Versatile Supramolecular Gene Vector Based on Host–Guest Interaction

It is a great challenge to arrange multiple functional components into one gene vector system to overcome the extra- and intracellular obstacles for gene therapy. In this study, we developed a supramolecular approach for constructing a versatile gene delivery system composed of adamantyl-terminated functional polymers and a β-cyclodextrin based polymer. Adamantyl-functionalized low molecular weight PEIs (PEI-Ad) and PEG (Ad-PEG) as well as poly­(β-cyclodextrin) (PCD) were synthesized by one-step chemical reactions. The supramolecular inclusion complex formed from PCD to assemble LMW PEI-Ad₄ via host–guest interactions can condense plasmid DNA to form nanopolyplexes by electrostatic interactions. The supramolecular polyplexes can be further PEGylated with Ad-PEG to form inclusion complexes, which showed increased salt and serum stability. In vitro experiments revealed that these supramolecular assembly polyplexes had good cytocompatibility and showed high transfection activity close to that of the commercial ExGen 500 at high dose of DNA. Also, the supramolecular vector system exhibited about 60% silencing efficiency as a siRNA vector. Thus, a versatile effective supramolecular gene vector based on host–guest complexes was fabricated with good cytocompatbility and transfection activity

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

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

Reversible Addition–Fragmentation Chain Transfer Synthesis of a Micelle-Forming, Structure Reversible Thermosensitive Diblock Copolymer Based on the <i>N</i>‑(2-Hydroxy propyl) Methacrylamide Backbone

A diblock copolymer composed of <i>N</i>-(2-hydroxy propyl) methacrylamide (HPMAm) as hydrophilic block and <i>N</i>-(2-hydroxy propyl) methacrylamide dilactate (HPMAm-Lac₂) as thermosensitive block was synthesized by reversible addition–fragmentation chain transfer (RAFT) polymerization. To this end, HPMAm was first polymerized with 4-cyano-4-[(dodecylsulfanylthiocarbonyl)-sulfanyl]­pentanoic acid as the chain transfer agent and azobisisobutyronitrile (AIBN) as the initiator. The polymerization showed a linear increase in <i>M</i>_n as a function of monomer conversion. The living p­(HPMAm) chain (7 kDa) was subsequently extended with HPMAm-Lac₂ yielding a diblock copolymer (total <i>M</i>_n of 22 kDa). The copolymer showed reversible thermosensitivity in aqueous solution and self-assembled into micelles with a size of 58 nm (PDI 0.13) above its critical micelle temperature (CMT, 2.1 °C) and concentration (CMC, 0.044 mg/mL) and was soluble below the CMT. Paclitaxel, a hydrophobic chemotherapeutic drug, was encapsulated in the micelles with a loading capacity of 16.1 ± 1.2%. Hydrolysis of the dilactate side groups of the p­(HPMAm-Lac₂) block converted the copolymer to the fully hydrophilic p­(HPMAm) homopolymer, resulting in dissociation of the micelles. In conclusion, the livingness and versatility of RAFT polymerization provide possibilities to synthesize block copolymers with HPMAm and derivatives thereof

Methyleneation of Peptides by <i>N</i>,<i>N</i>,<i>N</i>,<i>N</i>‑Tetramethylethylenediamine (TEMED) under Conditions Used for Free Radical Polymerization: A Mechanistic Study

Free radical polymerization is often used to prepare protein and peptide-loaded hydrogels for the design of controlled release systems and molecular imprinting materials. Peroxodisulfates (ammonium peroxodisulfates (APS) or potassium peroxodisulfates (KPS)) with <i>N</i>,<i>N</i>,<i>N</i>,<i>N</i>-tetramethylethylenediamine (TEMED) are frequently used as initiator and catalyst. However, exposure to these free radical polymerization reagents may lead to modification of the protein and peptide. In this work, we show the modification of lysine residues by ammonium peroxodisulfate (APS)/TEMED of the immunostimulant thymopentin (TP5). Parallel studies on a decapeptide and a library of 15 dipeptides were performed to reveal the mechanism of modification. LC–MS of APS/TEMED-exposed TP5 revealed a major reaction product with an increased mass (+12 Da) with respect to TP5. LC–MS² and LC–MS³ were performed to obtain structural information on the modified peptide and localize the actual modification site. Interpretation of the obtained data demonstrates the formation of a methylene bridge between the lysine and arginine residue in the presence of TEMED, while replacing TEMED with a sodium bisulfite catalyst did not show this modification. Studies with the other peptides showed that the TEMED radical can induce methyleneation on peptides when lysine is next to arginine, proline, cysteine, aspargine, glutamine, histidine, tyrosine, tryptophan, and aspartic acid residues. Stability of peptides and protein needs to be considered when using APS/TEMED in <i>in situ</i> polymerization systems. The use of an alternative catalyst such as sodium bisulfite may preserve the chemical integrity of peptides during in situ polymerization

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

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

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

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

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