Synthesis and characterization of injectable, thermally and chemically gelable, amphiphilic poly(N-isopropylacrylamide)-based macromers

In this study, we synthesized and characterized a series of macromers based on poly( N-isopropylacrylamide) that undergo thermally induced physical gelation and, following chemical modification, can be chemically cross-linked. Macromers with number average molecular weights typically ranging from 2000-3500 Da were synthesized via free radical polymerization from, in addition to N-isopropylacrylamide, pentaerythritol diacrylate monostearate, a bifunctional monomer containing a long hydrophobic chain, acrylamide, a hydrophilic monomer, and hydroxyethyl acrylate, a hydrophilic monomer used to provide hydroxyl groups for further chemical modification. Results indicated that the hydrophobic-hydrophilic balance achieved by varying the relative concentrations of comonomers used during synthesis was an important parameter in controlling the transition temperature of the macromers in solution and stability of the resultant gels. Storage moduli of the macromers increased over 4 orders of magnitude once gelation occurred above the transition temperature. Furthermore, chemical cross-linking of these macromers resulted in gels with increased stability compared to uncross-linked controls. These results demonstrate the feasibility of synthesizing poly( N-isopropylacrylamide)-based macromers that undergo tandem gelation and establish key criteria relating to the transition temperature and stability of these materials. The data suggest that these materials may be attractive substrates for tissue engineering and cellular delivery applications as the combination of mechanistically independent gelation techniques used in tandem may offer superior materials with regard to gelation kinetics and stability.     

Stimuli-responsive zwitterionic block copolypeptides: poly(N-isopropylacrylamide)-block-poly(lysine-co-glutamic acid)

Synthesis of novel zwitterionic block copolypeptides, poly(N-isopropylacrylamide)-block-poly(L-glutamic acid-co-L-lysine) [PNiPAM(n)(PLG(x)-co-PLLys(y))m , where n is the number-average degree of polymerization (DP(n)) of PNiPAM block, x and y are the mole fraction of glutamic acid and lysine residues, respectively, and m is the total DP(n) of the peptide block], and their stimuli-responsiveness to temperature and pH variation in aqueous solutions are described. Initiated with the amino-terminated poly(N-isopropylacrylamide) (PNiPAM(n)-NH2), ring-opening polymerization (ROP) of a mixture of gamma-benzyl-L-glutamate N-carboxyanhydride (BLG-NCA), and Boc-L-lysine N-carboxyanhydride (BLLys-NCA) afforded the block copolypeptides PNiPAM(n)(PBLG(x)-co-PBLLys(y))m, with a poly(N-isopropylacrylamide) block together with a random copolypeptide block, which was then deprotected with HBr/trifluoroacetic acid into the double hydrophilic block copolypeptides, PNiPAM(n)(PLG(x)-co-PLLys(y))m. Their block ratios and lengths, as well as the amino acid residue ratios in the random copolypeptide block are varied (n = 360, x = 0.4-0.5, y = 0.4-0.6, and m = 220-252). The secondary structures of the copolypeptides in aqueous solution at different pH conditions were examined. Phase transitions in aqueous solutions induced by both pH and temperature variation were investigated by (1)H NMR spectroscopy. The transitions induced by temperature were also explored by turbidity measurements using UV/vis spectroscopy for their lower critical aggregation temperature (LCAT) determination. Furthermore, these aggregation processes were followed by dynamic light scattering measurements.     

Synthesis and characterization of thermo- and pH-responsive double-hydrophilic diblock copolypeptides

Synthesis of novel double-hydrophilic diblock copolypeptides (BCPs), poly(l-glutamic acid)-block-poly(N-isopropylacrylamide) (PLGnPNm), and their thermoresponsive properties in aqueous solutions at different pH values are described. The diblock copolypeptides were synthesized by a combination of ring-opening polymerization (ROP) of gamma-benzyl-l-glutamate N-carboxyanhydrides (BLG-NCA) and reversible addition-fragmentation chain transfer (RAFT) polymerization of N-isopropylacrylamide (NiPAM). A new class of RAFT agents (CTA-2 and CTA-3) with amino-functional groups was designed for this purpose. Two different strategies, i.e., macrochain transfer agent (CTA) and macroinitiator routes, were utilized and compared on the control of the chemical structures of the resulting BCPs. Their block ratios and lengths are broadly varied (n = 21-600 and m =180-442). Their thermally switchable aggregation behaviors in aqueous solutions were investigated at the microscopic level by 1H NMR spectroscopy and at the macroscopic level by turbidity measurements using UV/vis spectroscopy. The latter was also utilized for their lower critical aggregation temperature (LCAT) determination. The effects of block lengths and ratios as well as solution pH values on the collapse of NiPAM chain and aggregation process of BCPs were examined. This aggregation process was also followed by dynamic light scattering (DLS) measurements, and the thermally induced aggregate structures were investigated by transmission electron microscopy (TEM).     

Water-dispersible superparamagnetic microspheres adorned with two types of surface chains

Water-dispersible superparamagnetic polymer/γ-Fe(2)O(3) composite microspheres adorned with two types of surface polymer chains are prepared and characterized. To prepare these spheres, we first synthesize uniform γ-Fe(2)O(3) nanoparticles that are covered by poly(2-cinnamoyloxyethyl methacrylate)-block-poly(acrylic acid) (PCEMA-b-PAA). These nanoparticles are then mixed with a PCEMA homopolymer in CHCl(3) to form an oil phase. The oil phase is dispersed into water under vigorous stirring with the help of two diblock copolymer surfactants, PGMA-b-PCEMA and PSGMA-b-PCEMA. Here PGMA and PSGMA denote poly(glyceryl monomethacrylate) and succinated PGMA, respectively. Solid microspheres with cores composed of PCEMA and PCEMA-b-PAA-covered γ-Fe(2)O(3) nanoparticles are obtained after CHCl(3) evaporation and PCEMA photo-cross-linking. Under certain conditions, the coronal PGMA and PSGMA chains become segregated, thus producing surface bumps, ridges, and valleys. The PSGMA chains preferentially cover the protruding regions. The PSGMA carboxyl groups are used to immobilize bovine serum albumin (BSA). The immobilized BSA retains its activity and binds with anti-BSA. These spheres should be useful in immunoassays.     

Noninvasive imaging of quantum dots in mice

Quantum dots having four different surface coatings were tested for use in in vivo imaging. Localization was successfully monitored by fluorescence imaging of living animals, by necropsy, by frozen tissue sections for optical microscopy, and by electron microscopy, on scales ranging from centimeters to nanometers, using only quantum dots for detection. Circulating half-lives were found to be less than 12 min for amphiphilic poly(acrylic acid), short-chain (750 Da) methoxy-PEG or long-chain (3400 Da) carboxy-PEG quantum dots, but approximately 70 min for long-chain (5000 Da) methoxy-PEG quantum dots. Surface coatings also determined the in vivo localization of the quantum dots. Long-term experiments demonstrated that these quantum dots remain fluorescent after at least four months in vivo.     

Development of a temperature-sensitive composite hydrogel for drug delivery applications

To develop materials with improved controllability and specificity, we have investigated composite hydrogels with temperature-sensitive properties using photo cross-linking. Specifically, our novel composite materials are composed of nanoparticles made of poly(N-isopropylacrylamide) (PNIPAAm), temperature-sensitive hydrogels, and a photo cross-linker, poly(ethylene glycol) diacrylate (PEGDA). PNIPAAm particles were synthesized by emulsion polymerization and by varying concentration of four main factors: monomers (N-isopropylacrylamide), cross-linkers (N,N'-methylenebisacrylamide), surfactants (sodium dodecyl sulfate, SDS), and initiators (potassium persulfate). We found that the surfactant, SDS, was the most important factor affecting the particle size using the factorial design analysis. Additionally, both nano- and micro-PNIPAAm particles had excellent loading efficiency (>80% of the incubated bovine serum albumin (BSA)), and their release kinetics expressed an initial burst effect followed by a sustained release over time. Furthermore, BSA-loaded PNIPAAm nanoparticles were used to form three-dimensional gel networks by means of a photocuring process using a photo cross-linker, PEGDA, and a photoinitiator, Irgacure-2959 (I-2959). Results from scanning electron microscopy and in vitro BSA release studies from these hydrogels demonstrated that PNIPAAm nanoparticles were embedded inside the PEG polymeric matrix and the composite material was able to release BSA in response to changes in temperature. These PNIPAAm nanoparticle hydrogel networks may have advantages in applications of controlled drug delivery systems because of their temperature sensitivity and their ability of in situ photopolymerization to localize at the specific region in the body.     

Acid-labile core cross-linked micelles for pH-triggered release of antitumor drugs

Micelles of a model amphiphilic block copolymer, poly(hydroxyethyl acrylate)-block-poly(n-butyl acrylate) (PHEA-b-PBA), synthesized via the RAFT polymerization were cross-linked by copolymerization of a degradable cross-linker from the living RAFT-end groups of PBA chains, yielding a cross-linked core without affecting significantly the original micelle size. The cross-linker incorporation into the micelles was evidenced via physicochemical analysis of the copolymer unimers formed upon acidic cleavage of the cross-linked micelles. High doxorubicin loading capacities (60 wt %) were obtained. Hydrolysis of less than half of the cross-links in the core was found to be sufficient to release doxorubicin faster at acidic pH compared to neutral pH. The system represents the first example of core-cross-linked micelles that can be destabilized (potentially both above and below CMC) by the pH-dependent cleavage of the cross-links and the subsequent polarity change in the core to enable the release of hydrophobic drugs entrapped inside the micelle.     

Water-soluble fluorescent diblock nanospheres

The hydroxyl groups of poly(tert-butyl acrylate)-block-poly(2-hydroxyethyl methacrylate) or PtBA-b-PHEMA were reacted with succinic anhydride to introduce some carboxyl groups into the PHEMA block. Such carboxyl groups were then reacted with Texas-red cadverine (TX-NH(2)) to incorporate dye molecules. The TX-bearing diblocks formed probably spherical micelles in block-selective solvent DMF/toluene containing 2% DMF. "Permanent" micelles or nanospheres were prepared after cross-linking the TX-bearing PHEMA core block. Such nanospheres were made water soluble by cleaving the tert-butyl groups from the PtBA coronas. Water-soluble nanospheres with high TX numbers and fluorescence quantum yields may find applications in fluorescence in situ hybridization assays.     

Synthesis of various glycopolymer architectures via RAFT polymerization: from block copolymers to stars

Well-defined linear poly(acryloyl glucosamine) (PAGA) exhibiting molar masses ranging from 3 to 120 K and low polydispersities have been prepared via reversible addition-fragmentation chain transfer polymerization (RAFT) in aqueous solution without recourse to protecting group chemistry. The livingness of the process was further demonstrated by successfully chain-extending one of these polymers with N-isopropylacrylamide affording narrow dispersed thermosensitive diblocks. This strategy of polymerization was finally extended to the preparation of glycopolymer stars from Z designed non-water-soluble trifunctional RAFT agent. After the growth of very short blocks of poly(hydroxyethyl acrylate) ((-)DP(n)(branch) = 10), AGA was polymerized in aqueous solution in a controlled manner affording well-defined 3-arm glycopolymer stars.     

Effects of nanoparticles on the compatibility of PEO-PMMA block copolymers

The compatibility of six kinds of designed poly(ethylene oxide)-block-poly(methyl methacrylate) (PEO-b-PMMA) copolymers was studied at 270, 298 and 400 K via mesoscopic modeling. The values of the order parameters depended on both the structures of the block copolymers and the simulation temperature, while the values of the order parameters of the long chains were higher than those of the short ones; temperature had a more obvious effect on long chains than on the short ones. Plain copolymers doped with poly(ethylene oxide) (PEO) or poly(methyl methacrylate) (PMMA) homopolymer showed different order parameter values. When a triblock copolymer had the same component at both ends and was doped with one of its component polymers as a homopolymer (such as A5B6A5 doped with B6 or A5 homopolymer), the value of its order parameter depended on the simulation temperature. The highest order parameter values were observed for A5B6A5 doped with B6 at 400 K and for A5B6A5 doped with A5 at 270 K. A study of copolymers doped with nanoparticles showed that the mesoscopic phase was influenced by not only the properties of the nanoparticles, such as the size and density, but also the compositions of the copolymers. Increasing the size of the nanoparticles used as a dopant had the most significant effect on the phase morphologies of the copolymers.     

Amphiphilic amylose-g-poly(meth)acrylate copolymers through "click" onto grafting method

Periodate oxidation and subsequent reductive amination with propargylamine was adopted for the controlled functionalization of amylose with alkyne groups, whereas ATRP polymerization was exploited to obtain end-(α)- or end-(ω)-azide functionalized poly(meth)acrylates to be used as "click" reagents in Cu(I) catalyzed azide-alkyne [3 + 2] dipolar cycloaddition. Amylose was effectively grafted with poly(n-butyl acrylate), poly(n-butyl methacrylate), poly(n-hexyl methacrylate), and poly(dimethylaminoethyl methacrylate) with this strategy. Their structure and composition were confirmed by FT-IR, NMR spectroscopies, and thermogravimetric analysis (TGA). Dynamic and static light scattering analyses, as well as TEM microscopy showed that the most amphiphilic among these hybrid graft copolymers self-assembled in water, yielding nanoparticles with ca. 30 nm diameter.     

Method for tracking nanogel particles in vivo and in vitro

Hydrogels made of N-isopropylacrylamide (NIPA) can be synthesized in the form of highly monodispersed nanoparticles. After synthesis, NIPA hydrogel nanoparticles (nanogels) can be labeled by Alexa Fluor 488 carboxylic acid, 2,3,5,6-tetrafluorophenyl ester through amine-terminated functional groups. This choice of dye is complementary to other biological labeling methods for in vivo studies. When the nanogel/dye nanoparticles are injected into rabbits, they can be imaged via tissue sectioning and confocal microscopy, while nanoparticle concentration can be determined by fluorescent microplate assays. Time-course persistence of nanoparticles in the circulatory system can be readily tracked by direct assay of plasma and urine samples using 485 nm excitation and 538 emission wavelengths to keep background fluorescence to nearly the same level as that found using an empty well. Depending upon how the nanoparticles are injected, circulatory system concentrations can reach high concentrations and diminish to low levels or gradually increase and gradually decrease over time. Injection in the femoral artery results in a rapid spike in circulating nanogel/dye concentration, while injection into the renal artery results in a more gradual increase.     

Response of cells on surface-induced nanopatterns: fibroblasts and mesenchymal progenitor cells

Ultrathin films of a poly(styrene)-block-poly(2-vinylpyrindine) diblock copolymer (PS-b-P2VP) and poly(styrene)-block-poly(4-vinylpyrindine) diblock copolymer (PS-b-P4VP) were used to form surface-induced nanopattern (SINPAT) on mica. Surface interaction controlled microphase separation led to the formation of chemically heterogeneous surface nanopatterns on dry ultrathin films. Two distinct nanopatterned surfaces, namely, wormlike and dotlike patterns, were used to investigate the influence of topography in the nanometer range on cell adhesion, proliferation, and migration. Atomic force microscopy was used to confirm that SINPAT was stable under cell culture conditions. Fibroblasts and mesenchymal progenitor cells were cultured on the nanopatterned surfaces. Phase contrast and confocal laser microscopy showed that fibroblasts and mesenchymal progenitor cells preferred the densely spaced wormlike patterns. Atomic force microscopy showed that the cells remodelled the extracellular matrix differently as they migrate over the two distinctly different nanopatterns.     

Are the interactions between recombinant prion proteins and polymeric surfaces related to the hydrophilic/hydrophobic balance?

New non-fouling tubes are developed and their influence on the adhesion of neuroproteins is studied. Recombinant prion proteins are considered as a single component representative of hydrophobic proteins. Samples are stored for 24?h at 4?°C in tubes coated with two different coatings: poly(N-isopropylacrylamide) as a hydrophilic surface and a plasma-fluorinated coating as a hydrophobic one. The protein adhesion is monitored by ELISA tests, XPS and confocal microscopy. It appears that the highest recovery of recombinant prion protein in the liquid phase is obtained with the hydrophilic surface while the hydrophobic character of the storage tube induces an important amount of biological loss. However, the recovery is not complete even for tubes coated with poly(N-isopropylacrylamide).     

Simple and tunable Förster resonance energy transfer-based bioprobes for high-throughput monitoring of caspase-3 activation in living cells by using flow cytometry

Sensing systems based on F?rster resonance energy transfer (FRET) can be used to monitor enzymatic reactions, protein-protein interactions, changes in conformation, and Ca2+ oscillations in studies on cellular dynamics. We developed a series of FRET-based chimeric bioprobes, each consisting of fluorescent protein attached to a fluorescent dye. Green and red fluorescent proteins were used as donors and a series of Alexa Fluor dyes was used as acceptors. The basic fluorescent proteins were substituted with appropriate amino acids for recognition of the target (caspase-3) and subjected to site-directed modification with a fluorescent dye. Variants that retained similar emission profiles to the parent proteins were readily derived for use as FRET-based bioprobes with various fluorescent patterns by incorporating various fluorescent proteins and dyes, the nature of which could be adjusted to experimental requirements. All the constructs prepared functioned as bioprobes for quantitative measurement of caspase-3 activity in vitro. Introduction of the bioprobes into cells was so simple and efficient that activation of caspase-3 upon apoptosis could be monitored by means of cytometric analysis. FRET-based bioprobes are valuable tool for high-throughput flow-cytometric analysis of many cellular events when used in conjunction with other fluorescent labels or markers. Statistical dynamic studies on living cells could provide indications of paracrine signaling.     

Synthesis, characterization, and bioavailability of mannosylated shell cross-linked nanoparticles

Saccharide-functionalized shell cross-linked (SCK) polymer micelles designed as polyvalent nanoscaffolds for selective interactions with receptors on Gram negative bacteria were constructed from mixed micelles composed of poly(acrylic acid-b-methyl acrylate) and mannosylated poly(acrylic acid-b-methyl acrylate). The mannose unit was conjugated to the hydrophilic chain terminus of the amphiphilic diblock copolymer precursor, from which the SCK nanoparticles were derived, by the growth of the diblock copolymer from a mannoside functionalized atom transfer radical polymerization (ATRP) initiator. Mixed micelle formation between the amphiphilic diblock copolymer and mannosylated amphiphilic diblock copolymer was followed by condensation-based cross-linking between the acrylic acid residues present in the periphery of the polymer micelles to afford SCK nanoparticles. SCKs presenting variable numbers of mannose functionalities were prepared from mixed micelles of controlled stoichiometric ratios of mannosylated and nonmannosylated diblock copolymers. The polymer micelles and SCKs were characterized by dynamic light scattering (DLS), electrophoretic light scattering, atomic force microscopy (AFM), transmission electron microscopy (TEM), and analytical ultracentrifugation (AU). Surface availability and bioactivity of the mannose units were evaluated by interactions of the nanostructures with the model lectin Concanavalin A via DLS studies, with red blood cells (rabbit) via agglutination inhibition assays and with bacterial cells (E. coli) via TEM imaging.     

Photo-cross-linked PLA-PEO-PLA hydrogels from self-assembled physical networks: mechanical properties and influence of assumed constitutive relationships

Poly(lactide)-block-poly(ethylene oxide)-block-poly(lactide) (PLA-PEO-PLA) triblock copolymers are known to form physical hydrogels in water as a result of the polymer's amphiphilicity. Their mechanical properties, biocompatibility, and biodegradability have made them attractive for use as soft tissue scaffolds. However, the network junction points are not covalently cross-linked, and in a highly aqueous environment these hydrogels adsorb more water, transform from gel to sol, and lose the designed mechanical properties. In this article, a hydrogel was formed by the use of a novel two-step approach. In the first step, the end-functionalized PLA-PEO-PLA triblock was self-assembled into a physical hydrogel through hydrophobic micelle network junctions, and in the second step, this self-assembled physical network structure was locked into place by photo-cross-linking the terminal acrylate groups. In contrast with physical hydrogels, the photo-cross-linked gels remained intact in phosphate-buffered solution at body temperature. The swelling, degradation, and mechanical properties were characterized, and they demonstrated an extended degradation time (approximately 65 days), an exponential decrease in modulus with degradation time, and a tunable shear modulus (1.6-133 kPa). We also discuss the various constitutive relationships (Hookean, neo-Hookean, and Mooney-Rivlin) that can be used to describe the stress-strain behavior of these hydrogels. The chosen model and assumptions used for data fitting influenced the obtained modulus values by as much as a factor of 3.5, which demonstrates the importance of clearly stating one's data fitting parameters so that accurate comparisons can be made within the literature.     

Synthesis and characterization of biocompatible thermo-responsive gelators based on ABA triblock copolymers

The synthesis of biocompatible, thermo-responsive ABA triblock copolymers in which the outer A blocks comprise poly(N-isopropylacrylamide) and the central B block is poly(2-methacryloyloxyethyl phosphorylcholine) is achieved using atom transfer radical polymerization with a commercially available bifunctional initiator. These novel triblock copolymers are water-soluble in dilute aqueous solution at 20 degrees C and pH 7.4 but form free-standing physical gels at 37 degrees C due to hydrophobic interactions between the poly(N-isopropylacrylamide) blocks. This gelation is reversible, and the gels are believed to contain nanosized micellar domains; this suggests possible applications in drug delivery and tissue engineering.     

Dual peptide nucleic acid- and peptide-functionalized shell cross-linked nanoparticles designed to target mRNA toward the diagnosis and treatment of acute lung injury

In this work, multifunctional biosynthetic hybrid nanostructures were prepared and studied for their potential utility in the recognition and inhibition of mRNA sequences for inducible nitric oxide synthase (iNOS), which are overexpressed at sites of inflammation, such as in cases of acute lung injury. Shell cross-linked knedel-like polymer nanoparticles (SCKs) that present peptide nucleic acids, for binding to complementary mRNAs, and cell penetrating peptides (CPPs), to gain cell entry, along with fluorescent labels and sites for radiolabeling, were prepared by a series of robust, efficient, and versatile synthetic steps that proceeded from monomers to polymers to functional nanoparticles. Amphiphilic block graft copolymers having combinations of methoxy- and thioacetyl-terminated poly(ethylene glycol) (PEG) and DOTA-lysine units grafted from the backbone of poly(acrylic acid) (PAA) and extending with a backbone segment of poly(octadecyl acrylate-co-decyl acrylate) (P(ODA-co-DA)) were prepared by a combination of reversible addition-fragmentation chain transfer (RAFT) polymerization and chemical modification reactions, which were then used as the building blocks for the formation of well-defined SCKs decorated with reactive thiols accessible to the surface. Fluorescent labeling with Alexa Fluor 633 hydrazide was then accomplished by amidation with residual acrylic acid residues within the SCK shells. Finally, the PNAs and CPP units were covalently conjugated to the SCKs via Michael addition of thiols on the SCKs to maleimide units on the termini of PNAs and CPPs. Confirmation of the ability of the PNAs to bind selectively to the target iNOS mRNAs when tethered to the SCK nanoparticles was determined by in vitro competition experiments. When attached to the SCKs having a hydrodynamic diameter of 60 ± 16 nm, the K(d) values of the PNAs were ca. an order of magnitude greater than the free PNAs, while the mismatched PNA showed no significant binding.     

Thermoresponsive self-assemblies of cyclic and branched oligosaccharide-block-poly(N-isopropylacrylamide) diblock copolymers into nanoparticles

This paper discusses the thermoresponsive nanoparticles obtained by self-assemblies of nonlinear oligosaccharide-based diblock copolymer systems. These diblock copolymers were synthesized by Cu(I)-catalyzed 1,3-dipolar azide/alkyne cycloaddition ("click" reaction) of propargyl-functionalized β-cyclodextrin (βCyD) and xyloglucooligosaccharide (XGO) with poly(N-isopropylacrylamide) (PNIPAM) having a terminal azido group prepared by atom transfer radical polymerization (ATRP). Elastic and quasi-elastic light scattering analysis of the dibock copolymers in H(2)O indicated that thermodynamic phase transitions of the PNIPAM blocks at their cloud points (T(cp)s ≈ 34 °C), around lower critical solution temperatures (LCSTs), triggered their self-assemblies into the nanoparticles. These nanoparticles had narrow size distributions and small interphases (i.e., sharp boundaries). The mean hydrodynamic radii (R(h)s) of the βCyD and XGO-based nanoparticles were determined to be around 150 and 250 nm upon slow heating (i.e., step-by-step heating), and 364 and 91.5 nm upon fast heating, respectively, depending on a predominance of the interchain association or the intrachain contraction. Transmission electron microscope (TEM) and field emission gun-scanning electron microscopy (FEG-SEM) images of the nanoparticles clearly showed compact spherical nanoparticles whose cores are mainly made with the PNIPAM blocks, whereas the rough shells consist in the oligosaccharidic blocks.