Enzymatically degradable mussel-inspired adhesive hydrogel

Mussel-inspired adhesive hydrogels represent innovative candidate medical sealants or glues. In the present work, we describe an enzyme-degradable mussel-inspired adhesive hydrogel formulation, achieved by incorporating minimal elastase substrate peptide Ala-Ala into the branched poly(ethylene glycol) (PEG) macromonomer structure. The system takes advantage of neutrophil elastase expression upregulation and secretion from neutrophils upon recruitment to wounded or inflamed tissue. By integrating adhesive degradation behaviors that respond to cellular cues, we expand the functional range of our mussel-inspired adhesive hydrogel platforms. Rapid (<1 min) and simultaneous gelation and adhesion of the proteolytically active, catechol-terminated precursor macromonomer was achieved by addition of sodium periodate oxidant. Rheological analysis and equilibrium swelling studies demonstrated that the hydrogel is appropriate for soft tissue-contacting applications. Notably, hydrogel storage modulus (G') achieved values on the order of 10 kPa, and strain at failure exceeded 200% strain. Lap shear testing confirmed the material's adhesive behavior (shear strength: 30.4 ± 3.39 kPa). Although adhesive hydrogel degradation was not observed during short-term (27 h) in vitro treatment with neutrophil elastase, in vivo degradation proceeded over several months following dorsal subcutaneous implantation in mice. This work represents the first example of an enzymatically degradable mussel-inspired adhesive and expands the potential biomedical applications of this family of materials.     

Novel amphiphilic poly(epsilon-caprolactone)-g-poly(L-lysine) degradable copolymers

As part of the search of novel degradable polymers, amphiphilic and cationic poly(epsilon-caprolactone)-g-poly(l-lysine) (PCL-g-PlL) copolymers have been synthesized following a grafting "onto" or a grafting "from" method both applied to a macropolycarbanionic PCL derivative. The first approach led to PCL-g-PZlL containing 36% of epsilon-caprolactone and 64% of N-epsilon-Z-l-lysine units, by reaction of activated poly(N-epsilon-Z-l-lysine) on the macropolycarbanion derived from PCL. The second route was based on the anionic ring opening polymerization of N-carboxyanhydride of N-epsilon-benzyloxycarbonyl-l-lysine initiated by the macropolycarbanion derived from PCL and led to a similar copolymer containing 45% of of epsilon-caprolactone and 55% of N-epsilon-Z-l-lysine units. After deprotection of the lysine units, PCL-g-PlL copolymers were obtained. These copolymers are water-soluble and form nanometric micelle-like objects with mean diameters between 60 and 500 nm in distilled water depending on the synthesis route.     

Characterization of protein release from hydrolytically degradable poly(ethylene glycol) hydrogels

We present a novel fully hydrophilic, hydrolytically degradable poly(ethylene glycol) (PEG) hydrogel suitable for soft tissue engineering and delivery of protein drugs. The gels were designed to overcome drawbacks associated with current PEG hydrogels (i.e., reaction mechanisms or degradation products that compromise protein stability): the highly selective and mild cross‐linking reaction allowed for encapsulating proteins prior to gelation without altering their secondary structure as shown by circular dichroism experiments. Further, hydrogel degradation and structure, represented by mesh size, were correlated to protein release. It was determined that polymer density had the most profound effect on protein diffusivity, followed by the polymer molecular weight, and finally by the specific chemical structure of the cross‐linker. By examining the diffusion of several model proteins, we confirmed that the protein diffusivity was dependent on protein size as smaller proteins (e.g., lysozyme) diffused faster than larger proteins (e.g., Ig). Furthermore, we demonstrated that the protein physical state was preserved upon encapsulation and subsequent release from the PEG hydrogels and contained negligible aggregation or protein–polymer adducts. These initial studies indicate that the developed PEG hydrogels are suitable for release of stable proteins in drug delivery and tissue engineering applications. Biotechnol. Bioeng. 2011; 108:197–206. © 2010 Wiley Periodicals, Inc.     

Poly(ethylene oxide sulfide): new poly(ethylene glycol) derivatives degradable in reductive conditions

Poly(ethylene oxide sulfide) (PEOS), polymers consisting of an internal ethylene oxide oligomer and disulfide linkage, were synthesized and characterized. The degree of polymerization was dependent upon temperature, dimethyl sulfoxide condition, and monomer hydrophobicity. The stability of PEOS was measured by the size exclusion chromatography method after the incubation both with and without 5 mM glutathione. The disulfide bond was stable in the extracellular condition but completely degraded in 2 h in the reductive cytosolic condition. Hydrophilic PEOS polymers showed no cytotoxicity on the HepG2 cell line. On the basis of these properties, PEOS can be applied in many drug delivery fields.     

Facilitation of gene transfection with well-defined degradable comb-shaped poly(glycidyl methacrylate) derivative vectors

Recently, we reported that ethanolamine (EA)-functionalized poly(glycidyl methacrylate) (PGMA) vectors (PGEAs) can produce good transfection efficiency, while exhibiting very low toxicity. Further improvement in degradability and transfection efficiency of the PGEA vectors will facilitate their application in gene therapy. Comb-shaped cationic copolymers have been of interest and importance as nonviral gene carriers. Herein, the degradable high-molecular-weight comb-shaped PGEA vectors (c-PGEAs) composed of the low-molecular-weight PGEA backbone and side chains were prepared by a combination of atom transfer radical polymerization (ATRP) and ring-opening reactions. The PGEA side chains were linked with the PGEA backbones via hydrolyzable ester bonds. Such comb-shaped c-PGEA vectors possessed the degradability, good pDNA condensation ability, low cytotoxicity, and good buffering capacity. More importantly, the comb-shaped c-PGEA vectors could enhance the gene expression levels. Moreover, the PGEA side chains of c-PGEA could also be copolymerized with some poly(poly(ethylene glycol)ethyl ether methacrylate) species to further improve the gene delivery system.     

Synthesis and characterization of injectable poly(N-isopropylacrylamide-co-acrylic acid) hydrogels with proteolytically degradable cross-links

Hydrogels composed of N-isopropylacrylamide (NIPAAm) and acrylic acid (AAc) were prepared by redox polymerization with peptide cross-linkers to create an artificial extracellular matrix (ECM) amenable for testing hypotheses regarding cell proliferation and migration in three dimensions. Peptide degradable cross-linkers were synthesized by the acrylation of the amine groups of glutamine and lysine residues within peptide sequences potentially cleavable by matrix metalloproteinases synthesized by mammalian cells (e.g., osteoblasts). With the peptide cross-linker, loosely cross-linked poly(N-isopropylacrylamide-co-acrylic acid) [P(NIPAAm-co-AAc)] hydrogels were prepared, and their phase transition behavior, lower critical solution temperature (LCST), water content, and enzymatic degradation properties were investigated. The peptide-cross-linked P(NIPAAm-co-AAc) hydrogels were pliable and fluidlike at room temperature and could be injected through a small-diameter aperture. The LCST of peptide-cross-linked hydrogel was influenced by the monomer ratio of NIPAAm/AAc but not by cross-linking density within the polymer network. A peptide-cross-linked hydrogel with a 97/3 molar ratio of NIPAAm/AAc exhibited a LCST of approximately 34.5 degrees C. Swelling was influenced by NIPAAm/AAc monomer ratio, cross-linking density, and swelling media; however, all hydrogels maintained more than 90% water even at 37 degrees C. In enzymatic degradation studies, breakdown of the peptide-cross-linked P(NIPAAm-co-AAc) hydrogels was dependent on both the concentration of collagenase and the cross-linking density. These results suggest that peptide-cross-linked P(NIPAAm-co-AAc) hydrogels can be tailored to create environmentally-responsive artificial extracellular matrixes that are degraded by proteases.     

Thermogelling poly(ethylene oxide-b-propylene oxide-b-ethylene oxide) disulfide multiblock copolymer as a thiol-sensitive degradable polymer

We report a reverse thermogelling poly(ethylene oxide-b-propylene oxide-b-ethylene oxide) disulfide multiblock copolymer as a thiol-sensitive biodegradable polymer. The poly(ethylene oxide-b-propylene oxide-b-ethylene oxide) aqueous solutions studied in this research underwent sol-gel-sol or sol-gel-sol-gel transition depending on the molecular weight and concentration of the polymer, whereas the corresponding disulfide multiblock copolymer aqueous solutions underwent sol-gel transition as the temperature increased in a range of 0-60 degrees C. The hydrophobic dye solubilization and dynamic light scattering of the polymer aqueous solution suggest that the poly(ethylene oxide-b-propylene oxide-b-ethylene oxide)s undergo unimer (3 nm) to micelle (12 nm) transition, whereas the disulfide multiblock copolymers undergo unimer (6 nm) to aggregated polymer (600 nm) transition as the temperature increases. The gel duration increased from 6 h (poly(ethylene oxide-b-propylene oxide-b-ethylene oxide)) to more than 12 days (the corresponding disulfide multiblock copolymer) in phosphate buffer saline, and the gel duration of the latter depended on the glutathione concentration of the medium. The model drug, paclitaxel, was released from the in-situ-formed poly(ethylene oxide-b-propylene oxide-b-ethylene oxide) disulfide multiblock copolymer gel in a glutathione concentration-sensitive manner.     

Highly efficient gene transfer with degradable poly(ester amine) based on poly(ethylene glycol) diacrylate and polyethylenimine in vitro and in vivo

BACKGROUND: Polyethylenimine (PEI) is toxic although it is one of the most successful and widely used gene delivery polymers with the aid of the proton sponge effect. Therefore, development of new novel gene delivery carriers having high efficiency with less toxicity is necessary. METHODS: In this study, a degradable poly(ester amine) carrier based on poly(ethylene glycol) diacrylate (PEGDA) and low molecular weight linear PEI was prepared. Furthermore, we compared the gene expression of the polymer/DNA complexes using two delivery methods: intravenous administration as an invasive method and aerosol as a non-invasive method. RESULTS: The synthesized polymer had a relatively small molecular weight (MW = 7980) with 25 h half-life in vitro. The polymer/DNA complexes were formed at an N/P ratio of 9. The particle sizes and zeta-potentials of the complexes were dependent on N/P ratio. Compared to PEI 25K, the newly synthesized polymer exhibited high transfection efficiency with low toxicity. Poly(ester amine)-mediated gene expression in the lung and liver was higher than that of the conventional PEI carrier. Interestingly, non-invasive aerosol delivery induced higher gene expression in all organs compared to intravenous method in an in vivo mice study. Such an expressed gene via a single aerosol administration in the lung and liver remained unchanged for 7 days. CONCLUSIONS: Our study demonstrates that poly(ester amine) may be applied as an useful gene carrier.     

Synthesis and properties of degradable poly(urethane urea)s to be used for ligament reconstructions

In the present study we describe the synthesis, wet spinning, mechanical testing, and degradation of poly(urethane urea)s (PUURs) intended for clinical use in anterior cruciate ligament (ACL) reconstruction. The effects of soft segment chemical composition and molar mass and the kind of diamine chain extender on the material properties were investigated. It was found that the fibers made of PUUR with polycaprolactone diol (PCL530) as soft segment and MDI/1,3-DAP as hard segment (PCL530-3) have high tensile strength and high modulus and when degraded keep their tensile strength for the time demanded for the application. In conclusion, from a chemical and mechanical point of view PUUR fibers of PCL530-3, ARTELON, are suitable for designing a degradable ACL device.     

Design and biophysical characterization of bioresponsive degradable poly(dimethylaminoethyl methacrylate) based polymers for in vitro DNA transfection

Water-soluble, degradable polymers based on poly(N,N-dimethylaminoethyl methacrylate) (PDMAEMA) with low cytotoxicity and good p-DNA transfection efficiency are highlighted in this article. To solve the nondegradability issue of PDMAEMA, new polymers based on DMAEMA and 5,6-benzo-2-methylene-1,3-dioxepane (BMDO) for gene transfection were synthesized. A poly(ethylene oxide) (PEO) azo-initiator was used as free-radical initiator. PEGylation was performed to improve water solubility and to reduce cytotoxicity of the polymers. The resulting polymers contain hydrolyzable ester linkages in the backbone and were soluble in water even with very high amounts of ester linkages. These degradable copolymers showed significantly less toxicity with a MTT assay using L929 cell lines and demonstrated promising DNA transfection efficiency when compared with the gold standard poly(ethyleneimine). Bioresponsive properties of the corresponding quaternized DMAEMA based degradable polymers were also studied. Although the quaternized DMAEMA copolymers showed enhanced water solubility, they were inferior in gene transfection and toxicity as compared to the unquaternized copolymers.     

Monitoring the in vitro enzyme-mediated degradation of degradable poly(ester amide) for controlled drug delivery by LC-ToF-MS

To scrutinize materials for specific biomedical applications, we need sensitive and selective analytical methods that can give more insight into the process of their biodegradation. In the present study, the enzymatic degradation of multiblock poly(ester amide) based on natural amino acids, such as lysine and leucine, was performed with serine proteases (α-chymotrypsin (α-CT) and proteinase K (PK)) in phosphate-buffered saline solution at 37 °C for 4 weeks. Fully and partially degraded water-soluble products were analyzed by liquid chromatography hyphenated with time-of-flight mass spectrometry using an electrospray interface (LC-ESI-ToF-MS). Tracking the release of monomeric and oligomeric products into the enzyme media during the course of enzymatic degradation revealed the preferences of α-CT and PK toward ester and amide bonds: both α-CT and PK showed esterase and amidase activity. Although within the experimental time frame up to 30 and 15% weight loss was observed in case of α-CT and PK, respectively, analysis by size exclusion chromatography showed no change in the characteristic molecular-weight averages of the remaining polymer. This suggests that the enzymatic degradation occurs at the surface of this biomaterial. A sustained and linear degradation over a period of 4 weeks supports the potential of this class of poly(ester amide)s for drug delivery applications.     

Monoclonal antibodies specific for poly(dG) X poly(dC) and poly(dG) X poly(dm5C)

Most duplex DNAs that are in the "B" conformation are not immunogenic. One important exception is poly(dG) X poly(dC), which produces a good immune response even though, by many criteria, it adopts a conventional right-handed helix. In order to investigate what features are being recognized, monoclonal antibodies were prepared against poly(dG) X poly(dC) and the related polymer poly(dG) X poly(dm5C). Jel 72, which is an immunoglobulin G, binds only to poly(dG) X poly(dC), while Jel 68, which is an immunoglobulin M, binds approximately 10-fold more strongly to poly(dG) X poly(dm5C) than to poly(dG) X poly(dC). For both antibodies, no significant interaction could be detected with any other synthetic DNA duplexes including poly[d(Gm5C)] X poly[d(Gm5C)] in both the "B" and "Z" forms, poly[d(Tm5Cm5C)] X poly[d(GGA)], and poly[d(TCC)] X poly[d(GGA)], poly(dI) X poly(dC), or poly(dI) X poly(dm5C). The binding to poly(dG) X poly(dC) was inhibited by ethidium and by disruption of the DNA duplex, confirming that the antibodies were not recognizing single-stranded or multistranded structures. Furthermore, Jel 68 binds significantly to phage XP-12 DNA, which contains only m5C residues and will precipitate this DNA in the absence of a second antibody. The results suggest that (dG)n X (dm5C)n sequences in natural DNA exist in recognizably distinct conformations.     

Complete disproportionation of duplex poly(dT)*poly(dA) into triplex poly(dT)*poly(dA)*poly(dT) and poly(dA) by coralyne

Coralyne is a small crescent-shaped molecule known to intercalate duplex and triplex DNA. We report that coralyne can cause the complete and irreversible disproportionation of duplex poly(dT)·poly(dA). That is, coralyne causes the strands of duplex poly(dT)·poly(dA) to repartition into equal molar equivalents of triplex poly(dT)·poly(dA)·poly(dT) and poly(dA). Poly(dT)·poly(dA) will remain as a duplex for months after the addition of coralyne, if the sample is maintained at 4°C. However, disproportionation readily occurs upon heating above 35°C and is not reversed by subsequent cooling. A titration of poly(dT)·poly(dA) with coralyne reveals that disproportionation is favored by as little as one molar equivalent of coralyne per eight base pairs of initial duplex. We have also found that poly(dA) forms a self-structure in the presence of coralyne with a melting temperature of 47°C, for the conditions of our study. This poly(dA) self-structure binds coralyne with an affinity that is comparable with that of triplex poly(dT)·poly(dA)·poly(dT). A Job plot analysis reveals that the maximum level of poly(dA) self-structure intercalation is 0.25 coralyne molecules per adenine base. This conforms to the nearest neighbor exclusion principle for a poly(dA) duplex structure with A·A base pairs. We propose that duplex disproportionation by coralyne is promoted by both the triplex and the poly(dA) self-structure having binding constants for coralyne that are greater than that of duplex poly(dT)·poly(dA).     

Nucleosome reconstitution of core-length poly(dG).poly(dC) and poly(rG-dC).poly(rG-dC)

The double-stranded polypurine.polypyrimidines poly(dG).poly(dC) and poly[d(A-G)].poly[d(T-C)] and the mixed ribose-deoxyribose polynucleotide poly(rG-dC).poly(rG-dC) have been successfully reconstituted into nucleosomes. The radioactively labeled particles comigrate in gel electrophoresis and sucrose density gradient experiments with authentic nucleosomes derived from chicken erythrocyte chromatin. These results show that nucleosomes are able to accommodate a wider variety of polynucleotides than was previously believed.     

Hydrolytically degradable poly(ethylene glycol) hydrogel scaffolds as a cell delivery vehicle: Characterization of PC12 cell response

The central nervous system (CNS) has a low intrinsic potential for regeneration following injury and disease, yet neural stem/progenitor cell (NPC) transplants show promise to provide a dynamic therapeutic in this complex tissue environment. Moreover, biomaterial scaffolds may improve the success of NPC‐based therapeutics by promoting cell viability and guiding cell response. We hypothesized that a hydrogel scaffold could provide a temporary neurogenic environment that supports cell survival during encapsulation, and degrades completely in a temporally controlled manner to allow progression of dynamic cellular processes such as neurite extension. We utilized PC12 cells as a model cell line with an inducible neuronal phenotype to define key properties of hydrolytically degradable poly(ethylene glycol) hydrogel scaffolds that impact cell viability and differentiation following release from the degraded hydrogel. Adhesive peptide ligands (RGDS, IKVAV, or YIGSR), were required to maintain cell viability during encapsulation; as compared to YIGSR, the RGDS, and IKVAV ligands were associated with a higher percentage of PC12 cells that differentiated to the neuronal phenotype following release from the hydrogel. Moreover, among the hydrogel properties examined (e.g., ligand type, concentration), total polymer density within the hydrogel had the most prominent effect on cell viability, with densities above 15% w/v leading to decreased cell viability likely due to a higher shear modulus. Thus, by identifying key properties of degradable hydrogels that affect cell viability and differentiation following release from the hydrogel, we lay the foundation for application of this system towards future applications of the scaffold as a neural cell delivery vehicle. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1255–1264, 2013     

Monohydroxylated poly(3-hydroxyoctanoate) oligomers and its functionalized derivatives used as macroinitiators in the synthesis of degradable diblock copolyesters

The presence of a hydroxyl group at the end of poly(3-hydroxyoctanoate) oligomers, noted PHO oligomers, is required to prepare diblock copolymers with improved properties by ring-opening polymerization of cyclic monomer as epsilon-caprolactone. Several chemical methods such as basic hydrolysis, acid-catalyzed reaction with APTS, and methanolysis were used to prepare well-defined low molar masses PHO oligomers. The methanolysis reaction was allowed to proceed for 10-60 min to produce PHO oligomers with Mn values ranging from 20,000 to 800 g mol-1 with low polydispersity index. Detailed analysis of the MALDI-TOF mass spectra of the obtained oligomers has revealed the presence of linear structures bearing methyl ester on one side and hydroxyl end group on the other side. The same procedure was applied to poly(3-hydroxyoctanoate-co-3-hydroxyundecenoate), PHOU, a poly(3-hydroxyalkanoate) containing unsaturated units in its side chains. These oligomers were further used to initiate the polymerization of epsilon-caprolactone by varying the PHO (or PHOU) and PCL lengths. By copolymerization with epsilon-caprolactone, the properties of PHO or PHOU have been improved. The crystallinity of the obtained copolymers was modified by controlling the length of the two different blocks. The unsaturations in the side chains of the PHOU block were oxidized in acid carboxylic functions to obtain a novel artificial biopolyester. Moreover, degradation was followed to study the influence of carboxylic groups on the hydrolysis of the copolymers.     

High pressure fourier transform infrared spectroscopy of poly(dA)poly(dT), poly(dA) and poly(dT)

The effect of hydrostatic pressure upon the DNA duplex, poly(dA)poly(dT), and its component single strands, poly(dA) and poly(dT) has been studied by fourier-transform infrared spectroscopy (FT-IR). The spectral data indicate that at 28 degrees C and pressures up to 12 kbar (1200 MPa) all three polymers retain the B conformation. Pressure causes the band at 967 cm(-1), arising from water-deoxyribose interactions, to shift to higher frequencies, a result consistent with increased hydration at elevated pressures. A larger pressure-induced frequency shift in this band is observed in the single stranded polymers than in the double stranded molecule, suggesting that the effect of pressure on the hydration of single strands may be greater than upon a double stranded complex. A pressure-dependent hypochromicity in the bands attributed to base stacking indicates that pressure facilitates the base stacking in the three polymers, in agreement with previous assessments of the importance of stacking in the stabilization of DNA secondary structure at ambient and high pressures.     

Laser Raman spectroscopic studies of poly(r-cytidylic acid) and poly(r-adenylic acid) complexes with poly(L-lysine) and poly(L-arginine)

Complexes of polyribocytidylic acid and polyriboadenylic acid with poly(L -lysine) and poly(L -arginine) were studied by Raman spectroscopy. The backbones of both polynucleotides are distorted by poly(L -arginine). On the other hand, poly(L -lysine) could distort the backbone of polyriboadenylic acid but not that of polyribocytidylic acid. In general, poly(L -arginine) can increase the order of the base stacking, while poly(L -lysine) causes disordering in the base stacking.     

Structure of poly (I).poly (A).poly (I)

The molecular structure of poly (I).poly (A).poly (I) has been determined and refined using the continuous intensity data on layer lines in the x-ray diffraction pattern obtained from an oriented fiber of this polymorphic RNA complex. The polymer forms a 12-fold right-handed triple-helix of pitch 39.7A and each base-triplet is stabilized by quasi Crick-Watson-Hoogsteen hydrogen bonds. The ribose rings in all the three strands have C3'-endo conformations. The final R-value for this best structure is 0.24 and the x-ray fit is significantly superior to all the alternative structures where the different chains might have different furanose conformations. This all-purine triple-helix, counter-intuitively, has a diameter roughly 3A shorter than that of DNA and RNA triple-helices containing a homopurine and two complementary homopyrimidine strands. Its compact, grooveless cylindrical shape is consistent with the lack of lateral organization.     

Proton exchange and base-pair kinetics of poly(rA).poly(rU) and poly(rI).poly(rC)

Proton exchange of poly(rA).poly(rU) and poly(rI).poly(rC) has been studied by nuclear magnetic resonance line broadening and saturation transfer from H2O. Five exchangeable peaks are observed. They are assigned to the imino, amino and 2'-OH ribose protons. The aromatic spectrum is also assigned. Contrary to previous observations, we find that the exchange of the imino proton is strongly buffer sensitive. This property is used to derive the base-pair lifetime, which is in the range of milliseconds at 27 degrees C, 100 times smaller than published values. The enthalpy for the base-opening reaction (-86 kJ/mol) and the insensitivity of the reaction to magnesium suggest that the open state involves a small number of base-pairs. The similarities in the exchange from the two duplexes indicate that the same open state is responsible for exchange of purine and pyrimidine imino protons. For the lifetime of the open state and for the base-pair dissociation constant, we obtain only lower limits. At 27 degrees C they are three microseconds and 10(-3), respectively. The analysis that yields the much larger values published previously is based on the assumption that amino protons exchange only from open base-pairs. But theory and preliminary experiments indicate that it may occur from the closed duplex. The exchange of amino protons is slower than that of the imino protons. Exchange of the 2'-OH protons from the duplexes is much slower than from single-stranded poly(rU), and it is accelerated by magnesium. This could indicate hydrogen-bonding to backbone phosphate. Discrepancies between our results and those of previous studies are discussed.