Diffusion of small molecules inside a peptide hydrogel

Article abstract of DOI:10.1039/c1cc13943f

A set of four phenylalanine analogues experiences diffusion retardation when transferred from phosphate-buffered saline into a peptide hydrogel of the same pH and ionic strength. The extent of retardation increases linearly with logP_oct, their

Full text of DOI:10.1039/c1cc13943f

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COMMUNICATION
Diffusion of small molecules inside a peptide hydrogelw
Yue Feng,a Manfai Lee,a Marc Tarabanb and Y. Bruce Yu*ab
Received 1st July 2011, Accepted 11th August 2011
DOI: 10.1039/c1cc13943f
A set of four phenylalanine analogues experiences diffusion
retardation when transferred from phosphate-buffered saline
into a peptide hydrogel of the same pH and ionic strength. The
extent of retardation increases linearly with logPoct, their
lipophilicity.
Table 1 Sequences, molecular weight of undecapeptides K11 and E11
Peptides
Sequences
MW/Da
K11
E11
acetyl-KWKAKAKAKWK-amide
acetyl-EWEAEAEAEWE-amide
1413
1419
A, alanine; E, glutamic acid; K, lysine; W, tryptophan. The N- and
C-termini of each peptide are acetylated (acetyl-) and amidated
(-amide), respectively. Each peptide contains two tryptophans for
concentration determination through UV spectroscopy.11
Peptide hydrogels are finding increasing applications as cell
growth and differentiation media1–4 with commercial products
on the market (e.g., PuraMatrixTM). Compared with collagens
extracted from animal sources, synthetic peptides have the
benefits of a lot-to-lot consistency and the absence of antigens
and pathogens. We previously developed a mixing-induced
approach to the assembly of peptide hydrogels.5–7 In this
approach, a hydrogel is assembled by mixing two oppositely
charged oligo-peptides. As potential media for cell growth and
differentiation, this type of hydrogels has several suitable
features: the two peptides can be pre-dissolved in physiological
buffers with gelation induced by mixing the two peptide
solutions at the desired temperature;5 the hydrogels can
recover rapidly from shear-induced breakdowns and thus are
injectable;6 proteins inside the hydrogels retain their native
conformation,7 which is important for biocompatibility.
Aside from biocompatibility, another requirement for cell
growth and differentiation media is transport properties: the
matrix should allow an efficient diffusion of nutrients and
metabolites. Extensive work has been done on the diffusion of
proteins in peptide hydrogels for controlled release.8–10 In this
project, the diffusion of small molecules inside a mixing-induced
peptide hydrogel is evaluated. The focus is on how molecular
characteristics, such as charge and lipophilicity, affect diffusion.
The hydrogel was assembled from a pair of oppositely
charged undecapeptides, K11 and E11, whose sequences are
shown in Table 1. The sequence of the positive peptide module,
K11, alternates between positively charged and neutral amino acids,
and the sequence of the negative peptide module, E11,
alternates between negatively charged and neutral amino acids.
Gelation in phosphate-buffered saline (PBS) and in H2O,
both of pH 7.4, was monitored using dynamic rheometry at
25 1C. Gelation in PBS was slower but reached a higher elastic
modulus G0 value (Fig. 1 and Fig. S21, ESIw). The PBS gel also
has a higher strain yield value (B3%) than the H2O gel
(B1%) (Fig. S22, ESIw).
To investigate small molecule diffusion inside this hydrogel, a
set of four phenylalanine analogues, 1, 2, 3 and 4 (Scheme 1), was
used as diffusants. Each compound contains a –CF3 group so that
it can be traced by 19F NMR without any interference from the
hydrogel. 1 was purchased from Chem-Impex, Inc (Wood Dale,
IL, USA). C-terminal amidation of 1 gives 2; N-terminal formyla-
tion of 1 gives 3; N-terminal formylation of 2 gives 4. The purity
and molecular weight of each compound were verified, respec-
tively, using analytical HPLC and mass spectrometry (ESIw).
In terms of the charge status, 4 is analogous to the solvent
(water) as both are neutral; 3 is analogous to E11 as both
contain negative charges carried by the carboxylic group
(–COOꢀ); 2 is analogous to K11 as both contain positive
charges carried by the amino group (–NH3+); 1 is analogous
to the hydrogel as both contain equal amounts of positive and
+
negative charges from –NH3 and –COOꢀ.
Diffusion of each compound was investigated in four types of
media: solvent, solution of 8 mM K11 peptide, solution of 8 mM
E11 peptide, and the hydrogel which contains 8 mM K11 and
8 mM E11. Pairing of diffusants and media is shown in Scheme 2.
The diffusion coefficient (D) was measured at 25 1C using
pulsed field gradient (PFG) NMR spectroscopy (bipolar pulse
longitudinal eddy current delay, BPP-LED).12 D of 1, 2, 3 and
4 was measured using the 19F signal. D of H2O was measured
as a reference point.
D values of four diffusants in four types of media are plotted
in Fig. 2, along with D of H2O. There are two series: a PBS
series and a D2O series. As each compound transfers from
solvents to peptide solutions, there is a diffusion retardation,
i.e., D(X in peptide solution) o D(X in solvent) (X = 1, 2, 3, 4
and H2O); as each compound transfers from solvents to
hydrogels, there is a diffusion retardation, i.e., D(X in hydrogel)
o D(X in solvent); and as each compound transfers from D2O
a Department of Pharmaceutical Sciences, University of Maryland,
Baltimore, MD 21201, USA. E-mail: byu@rx.umaryland.edu;
Fax: +1 4017065017; Tel: +1 4017067514
b Fischell Department of Bioengineering, University of Maryland,
College Park, MD 20742, USA
w Electronic supplementary information (ESI) available. See DOI:
10.1039/c1cc13943f
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 10455–10457 10455

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Fig. 1 Elastic modulus vs. time for the hydrogel assembled by mixing
K11 and E11 in PBS (K) and H2O (J).
Fig. 2 Diffusion coefficients of compounds in various media.
(A) In four PBS-based media; (B) in four D2O-based media. The pH
of each medium was 7.4. E, H2O; ’, 1; K, 2; %, 3; ., 4. Solid
symbols: in PBS media; hollow symbols: in D2O media. Error bars for
D(H2O) are based on 4 repeated measurements.
retardation in a given medium should be based on normalized
diffusion retardation, Re, defined as:
Re = 1 ꢀ D (X in medium)/D (X in solvent) ꢁ 100% (1)
with X = 1, 2, 3, 4 and H2O; and medium = solvent, K11
solution, E11 solution and hydrogel. Table 2 gives Re values
for 1, 2, 3, 4 and H2O in different media. In PBS or D2O, Re is
0 by definition.
With two exceptions, hydrogels cause larger diffusion
retardation than peptide solutions, i.e.
Scheme 1 Synthesis of fluorinated phenylalanine analogues.
Re(X in hydrogel) 4 Re(X in peptide solution)
(2)
Considering that a hydrogel contains twice as much peptide
(8 mM K11 + 8 mM E11) as a peptide solution (8 mM K11 or
8 mM E11), this is to be expected. In fact, for H2O, Re satisfies
the following additive relationship in both PBS and D2O
series:
Re(H2O in hydrogel) E Re(H2O in K11 solution)
+ Re(H2O in E11 solution)
(3)
However, such additivity does not hold for the non-solvent
diffusants 1, 2, 3 and 4. The implication is that the interactions
between these diffusants and peptides are quite different in
hydrogels and in solutions.
Scheme 2 Pairing of four diffusants with four media. + denotes positive
charge carried by –NH3+; ꢀdenotes negative charge carried by –COOꢀ; 0
denotes a molecule or a medium devoid of –NH3+ or –COOꢀ, e.g., solvent
and 4 are denoted as (0, 0) as they contain neither –NH3+ nor –COOꢀ.
As 1, 2, 3 and 4 differ in their charge status and the two
peptides carry multiple charges, one might expect 2 and 3 to
have larger Re than 1 and 4, because 2 and 3 carry the net
to PBS, there is a diffusion retardation, i.e., D(X in PBS) o
D(X in D2O). Clearly, diffusion retardation is caused by the
interaction between diffusants and non-solvent components of
the media (peptides, hydrogel matrix and salts). The question
is, in a given medium, does diffusion retardation correlate with
molecular characteristics of the diffusants? This is answered by
comparing diffusion retardation of the four closely related
diffusants, 1, 2, 3 and 4.
Table 2 Re (in %) of 1, 2, 3, 4 and H2O in various media. The charge
status symbols are the same as in Scheme 2
1
2
3
4
H2O
(+,ꢀ) (+, 0) (0,ꢀ) (0, 0) (0, 0)
Media
Diffusants
PBS series (pH 7.4) PBS (0, 0)
0
0
7.2
0
0
0
K11 sol (+, 0) 7.2
E11 sol (0,ꢀ)
8.2 9.2 3.3
2.2 11.2 3.7
8.7 11.2
Hydrogel (+,ꢀ) 13.2 14.2 11.6 14.7 7.5
D2O series (pH 7.4) D2O (0, 0)
K11 sol (+, 0) 3.9
3.0
Even in PBS and D2O, 1, 2, 3 and 4 have slightly different D
values, probably due to the difference in hydrodynamic radii
as a result of the difference in hydration and counter-ion
paring. Thus, a meaningful comparison of their diffusion
0
0
0
0
0
4.1 12.0 3.7 1.7
8.5
7.9
E11 sol (0,ꢀ)
4.5 5.2 1.6
7.2 8.4 3.4
Hydrogel (+,ꢀ) 7.7
c
10456 Chem. Commun., 2011, 47, 10455–10457
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Table 3 Poct of 1, 2, 3 and 4 at pH 7.4
the decline of electrostatic attraction; Re of 4 (neutral, highest
P
cot) increases, attesting rise of hydrophobic interaction; Re of
Compounds
Poct (1-octanol/PBS)
Poct (1-octanol/water)
2 (charged, 2nd highest Pcot) increases, attesting that hydro-
phobic interaction has become more important than electro-
static attraction. However, hydrophobic interaction does not
fully dominate diffusion retardation and there is no mono-
tonous increase of Re with logPoct in PBS solutions. One
possible explanation to the difference between E11 and K11
solutions is that E11 and K11 have different lipophilicity and
conformation, which leads to difference in Re values.
1 (zwitter ion)
2 (cation)
3 (anion)
0.84
4.87
0.02
13.01
0.76
1.92
0.01
13.65
4 (neutral)
charge. However, this is true only in the D2O solution of E11
(anion) in which 2 (cation) has the largest Re; and in the D2O
solution of K11 (cation) in which 3 (anion) has the largest Re.
In all other media, Re of 2 and 3 is not higher than Re of 1 and
4. This result suggests that only in D2O solutions, do electro-
static attraction between diffusants and peptides dominate
diffusion retardation. In other media, non-electrostatic inter-
actions become important.
In hydrogels, electrostatic attraction is primarily between
the two oppositely charged peptides as they both carry multiple
charges. As a result, electrostatic attraction between peptides
and diffusants is diminished to such an extent that hydro-
phobic interaction between peptides and diffusants dominates,
leading to Re increasing monotonously with logPoct. Any
residual electrostatic attraction between peptides and diffu-
sants in the D2O hydrogel is further abolished by the addition
of salts. Consequently, in the PBS hydrogel, Re has a linear
In addition to the charge status, another difference among
the four diffusants is their lipophilicity, which was quantified
by Poct, the 1-octanol/water partition coefficient. Poct values of
the four diffusants, measured in both PBS and H2O, are listed
in Table 3.
dependency on logPoct
.
This above analysis also sheds light on the gelation process
shown in Fig. 1. As a longer range interaction, electrostatic
attraction drives peptide association. In D2O, electrostatic
attraction is unscreened, leading to faster gelation. In PBS,
electrostatic interaction is screened, leading to slower gelation.
Finally, we wish to point out that the close structural
similarity of 1, 2, 3 and 4 is likely crucial in revealing the
linear relationship between Re and logPoct, as many con-
founding factors, such as size and conformation of the diffu-
sants, are eliminated.
When Re is plotted against logPoct (Fig. 3), a clear pattern
emerges: Re increases monotonously with logPoct in hydrogels
but not in solutions. Further, in the PBS hydrogel, Re has a
nearly perfect linear dependency on logPoct (R2 = 0.996).
It thus appears that both electrostatic attraction and hydro-
phobic interaction can retard the diffusion of a small molecule
in a medium. As to which one dominates, it depends on
whether other electrolytes (salts and peptides) are present.
We have three scenarios in this work.
In D2O solutions, electrostatic attraction dominates due to
the absence of other electrolytes. Thus, in the D2O solution of
K11 (cation), 3 (anion) has the largest Re; in the D2O solution
of E11 (anion), 2 (cation) has the largest Re. Further, these
two cases are the only two exceptions to eqn (2), attesting to
the prominence of electrostatic attraction in the absence of
other electrolytes.
When small molecules of similar size are transferred from
phosphate-buffered saline to a peptide hydrogel of the same
pH and ionic strength, their diffusion is retarded by 10–15%.
The extent of retardation has a linear dependency on logPoct
.
Overall, this type of mixing-induced peptide hydrogels has
excellent transport properties for small molecules.
We thank the NIH for financial support (EB004416).
Salt is known to screen electrostatic attraction and enhance
hydrophobic interaction.13 Due to the added salts in PBS
solutions, Re of 3 (charged, lowest Pcot) decreases, attesting
Notes and references
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Fig. 3 Re vs. logPoct. R2 is the goodness of linear fitting. ’, 1; K, 2;
%, 3; ., 4. Solid symbols: PBS media; hollow symbols: D2O media.
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 10455–10457 10457