 min. The results are shown
in Figures 33A-33C for IEIK13.
Example 5 : Rheological properties of peptide compositions as a function of both pH and ionic
strength
[00175] The present Example describes rheological properties of peptide compositions at
increased pH and ionic strength. In particular, the present Example describes effects of a
physiological medium, such as a cell culture medium, on rheological properties of certain
peptide compositions.
[00176] Effects of Dulbecco's modified Eagle's medium (DMEM) (pH 7.4) on rheological
properties of IEIK13, KLD12, and RADA16 compositions were evaluated using a rheometer
(AR500, TA Instruments) with 40 mm plates. DMEM is a cell culture medium that contains 6.4
g/L of NaCl, 3.4 g/L NaHCOs (sodium bicarbonate), minor amounts of other salts, various amino
acids, and 4.5 g/L of glucose. The pH of DMEM is 7.2 ± 0.2 and the osmolality is 335 ± 30
mOsm/Kg H20 . DMEM is close to human physiological fluids, for example, blood.
[00177] Before being mixed with the DMEM solution, 1% peptide compositions were kept in
4 °C for at least 48 hours. To perform experiments, 1mL of peptide composition was gently
pipetted and placed on the plate of the rheometer. 2 mL of the DMEM solution was gently
added around the peptide composition. The peptide composition was treated with the DMEM
for two minutes, then medium was removed, and the plates were placed at a measuring geometry
gap at around 450 mhi. Measurements were performed at 37°C after 2 min of relaxation.
Frequency tests were performed from 1 rad/s to 100 rad/s at 1 Pa of oscillation stress.
[00178] Rheological properties of 1% peptide compositions were measured before and after
the DMEM treatment for 2 minutes; results are presented in Figure 5A. The fold increase of
storage moduli after the DMEM treatment is shown in Figure 5B. As can be seen, peptide
compositions showed large increases of storage moduli after DMEM treatment. Fold differences
between RADA16, KLD12, and IEIK13 after the DMEM treatment were relatively small
compared to that before the DMEM treatment. Similarly, stiffer peptide compositions (e.g.,
IEIK13) showed lower-fold increase of storage modulus than less stiff peptide compositions
(e.g., RADA16) after the DMEM treatment. Critical intermolecular interactions were increased
after the DMEM treatment, which may determine final stiffness.
[00179] Using a DHR-1 rheomether (TA Instruments), rheological properties of peptide
compositions at selected concentrations were measured before and after the DMEM treatment.
Frequency sweep tests were performed from 1 rad/sec to 10 rad/sec at 1 Pa and the storage
moduli in the graphs were at 1 rad/sec. Rheological properties of RADA16 and IEIK13
compositions were increased with the DMEM treatment and/or pH elevation. Results are shown
in Figures 22A and 22B for RADA16 and Figure 23A and 23B for IEIK, respectively.
[00180] Using a DHR-1 rheomether (TA Instruments), rheological properties of peptide
compositions at selected ion strengths were evaluated 10 mins after the DMEM treatment.
Frequency sweep tests were performed from 1 rad/sec to 10 rad/sec at 1 Pa and the storage
modulus at 1 rad/sec was selected for data. Rheological properties of RADA16 compositions
were increased with ionic strength adjustment up to 0.7 M, while they were decreased with 0.7
M or higher. At 0.9 M or higher ionic strengths of NaCl, RADA16 compositions became
cloudy. Rheological properties of RADA16 did not change with the DMEM treatment (e.g. no
gelation). However, rheological properties of IEIK1 3 compositions were increased with the
DMEM treatments at selected ionic strengths. Results are shown in Figure 34-35.
[00181] IEIK13, KLD12, and RADA16 were dissolved in salt buffer (e.g. NaCl) and kept at
elevated pH level adjusted with alkali salt buffer (e.g. NaOH). The compositions had their pH
levels about 2.5-4.0 and lower ionic strength than their critical points. With respect to RADA16,
KLD13 and IEIK13, peptide compositions were still clear with 0.9% NaCl (i.e. ionic strength of
0.15 M) at pH 3.4 (adjusted with NaOH). Rheological properties of RADA16 with 0.9% NaCl at
pH 3.4 were stiffer than those of RADA16 control (i.e. no ionic strength and pH elevation) and
RADA16 with 0.9% NaCl (no pH elevation). Results are shown in Figure 40.
[00182] A Congo Red assay was performed to determine gel formation of peptide
compositions in a PBS (Phosphate buffered saline) solution (pH 7.4), as shown in Figure 1.
100 mΐ of each gel at selected concentrations were plated on a glass slide. After 30 seconds,
500 mΐ of 1% Congo Red solution was added around and on top of each of the composition
aliquots and then the excess Congo Red solution was wiped off prior to examination. RADA16,
IEIK13, and KLD12 were plated at selected concentrations of 0.5%>, 1.0%, 1.5%, 2.0% and
2.5%. Visual observation determined the success or failure of gelation at each concentration.
RADA16, IEIK13, and KLD gelled at all concentrations.
Example 6 : Cell viability
[00183] The present Example describes ability of certain peptide compositions to support cell
viability. In some embodiments, provided peptide compositions are characterized in that they
support high cell viability, particularly as compared with appropriate reference compositions.
[00184] A cell viability (cytotoxicity) assay was performed to measure the viability of
C57 BL/6 Mouse Mesenchymal Stem Cells (mMSCs) with IEIK13, KLD12 and RADA16
compositions as described herein. mMSCs are a frequently used cell line in hydrogel tissue
culture systems. Peptide compositions were prepared at a concentration of 2.5%, and then were
diluted to concentrations of 1.5%, 1.25%, 1.0%, 0.75%, and 0.50% with sucrose. The final
concentration of sucrose was 10%. Cells were washed and re-suspended in 10% sucrose to a
final concentration of 5 million cells/ml. Cells were centrifuged and the supernatant was
removed. The cells were re-suspended in peptide compositions with 10% sucrose. The protocol
was then followed for plating drop cultures and subsequent isolation as described in the
PuraMatrix ® Guidelines for Use (BD/Corning website). Results are shown in Figures 14-16 for
RADA16, IEIK13, and KLD12, respectively.
[00185] The cell viabilities in IEIK13 and KLD12 compositions at 0.5 % were similar to those
at 0.25%. Cell viability in RADA16 compositions at 0.5 % is significantly higher than that at
0.25%. However, cell viabilities significantly decreased when the concentrations of peptides
were over 0.75%. KLD12 and IEIK13 compositions showed similar or higher cell viability
compared to RADA16 at all tested concentrations within the range 0.25% to 1.5%. The order of
overall cell viability among these peptide compositions was KLD12 > IEIK13 > RADA16. The
tested peptide compositions with concentrations of 0.75% or less showed cell viabilities higher
than 80%.
Example 7 : Rheological properties of RADA16 compositions with different salts
[00186] The present Example describes, among other things, studies that achieved controlled
mechanical enhancement of self-assembling peptide gels while still maintaining gel reversibility
(e.g., without compromising gel formation and its mechanical integrity post mechanical
perturbation). These described studies also achieved control of gelation kinetics through mixing of
cations and anions at selected concentrations in combination with various self-assembling peptides,
most notably, RADARADARADARADA (or RADA-16).
[00187] The present Example, particularly when taken in context with the present specification,
confirms that parameters have been defined that permit peptide compositions to be specifically
formulated to have material and/or rheological characteristics particularly useful for certain
applications. For example, the technology described herein permits preparation of self-assembling
peptide compositions that are specifically tailored to function well as sealants (which may require or
benefit from, for example, enhanced stiffness), lubricants (which may require or benefit from, for
example, enhanced kinetics), drug mixtures (which may require or benefit from, for example,
reversibility and enhanced kinetics), injectables (which may require or benefit from, for example,
reversibility), etc.. Alternatively or additionally, technology described herein permits preparation of
peptide compositions and/or selection of parameters included in or applied to them, that can assist in
general handling and/or manufacture of useful peptide compositions and/or devices that include
them.
[00188] For example, as demonstrated herein, by systematically controlling the type, e.g. Na, K,
and Ca, and/or concentration of cation included in self-assembling peptide compositions, the
mechanical strength (i.e. the stiffness) can be adjusted while still maintaining reversibility and
gelation kinetics. Alternatively or additionally, by systematically controlling the type e.g. CI, S04,
PO4 ,and/or concentration of anion, the gelation kinetics can be controlled while maintaining
reversibility.
[00189] As demonstrated in the present Example, in some embodiments (and in particular, in
embodiments that utilize a RADA16 peptide), Ca will allow for greater enhanced stiffness in peptide
gels compared to Na and K. In addition, in some embodiments (and in particular, in embodiments
that utilize a RADA16 peptide), CI will allow for faster gelation kinetics in peptide gels compared to
SO4. Moreover, in some embodiments (and in particular, in embodiments that utilize a RADA16
peptide), CaCb will allow for optimally mechanically enhanced reversible gels at a concentration of
> 0.125 and < 0.500 M. In the particular studies reported in this Example, concentrations > 0.500 M
compromised the mechanical properties of certain gels, or rendered them unusable to post-gelation
mechanical perturbations.
[00190] In general, findings reported in the present Example demonstrate that, through the use of
a variety of salts and salt concentrations, attributes such as stiffness, gelation kinetics, and
reversibility of gelation can be determined by selection of parameters such as concentration of
peptide, identity (e.g., amino acid sequence) of peptide, concentration of cations/anions, identity of
cation/anion, etc . It has been observed that both cation and anion, independently and in
combination, can impact attributes. Teachings provided by the present disclosure, including the
present Example, provide a system for tailoring peptide mixtures in accordance with desired
attributes (e.g, performance characteristics), for example as may be appropriate for a particular
application or situation.
[00191] The present Example specifically demonstrates that certain particular types of cations
and/or anions, and/or concentrations thereof/ have desired beneficial effects; among other things, the
present Example defines such anions/cations and concentrations with respect to the exemplified
contexts, and moreover provides a framework permitting those skilled in the art to do the same
for other cases (e.g., other peptides, etc).
[00192] The present Example particularly and surprisingly identifies a problem with current
strategies for providing useful compositions of self-assembling peptides. That is, it has been
theorized that the capacity for self-assembly is dependent on the amount of charged groups
available for attack by ionic salts, which is the peptide's capacity for saturation [P. Chen. (2005).
"Self-assembly of ionic-complementary peptides: a physicochemical viewpoint." Colloids and
Surfaces]. However, the present Example documents that, in at least some cases, peptides are
not proportionally affected by salt concentration, and thus, the mechanical properties and the
reversibility do not increase linearly and are not rate dependent.
[00193] Figures 41A-41D depict the protocol used to follow peptide dissolution and to assess
effects of particular anions and/or cations, and/or their concentration, on certain RADA16
compositions. As shown, peptide powder in a vial was dissolved in deionized water with
vortexing and sonication. The particular peptide composition utilized in this Example was a 1%
composition of RADA16 that was mixed at a 1:1 ratio with a 2X salt solution to obtain a final
concentration of 0.5% RADA16 and the desired molar concentration of salt.
Salt Concentration Study
[00194] The protocol depicted in Figures 41A-41D was followed to prepare 0.5% solutions of
RADA16 with different concentrations of CaCl2. Mixed solutions were allowed to sit for a
relaxation period of about 24 hours. Vials were then inverted upside down so that gel properties
could be assessed. If the composition remained entirely in place when the vial containing it was
inverted, the composition was considered a fully functional gel. If more than half of the
composition remained in place when the vial containing it was inverted, it was considered a
semi-functional gel. If more than half of the composition, and particularly if substantially all of
the composition fell to the opposite end of the vial containing it upon inversion, it was
considered a non-functional gel.
[00195] Figures 42A-42G show images of results achieved, specifically depicting upright and
inverted vials for each of a variety of concentrations of CaCl2. As can be seen, for these 0.5%
RADA16 compositions, fully functional gel was formed with 0.250 M CaCl2 (Panel E), semi
functional gel was formed with 0.500 M CaCl2 (Panel F), and a non-functional gel was formed at
lM CaCl2 (Panel G).
Cation Selection Study
[00196] The protocol depicted in Figures 41A-41D was followed to prepare 0.5%> solutions of
RADA16 with 0.005, 0.05, 0.125, 0.25, 0.5, and 1M NaCl, KC1, and CaCl2 were prepared. The
anion, chloride (CI ), was kept the same to observe the effect of the cations, sodium (Na+),
potassium (K+), and calcium (Ca +) . Results are depicted in Figure 43, which provides a basic
understanding of how varying the cations of a salt solution affects viscoelastic properties and the
stiffness of the resulting compositions.
Mechanical Strength Study
[00197] The protocol depicted in Figures 41A-41D was followed to analyze stiffness of
peptide compositions containing either 2.5% RADA16 and no added salt or 2.5% RADA16 and
0.125 M CaCl2. Results are shown in Figure 44, which provides a basic understanding of the
viscoelastic properties of the resulting compositions. As can be seen, there is noticeable increase
in stiffness between the two solutions when a cation solution is mixed in.
Reversibility Study
[00198] The protocol depicted in Figures 41A-41D was followed to prepare solutions of 0.5%
RADA16 mixed with 0.125, 0.25, or 0.5 M CaCl2 were prepared. The compositions were
subjected to mechanical stress through vortexing and sonication, so that their structure was
thoroughly disrupted (e.g., randomized). Disrupted compositions were then placed at room
temperature for 48 hours to allow self-assembly to take place. Figures 45A and 45B present
results from this study, and reveal basic viscoelastic properties of the peptide compositions,
showing that that reversibility can be maintained even after perturbation of any structure in the
composition when 0.125 or 0.25 M CaCl2 is included. By contrast, a composition with 0.5 M
CaCl2 shows dramatically less stiffness (reflecting dramatically reduced ability to restore
structure) after disruption.
REFERENCES
[1] P. Chen. (2005). "Self-assembly of ionic-complementary peptides: a physicochemical
viewpoint." Colloids and Surfaces.
[2] Mishra A., et al. (2013). "Influence of metal salts on the hydrogelation properties of
ultrashort aliphatic peptides." RSC Advances .
[3] Shuguang, Z., et al. (1999). "Peptide self-assembly in functional polymer science and
Engineering." Reactive and Functional Polymers.
[4] Yanlian, Y., et al. (2009). "Designer self-assembling peptide nanomaterials." Nano Today.
[5] Zhaoyang, Y., et al. (2008). "Temperature and pH effects on biophysical and morphological
properties of self-assembling peptide RADA16-I." Journal of Peptide Science.
EQUIVALENTS
[00199] Those skilled in the art will recognize, or be able to ascertain using no more than
routine experimentation, many equivalents to the specific embodiments of the invention
described herein. The scope of the present invention is not intended to be limited to the above
Description, but rather is as set forth in the following claims:

CLAIMS
We claim:
1. An IEIK13 composition comprising:
an IEIK13 peptide at a concentration of at least 0.25%;
which composition has a pH within the range of about 2.5 to about 4.0.
2. The composition of claim 1, which composition is a solution.
3. The composition of claim 1, which composition is a gel.
4. The composition of claim 1, wherein the composition has ionic strength within the range
of about 0.0001 M to about 0.1 M.
5. The composition of claim 4, wherein the ionic strength is adjusted/given by common salts,
wherein the common salts are selected from the group consisting of NaCl, KC1, MgCl2,
CaCl2, and CaS0 4.
6. The composition of claim 4, wherein the ionic strength is given by common salts,
wherein the common salts are composed of one or more salt forming cations and one or
more salt forming anions, wherein the salt forming cations are selected from the group
consisting of ammonium, calcium, iron, magnesium, potassium, pyridinium, quaternary
ammonium, and sodium, wherein the salt forming anions are selected from the group
consisting of acetate, carbonate, chloride, citrate, cyanide, floride, nitrate, nitrite, and
phosphate.
7. The composition of claim 1, wherein the composition has storage modulus within the
range of about 100 to about 10000 Pa at 1 rad/sec of frequency and 1 Pa of oscillation
stress.
8. The composition of claim 1, wherein the composition is buffered with sodium hydroxide,
potassium hydroxide, calcium hydroxide, sodium carbonate, sodium acetate, sodium
sulfide, or DMEM.
9. The composition of claim 1, wherein:
the IEIK13 peptide is present at a concentration of less than 3% and/or
the composition has a pH within the range of about 3.0 to about 4.0.
10. The composition of claim 9, which composition is a gel that exhibits a storage modulus
of more than 500 Pa at 1 rad/sec of frequency and 1 Pa of oscillation stress.
11. The composition of claim 1, wherein the composition is buffered with a sodium
hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate, sodium acetate,
or sodium sulfide, so that the pH of the composition is regulated.
12. A liquid peptide composition comprising:
a peptide having a length within the range of about 6 to about 20 amino acids and an
amino acid sequence of alternating hydrophobic amino acid and hydrophilic amino acids,
which composition is characterized in that:
it has a viscosity within the range of about 1 Pa s to about 500,000 Pa s at room
temperature.
ithas a storage modulus at 1 rad/sec of frequency and 1 Pa of oscillation stress
within the range of about 1 to about 5000 Pa
it forms a gel within a time period about 0 to about 30s when exposed
to/maintained under pH within the range of about 2.5 to about 4.0 or and/or ionic
strength within the range of about 0.0001 M to about 1.5 M.
12A. The composition of claim 12, which is an aqueous composition.
13. The composition of claim 12, wherein the peptide comprises RADA16.
14. The composition of claim 12, wherein the peptide comprises IEIK13
15. The composition of claim 12, wherein the peptide comprises comprises KLD12.
16. The composition of claim 12, wherein the ionic strength is given by common salts,
wherein the common salts are selected from the group consisting of NaCl, KC1, MgCl2,
CaCl2, and CaS0 4.
17. The composition of claim 12, wherein the ionic strength is given by common salts,
wherein the common salts are composed of one or more salt forming cations and one or
more salt forming anions, wherein the salt forming cations are selected from the group
consisting of ammonium, calcium, iron, magnesium, potassium, pyridinium, quaternary
ammonium, and sodium, wherein the salt forming anions are selected from the group
consisting of acetate, carbonate, chloride, citrate, cyanide, floride, nitrate, nitrite, and
phosphate.
18. The composition of claim 12, wherein the composition is buffered with a sodium
hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate, sodium acetate,
or sodium sulfide, so that the pH of the composition is regulated.
19. A method comprising steps of:
selecting a peptide composition for application to a particular in vivo site, the method
comprising steps of:
determining one or more parameters selected from the group consisting of storage
modulus, viscosity, gelation time, shear-thinning property, and peptide nano-fiber re-assembly
time for the peptide composition; and
comparing the determined one or more parameters to a set of characteristics determined
to be appropriate for application to the particular in vivo site; and
choosing the peptide composition in light of the comparison; and
administering the chosen peptide composition to the site.