Heterofunctional Poly(ethylene glycol) (PEG) Macroinitiator Enabling Controlled Synthesis of ABC Triblock Copolymers

ABC triblock copolymers with a poly­(ethylene glycol) (PEG) midblock have attractive properties for biomedical applications because of PEG’s favorable properties regarding biocompatibility and hydrophilicity. However, easy strategies to synthesize polymers containing a PEG midblock are limited. In this study, the successful synthesis of a heterofunctional PEG macroinitiator containing both an azoinitiator and an atom transfer radical polymerization (ATRP) initiator is demonstrated. This novel PEG macroinitiator allows the development of elegant synthesis routes for PEG midblock-containing ABC copolymers that does not require protection of initiating sites or polymer end-group postmodification. Polymers with outer blocks composed of different monomers were synthesized to illustrate the versatility of this macroinitiator. <i>N</i>-Isopropylacrylamide (NIPAM) was included to obtain thermosensitive polymers, 2-(dimethylamino)­ethyl methacrylate (DMAEMA) provided pH-sensitive properties, and 2-hydroxyethyl acrylate (HEA) functioned as a noncharged hydrophilic block that also allows for postmodifications reactions. This synthesis approach can further contribute to the design of high-precision polymers with tailorable block compositions and polymer topologies, which is highly attractive for applications in nanotechnology

Thermoresponsive Injectable Hydrogels Cross-Linked by Native Chemical Ligation

Temperature-induced physical gelation was combined with native chemical ligation (NCL) as a chemical cross-linking mechanism to yield rapid network formation and mechanically strong hydrogels. To this end, a novel monomer <i>N</i>-(2-hydroxypropyl)­methacrylamide-cysteine (HPMA-Cys) was synthesized that copolymerizes with <i>N</i>-isopropylacrylamide (NIPAAm) to yield thermoresponsive polymers decorated with cysteine functionalities. Triblock copolymers consisting of a poly­(ethylene glycol) (PEG) middle block flanked by random blocks of NIPAAm and HPMA-Cys were successfully synthesized and characterized. Additionally, thioester cross-linkers were synthesized based on PEG and hyaluronic acid, respectively. Upon mixing the thermoresponsive polymer with PEG or hyaluronic acid cross-linker, cysteine and thioester functionalities react under physiological conditions to generate a native peptide bond. An immediate physical network was formed after elevation of the temperature to 37 °C due to the self-assembly of the pNIPAAm chains. This network was stabilized in time by covalent cross-linking due to NCL reaction between thioester and cysteine functionalities, resulting in hydrogels with up to 10 times higher storage moduli than without chemical cross-links. Finally, a collagen mimicking peptide sequence was successfully ligated to this hydrogel using the same reaction mechanism, showing the potential of this hydrogel for tissue engineering applications

Triggered Release of Doxorubicin from Temperature-Sensitive Poly(<i>N</i>‑(2-hydroxypropyl)-methacrylamide mono/dilactate) Grafted Liposomes

The objective of this study was to design temperature-sensitive liposomes with tunable release characteristics that release their content at an elevated temperature generated by high intensity focused ultrasound (HIFU) exposure. To this end, thermosensitive polymers of <i>N</i>-(2-hydroxypropyl)­methacrylamide mono/dilactate of different molecular weights and composition with a cholesterol anchor (chol-pHPMAlac) were synthesized and grafted onto liposomes loaded with doxorubicin (DOX). The liposomes were incubated at different temperatures and their release kinetics were studied. A good correlation between the release-onset temperature of the liposomes and the cloud point (CP) of chol-pHPMAlac was found. However, release took place at significantly higher temperatures than the CP of chol-pHPMAlac, likely at the CP, the dehydration and thus hydrophobicity is insufficient to penetrate and permeabilize the liposomal membrane. Liposomes grafted with chol-pHPMAlac with a CP of 11.5 °C released 89% DOX within 5 min at 42 °C while for the liposomes grafted with a polymer with CP of 25.0 °C, a temperature of 52 °C was needed to obtain the same extent of DOX release. At a fixed copolymer composition, an increase in molecular weight from 6.5 to 14.5 kDa decreased the temperature at which DOX was released with a release-onset temperature from 52 to 42 °C. Liposomes grafted with 5% chol-pHPMAlac exhibited a rapid release to a temperature increase, while at a grafting density of 2 and 10%, the liposomes were less sensitive to an increase in temperature. Sequential release of DOX was obtained by mixing liposomes grafted with chol-pHPMAlac having different CPs. Chol-pHPMAlac grafted liposomes released DOX nearly quantitatively after pulsed wave HIFU. In conclusion, the release of DOX from liposomes grafted with thermosensitive polymers of <i>N</i>-(2-hydroxypropyl)­methacrylamide mono/dilactate can be tuned to the characteristics and the grafting density of chol-pHPMAlac, making these liposomes attractive for local drug delivery using hyperthermia

Thermogelling and Chemoselectively Cross-Linked Hydrogels with Controlled Mechanical Properties and Degradation Behavior

Chemoselectively cross-linked hydrogels have recently gained increasing attention for the development of novel, injectable biomaterials given their limited side reactions. In this study, we compared the properties of hydrogels obtained by native chemical ligation (NCL) and its recently described variation termed oxo-ester-mediated native chemical ligation (OMNCL) in combination with temperature-induced physical gelation. Triblock copolymers consisting of cysteine functionalities, thermoresponsive <i>N</i>-isopropylacrylamide (NIPAAm) units and degradable moieties were mixed with functionalized poly­(ethylene glycol) (PEG) cross-linkers. Thioester or <i>N</i>-hydroxysuccinimide (NHS) functionalities attached to PEG reacted with cysteine residues of the triblock copolymers via either an NCL or OMNCL pathway. The combined physical and chemical cross-linking resulted in rapid network formation and mechanically strong hydrogels. Stiffness of the hydrogels was highest for thermogels that were covalently linked via OMNCL. Specifically, the storage modulus after 4 h reached a value of 26 kPa, which was over a 100 times higher than hydrogels formed by solely thermal physical interactions. Endothelial cells showed high cell viability of 98 ± 2% in the presence of OMNCL cross-linked hydrogels after 16 h of incubation, in contrast to a low cell viability (13 ± 7%) for hydrogels obtained by NCL cross-linking. Lysozyme was loaded in the gels and after 2 days more than 90% was released, indicating that the cross-linking reaction was indeed chemoselective as the protein was not covalently grafted to the hydrogel network. Moreover, the degradation rates of these hydrogels under physiological conditions could be tailored from 12 days up to 6 months by incorporation of a monomer containing a hydrolyzable lactone ring in the thermosensitive triblock copolymer. These results demonstrate a high tunability of mechanical properties and degradation rates of these in situ forming hydrogels that could be used for a variety of biomedical applications

Luminescent Gold Nanocluster-Decorated Polymeric Hybrid Particles with Assembly-Induced Emission

Ultrasmall gold atom clusters (<2 nm in diameter) or gold nanoclusters exhibit emergent photonic properties (near-infrared absorption and emission) compared to larger plasmonic gold particles because of the significant quantization of their conduction band. Although single gold nanocluster properties and applications are being increasingly investigated, little is still known about their behavior and properties when assembled into suprastructures, and even fewer studies are investigating their use for biomedical applications. Here, a simple synthetic pathway combines gold nanoclusters with thermosensitive diblock copolymers of poly­(ethylene glycol) (PEG) and poly­(<i>N</i>-isopropylacrylamide) (PNIPAm) to form a new class of gold-polymer, micelle-forming, hybrid nanoparticle. The nanohybrids’ design is uniquely centered on enabling the temperature-dependent self-assembly of gold nanoclusters into the hydrophobic cores of micelles. This nonbulk assembly not only preserves but also enhances the attractive near-infrared photonics of the gold nanoclusters by significantly increasing their native fluorescent signal. In parallel to the fundamental insights into gold nanocluster ordering and assembly, the gold-polymer nanohybrids also demonstrated great potential as fluorescent live-imaging probes in vitro. This innovative material design based on the temperature-dependent, self-assembly of gold nanoclusters within a polymeric micelle’s core shows great promise toward bioassays, nanosensors, and nanomedicine

Temperature triggered release of DOX from TSL.

<p>Temperature triggered release of DOX from TSL (DPPC:MSPC:DSPE-PEG2.000 86:10:4) in 20 mM HEPES buffer pH 7.4 (A) or 50% fetal bovine serum (B) at 37 and 42°C. Temperature triggered DOX release from TSL-Ba-ms in 20 mM HEPES buffer pH 7.4 (C) and 50% FBS (D) at 37 and 42°C.</p

T₁-maps and T₁-wt images of intratumoral injected TSL-Ba-ms and Ho-ms in a tumor in the auricle of a New Zealand White rabbit.

<p>T₁-maps (A-C) and T₁-wt images (D-E) of a tumor in the auricle of a New Zealand White rabbit before (A,D) and after (B) intratumoral co-injection of TSL-Ba-ms and Ho-ms. Finally, the tumor was heated in the range between 42 and 46°C for 15 minutes (C,E).</p

Characteristics of temperature senstitive liposomes (TSL) loaded with DOX and [Gd(HPDO3A)(H₂O)].

<p>Characteristics of temperature senstitive liposomes (TSL) loaded with DOX and [Gd(HPDO3A)(H₂O)].</p

T₁ and T₂* in the microsphere pellet and supernatant of alginate microspheres before and after mild hyperthermia.

<p>T₁ in the microsphere pellet (A) and supernatant (B) and T₂* in the microsphere pellet (C) of alginate microspheres before and after mild hyperthermia (HT; 42°C for 15 minutes). A region of interested (ROI) was positioned in the microsphere pellet (if present) or the supernatant to determine the T₁ and T₂*-values (Mean + standard deviation).</p

Experimental setup for the preparation of alginate microspheres.

<p><b>(A) Schematic representation of the experimental setup.</b> An alginate solution (3% w/v in 20 mM HEPES buffer pH 7.4 containing 8 NaCl/L) is transferred into a syringe and introduced in the spraying system via a syringe pump (1.5 mL/min) which are connected via a plastic tube. Alginate droplets are formed by passing nitrogen gas (0.8 Bar) through the nozzle. The droplets are collected in a crosslinking solution (containing 100 mM barium chloride or holmium chloride). Photos of experimental setup (B) and nozzle (C).</p