25 research outputs found
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 polyÂ(β-cyclodextrin) (PCD)
were synthesized by one-step chemical reactions. The supramolecular
inclusion complex formed from PCD to assemble LMW PEI-Ad<sub>4</sub> 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)Âmethacrylamide-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 polyÂ(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 polyÂ(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-Lac<sub>2</sub>) 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]Âpentanoic
acid as the chain transfer agent and azobisisobutyronitrile (AIBN)
as the initiator. The polymerization showed a linear increase in <i>M</i><sub>n</sub> as a function of monomer conversion. The living
pÂ(HPMAm) chain (7 kDa) was subsequently extended with HPMAm-Lac<sub>2</sub> yielding a diblock copolymer (total <i>M</i><sub>n</sub> 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 pÂ(HPMAm-Lac<sub>2</sub>) block
converted the copolymer to the fully hydrophilic pÂ(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–MS<sup>2</sup> and LC–MS<sup>3</sup> 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)Âmethacrylamide 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)Âmethacrylamide 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 polyÂ(ethylene glycol) (PEG) and polyÂ(<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
T<sub>1</sub>-maps and T<sub>1</sub>-wt images of intratumoral injected TSL-Ba-ms and Ho-ms in a tumor in the auricle of a New Zealand White rabbit.
<p>T<sub>1</sub>-maps (A-C) and T<sub>1</sub>-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)(H<sub>2</sub>O)].
<p>Characteristics of temperature senstitive liposomes (TSL) loaded with DOX and [Gd(HPDO3A)(H<sub>2</sub>O)].</p