A novel strategy for encapsulation and release of proteins: Hydrogels and microgels with acid-labile acetal cross-linkers

Article abstract of DOI:10.1021/ja026925r

A new acid-labile acetal cross-linker was synthesized and used to prepare protein-loaded hydrogels and microgels. This cross-linker undergoes an acid-catalyzed degradation with a half-life of 5.5 min at pH 5.0 and 24 h at pH 7.4. Protein-loaded hydrogels were synthesized with this cross-linker, and their release profiles were measured as a function of pH. Hydrogels made with the acetal cross-linker release their contents in a pH-dependent manner. The acetal cross-linker was also used to synthesize microgels with sizes between 1 and 10 μm, a range suitable for phagocytosis. The unique acid sensitivity of the acetal cross-linker should make it a useful synthetic intermediate in the design of acid-sensitive drug or gene delivery systems. Copyright

Full text of DOI:10.1021/ja026925r

Published on Web 10/04/2002
A Novel Strategy for Encapsulation and Release of Proteins: Hydrogels and
Microgels with Acid-Labile Acetal Cross-Linkers
Niren Murthy, Yi X. Thng, Stephany Schuck, Ming C. Xu, and Jean M. J. Fre´chet*
Center for New Directions in Organic Synthesis, Department of Chemistry, UniVersity of California,
Berkeley, California 94720-1460, and Lawrence Berkeley National Laboratory, Materials Sciences DiVision,
Berkeley, California 94720
Received May 15, 2002
Scheme 1. Acid-Degradable Protein-Loaded Hydrogela
Hydrogels and microgels have been intensely investigated as
protein delivery vehicles because of their excellent biocompatibility
and hydrophilicity.1 Numerous protein-loaded hydrogels and mi-
crogels have been synthesized and investigated. For example,
hydrogels or microgels composed of acrylamide and methylene
bisacrylamide, dextrans grafted with methacrylates, PEG-meth-
acrylates, and PLGA-PEG-methacrylates have been synthesized and
used to encapsulate proteins.2 These hydrogels are usually cross-
linked using ester, amide, or carbonate linkages that are most
susceptible to degradation via base-catalyzed hydrolysis.
For drug delivery applications, it would be particularly useful
to develop protein-loaded hydrogels and microgels that degrade
by acid-catalyzed hydrolysis. There are numerous drug delivery
targets that exist at acidic pHs, such as tumors, inflammatory tissues,
and the phagolysosomes of antigen presenting cells.3 Acid-
degradable hydrogels and microgels designed to undergo degrada-
tion in these acidic tissues should therefore be capable of selective
delivery of their therapeutic contents. At present, the only method
available for engineering acid sensitivity in hydrogels is through
the incorporation of cationic groups, which become protonated at
acidic pHs and cause hydrogel swelling.4 However, for protein
delivery, it would be preferable to develop acid-sensitive hydrogels
that were neutral, thus avoiding the potential toxicity of polycations
and the complications of electrostatic interaction with proteins.5
In this communication, a new acetal cross-linker is synthesized
and used to prepare acid-sensitive, acetal cross-linked, protein-
loaded hydrogels and microgels (see Scheme 1). At acidic pHs,
the pore size of the acetal cross-linked hydrogels increases, due to
the hydrolysis of the acetal, and entrapped macromolecules diffuse
out, whereas at neutral pHs the cross-linker remains largely intact,
and the release of entrapped macromolecules is significantly slower.
Benzaldehyde acetals are particularly well suited for this ap-
plication because their acid lability can be manipulated by introduc-
ing substituents in the para position, and it should therefore be
possible to engineer the hydrolysis kinetics of these gels for a wide
a Proteins are encapsulated by copolymerizing acrylamide (I) with the
range of applications.6 Therefore, the bisacrylamide acetal cross-
bisacrylamide acetal cross-linker (II). Acidic hydrolysis of the hydrogel
linker (II) with a p-methoxy substituent was chosen as the acid-
degradable linkage to ensure that microgels derived from it
hydrolyzed rapidly within the pH 5.0 environment that is encoun-
tered in phagolysosomes. Compound II was synthesized in two steps
with an overall yield of 40%. Reaction of N-(2-hydroxyethyl)-2,2,2-
trifluoroacetamide (4 equiv) with p-methoxybenzaldehyde in dry
THF in the presence of a catalytic amount of p-toluenesulfonic acid
afforded 70% yield of the acetal with two trifluoroacetamide groups.
This intermediate was then deprotected with 6 N NaOH, and the
results in protein release.
product was allowed to react with acryloyl chloride to afford the
desired compound, II, in 60% yield.
The hydrolysis of II was measured at pH 5.0 and at pH 7.4. At
pH 5.0, hydrolysis is rapid, with a half-life of 5.5 min, whereas at
pH 7.4, the half-life is 24 h. The acceleration of the hydrolysis
kinetics of this cross-linker from pH 7.4 to pH 5.0 is expected
because the rate of hydrolysis of benzaldehyde acetals is propor-
tional to the hydronium ion concentration, which increases 250-
fold between pH 7.4 and pH 5.0.6 The second-order hydrolysis rate
* To whom correspondence should be addressed. Phone: (510) 643-3077. Fax:
643-3079. E-mail: frechet@cchem.berkeley.edu.
constant of this cross-linker is 5610 min-1 mol-1
.
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J. AM. CHEM. SOC. 2002, 124, 12398-12399
10.1021/ja026925r CCC: $22.00 © 2002 American Chemical Society

C O M M U N I C A T I O N S
Figure 2. SEM image of microparticles synthesized by polymerization of
acrylamide (I) with the bisacrylamide acetal cross-linker (II).
Figure 1. pH-dependent release of FITC-Albumin from acid-degradable
hydrogels (data points are an average of three experiments).
shown in Figure 2; the particle size varies between 1 and 10 µm,
and the particles are therefore of a size appropriate for their
phagocytosis by antigen presenting cells.
The protein-loaded hydrogels and microgels based on acid
cleavable acetal cross-linkers are expected to release their contents
under the mild acidic conditions found in lysosomes, tumors, and
sites of inflammation. As a result, such cross-linkers should find
applications as lysosomal escape promoters and more generally in
the areas of tumor drug delivery, biomaterial coatings, and in the
development of vaccine and DNA delivery systems.
Protein-loaded hydrogels were prepared using II, acrylamide (I),
and a fluorescently labeled protein (FITC-Bovine Serum Albumin
(BSA)). Encapsulation was carried out by copolymerizing II (60
mg/mL) with I (200 mg/mL) in the presence of FITC-BSA (1 mg/
mL) in PBS buffer. This hydrogel was washed with PBS (pH 8.5)
buffer; analysis of the washing solution demonstrated essentially
quantitative gel entrapment of the FITC-BSA, suggesting that the
pore size of this gel, at this cross-linking ratio, is smaller than 3.48
nm (the Stokes radius of bovine albumin).2c As expected from the
molecular design of the gel, the rate of protein release was found
to be pH dependent. At pH 5.0, the acetal cross-links hydrolyze,
rapidly transforming the gel into a soluble polymer, and the
encapsulated protein is completely released within 2 h (see Figure
1). Gel electrophoresis of the released protein confirmed that it was
not significantly modified by either the encapsulation or the
hydrolysis procedures (see Supporting Information). At pH 7.4,
release of the entrapped protein is significantly slower as only 5%
of the encapsulated protein is released after 2 h, and 96 h was
required for the hydrogel to completely release its contents (Figure
1). The equilibrium water content, at this cross-linking ratio, is 5.1
mL of water per gram of gel.
The bisacrylamide acetal cross-linker was also used to synthesize
acid-degradable micron-sized hydrogels (microgels). Micron-sized
materials that decompose under acidic conditions are of great
interest because they can induce a colloid osmotic disruption of
phagosomes and deliver macromolecules into the cytoplasm of
antigen presenting cells.7
Microgel particles were prepared by inverse microemulsion
polymerization using I and II as co-monomers. Given the unusual
combination of monomers, several different polymerization pro-
cedures involving different combinations of organic phases and
blends of surfactant had to be tried.2a,8 Inverse emulsion polymer-
izations with toluene/chloroform as the continuous phase and
pluronic F-68 as the surfactant were unsuccessful, likely due to
the high solubility of II in toluene/chloroform. However, particles
could be obtained using hexane (4.0 g) as the continuous phase,
dioctyl sulfosuccinate (0.506 g) and Brij 30 (0.16 g) as the
surfactants, and an aqueous phase (5 mL, pH 8.4, 100 mM
phosphate buffer) containing 40 mg (0.5 mmol) of acrylamide and
22 mg (0.061 mmol) of II. An SEM image of these particles is
Acknowledgment. The Center for New Directions in Organic
Synthesis is supported by Bristol-Myers Squibb as Sponsoring
Member and Novartis as a Supporting Member. Financial support
of this research by the Biomolecular Program of the E. O. Lawrence
Berkeley National Laboratory (Department of Energy, Basic Energy
Sciences) is acknowledged with thanks. We would also like to thank
Dr. Thomas Rohr for help with SEM images and Amish Patel for
help with the protein gels.
Supporting Information Available: Experimental details of the
synthesis of II, hydrogel synthesis, and protein release studies (PDF).
This material is available free of charge via the Internet at http://
pubs.acs.org.
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