Fu-Sheng Du

Multi-responsive nanogels containing motifs of ortho ester, oligo(ethylene glycol) and disulfide linkage as carriers of hydrophobic anti-cancer drugs

A family of multi-responsive nanogels with different compositions and crosslinking degrees have been prepared by the miniemulsion copolymerization of monomethyl oligo(ethylene glycol) acrylate (OEGA) and an ortho ester-containing acrylic monomer, 2-(5,5-dimethyl-1,3-dioxan-2-yloxy) ethyl acrylate (DMDEA), with bis(2-acryloyloxyethyl) disulfide (BADS) as a crosslinker. These nanogels are thermoresponsive and labile in the weakly acidic or reductive environments. The thermoresponsive behaviors, acid-triggered hydrolysis, and reduction-induced degradation of these nanogels were studied by means of dynamic light scattering (DLS), transmission electron microscopy (TEM) and atomic force microscopy (AFM). The results indicate that the volume phase transition temperature (VPTT), thermally induced deswelling ratio, and acid-triggered swelling ratio of the nanogels are closely relevant to their compositions and crosslinking degrees. Although these nanogels could be reductively disrupted by dithiothreitol (DTT), single polymer chains with sizes smaller than 20 nm were not detected by DLS. This is probably due to the existence of some unbreakable linkages formed by chain transfer to the disulfide bond during the radical polymerization. These nanogels are capable of encapsulating hydrophobic compounds. The loading capability of the nanogels for Nile Red (NR), paclitaxel (PTX), and doxorubicin (DOX), and the release behaviors of the drug-loaded nanogels were investigated by UV-vis spectrometry and HPLC. As expected, drug release can be greatly accelerated by a cooperative effect of both acid-triggered hydrolysis and DTT-induced degradation. Finally, the PTX-loaded nanogels exhibit a concentration-dependent toxicity to MCF-7 cells while the intact unloaded nanogels are non-toxic, thereby they may be used as potential carriers for hydrophobic anticancer drugs.

Intelligent nucleic acid delivery systems based on stimuli-responsive polymers

Despite significant advances in the past two decades, gene therapy is still in the stage of clinical trials worldwide mainly due to the lack of safe and efficient delivery vehicles for therapeutic nucleic acids. Among the various attempts to develop clinically applicable gene therapy, polymer-based nucleic acid delivery systems have attracted great interest, especially for the exciting RNAi-based gene therapy. Regarding in vivo nucleic acid delivery, in particular via intravenous injection, there are many extra- and intracellular obstacles, some of which are conflicting. Virus-mimicking nucleic acid delivery systems that combine multiple and programmable functions are thought to be very promising for conquering these challenging barriers. In this review article, we highlight recent progress in stimuli-responsive polymers that have been applied in fabrication of non-viral multi-functional nucleic acid vehicles, which are categorized by the type of stimulus: reduction potential, pH, temperature, and others. In each section, intelligent pDNA delivery systems are introduced first, followed by summarizing various responsive polymer-based siRNA vehicles. Considering the great potential of RNAi-based gene therapy, we devote some space to the recent progress of multi-functional siRNA delivery systems. In addition, different requirements in designing polymer-based siRNA and pDNA carriers are also specified in this review.

Facile Synthesis of Acid-Labile Polymers with Pendent Ortho Esters

This work presents a facile approach for preparation of acid-labile and biocompatible polymers with pendent cyclic ortho esters, which is based on the efficient and mild reactions between cyclic ketene acetal (CKA) and hydroxyl groups. Three CKAs, 2-ethylidene-1,3-dioxane (EDO), 2-ethylidene-1,3-dioxolane (EDL), and 2-ethylidene-4- methyl-1,3-dioxolane (EMD) were prepared from the corresponding cyclic vinyl acetals by catalytic isomerization of the double bond. The reaction of CKAs with different alcohols and diols was examined using trace of p-toluenesulfonic acid as a catalyst. For the monohydroxyl alcohols, cyclic ortho esters were formed by simple addition of the hydroxyl group toward CKAs with ethanol showing a much greater reactivity than iso-propanol. When 1,2- or 1,3-diols were used to react with the CKAs, we observed the isomerized cyclic ortho esters besides the simple addition products. Biocompatible polyols, that is, poly(2-hydroxyethyl acrylate) (PHEA) and poly(vinyl alcohol) (PVA) were then modified with CKAs, and the degree of substitution of the pendent ortho esters can be easily tuned by changing feed ratio. Both the small molecule ortho esters and the CKA-modified polymers demonstrate the pH-dependent hydrolysis profiles, which depend also on the chemical structure of the ortho esters as well as the polymer hydrophobicity.

Reduction-degradable linear cationic polymers as gene carriers prepared by Cu(I)-catalyzed azide-alkyne cycloaddition.

Linear reduction-degradable cationic polymers with different secondary amine densities (S2 and S3) and their nonreducible counterparts (C2 and C3) were synthesized by Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) step-growth polymerization of the dialkyne-oligoamine monomers and the diazide monomers. These polymers were studied with a goal of developing a set of new gene carriers. The buffering capacity and DNA binding ability of these polymers were evaluated by acid-base titration, gel retardation, and ethidium bromide (EB) exclusion assay. The polymers with lower amine density exhibit a weaker DNA-binding ability but a stronger buffering capacity in the range of pH 5.1 and 7.4. Particle size and zeta-potential measurements demonstrate that the polymers with higher amine density condense pDNA to form polyplexes with smaller sizes, while the disulfide bond in the backbone shows a negative effect on the condensing capability of the polymers, resulting in the formation of polyplexes with large size and nearly neutral surface. The reduction-sensitive polyplexes formed by polymer S2 or S3 can be disrupted by dithiothreitol (DTT) to release free DNA, which has been proven by the combination of gel retardation, EB exclusion assay, particles sizing, and zeta potential measurements. Cell viability measurements by MTT assay demonstrate that the reduction-degradable polymers (S2 and S3) have little cytotoxicity while the nonreducible polymers (C2 and C3) show obvious cytotoxicity, in particular, at high N/P ratios. In vitro transfection efficiencies of these polymers were evaluated using EGFP and luciferase plasmids as the reporter genes. Polymers S3 and S2 show much higher efficiencies than the nonreducible polymers C3 and C2 in the absence of 10% serum; unexpectedly, the lowest transfection efficiency has been observed for polymer S3 in the presence of serum.

Combination of ATRA and ATRC for the synthesis of periodic vinyl copolymers.

A new approach to periodic vinyl copolymers via combination of atom transfer radical addition (ATRA) and atom transfer radical coupling (ATRC) is reported. The two examples are methyl methacrylate (MMA) and styrene (St) periodic copolymer (P(SMMS)) and acrylonitrile (AN) and St periodic copolymer (P(SAAS)). First, two monomer sequence units (MSU) with built-in sequence, SMMS and SAAS, are synthesized through the controlled ATRA of two ATRP initiators with St. Then, the ATRC of SMMS and SAAS are conducted at high radical conditions to generate two types of high-molecular-weight copolymers, P(SMMS) and P(SAAS). Though side reactions can not be totally avoided, characterizations of the polymer structure with a variety of means confirm that the main chain structures of P(SMMS) and (PSAAS) are predominantly with the periodic sequences from the MSUs. Attempts to suppress the side reactions are successful via the MNP-mediated ATRC of SMMS and SAAS.

Shell-sheddable, pH-sensitive supramolecular nanoparticles based on ortho ester-modified cyclodextrin and adamantyl PEG.

We report a new type of pH-sensitive supramolecular aggregates which possess a programmable character of sequential dePEGylation and degradation. As a platform of designing and building multifunctional supramolecular nanoparticles, a family of 6-OH ortho ester-modified β-cyclodextrin (β-CD) derivatives have been synthesized via the facile reaction between β-CD and cyclic ketene acetals with different alkyl lengths. These asymmetric acid-labile β-CD derivatives formed amphiphilic supramolecules with adamantane-modified PEG through host-guest interaction in polar solvents such as ethanol. The supramolecules can self-assemble in water to form acid-labile supramolecular aggregates. The results of TEM and light scattering measurements demonstrate that the size and morphology of the aggregates are influenced by the alkyl or PEG length and the host-guest feed ratio. By carefully balancing the alkyl and PEG lengths and adjusting the host-guest ratio, well-dispersed vesicles (50-100 nm) or sphere-like nanoparticles (200-500 nm) were obtained. Zeta potential measurements reveal that these supramolecular aggregates are capable of being surface-functionalized via dynamic host-guest interaction. The supramolecular aggregates were stable at pH 8.4 for at least 12 h as proven by the (1)H NMR and LLS measurements. However, rapid dePEGylation occurred at pH 7.4 due to the hydrolysis of the ortho ester linkages locating at the interface, which resulted in aggregation of the dePEGylated hydrophobic inner cores. Upon further decreasing the pH to 6.4, the hydrophobic cores were further degraded due to the acid-accelerated hydrolysis of the ortho esters. The incubation stability of the acid-labile supramolecular aggregates in neutral buffer could be improved by incorporating hydrophobic poly(ε-caprolactone) into the core of the aggregates.

Truncated peptides from melittin and its analog with high lytic activity at endosomal pH enhance branched polyethylenimine‐mediated gene transfection

Melittin is a commonly used cell‐penetrating peptide (CPP) for improving branched polyethylenimine (BPEI)‐mediated gene transfection. However, its application is limited owing to the cytotoxicity generated by the lytic activity at neutral pH. In the present study, we report two truncated peptides from melittin and florae with improved transfection efficiency.

Biocompatible acid-labile polymersomes from PEO-b-PVA derived amphiphilic block copolymers

A family of amphiphilic block copolymers with pendent ortho ester groups were synthesized by modifying the double hydrophilic block copolymer PEO114-b-PVA240 with 2-ethylidene-4-methyl-1,3-dioxolane (EMD) under mild conditions (30 °C). The degree of modification (DM) of the ortho ester groups can be tuned by simply varying the feed ratio of EMD to the hydroxyl groups in the PVA block. These block copolymers are stable in an anhydrous environment. Laser light scattering (LLS) and transmission electron microscopy (TEM) measurements revealed that in weakly basic aqueous buffer, these amphiphilic block copolymers self-assembled into aggregates with different size and morphology, ranging from solid-like spherical nanoparticles to polymersomes as the DM increased. The acid-triggered dissociation behaviour of the aggregates were studied by LLS, nile red (NR) fluorescence and TEM. The copolymer aggregates dissociated faster in a buffer with the lower pH; the dissociation rate of the aggregates became faster for the copolymers with lower DM. The polymersomes can load both hydrophilic biomacromolecules like lysozyme and hydrophobic anticancer drug doxorubicin (DOX). The drug-loaded polymersomes were stable in neutral phosphate buffer for at least 6 h with a payload leakage of less than 25% in 12 h at 37 °C; however, significant acid-triggered payload release was accomplished even at a mildly acidic pH (6.0). Finally, the DOX-loaded polymersomes exhibited a concentration-dependent toxicity to MCF-7 and HeLa cells while the copolymers themselves are non-toxic.

Oxidation and temperature dual responsive polymers based on phenylboronic acid and N-isopropylacrylamide motifs

Oxidation stress has been becoming an important target for the development of smart nanomedicines, triggering research interest in oxidation responsive polymers. Herein, we report a new type of temperature/oxidation dual responsive copolymer. They were synthesized by the sequential atom transfer radical copolymerization (ATRP) of N-isopropylacrylamide (NIPAM, M1) and a phenylboronic pinacol ester-containing acrylate (M2) and dialysis against water to remove the pinacol protecting groups. The copolymers with a small amount of phenylboronic acid units (<7%) were soluble in cold neutral phosphate buffer but showed lower critical solution temperatures in the range of 12–31 °C. The thermally induced phase transition profiles depended on both the composition and concentration of the polymers. The cloud points of the copolymers were shifted to higher temperatures upon H2O2 induced oxidation of the phenylboronic acid and the subsequent 1,6-elimination. Using PEG-Cl as the macroinitiator to initiate ATRP of M1 and M2, three block copolymers composed of a PEG block and a temperature/oxidation dual responsive segment were prepared after dialysis in water. These block copolymers had a similar ratio of NIPAM to phenylboronic acid units but different molecular weights. We have studied their thermal self-assembly and H2O2 triggered decomposition by laser light scattering, 1H NMR, and transmission electron microscopy. Upon a fast heating protocol, these block copolymers formed stable micelle-like nanoparticles (at 37 °C) that were capable of encapsulating doxorubicin (DOX) and showed H2O2 triggered release. The naked nanoparticles were cytocompatible, however the DOX-loaded ones exhibited concentration dependent cytotoxicity, in particular to cancer cells.

Oxidation-Responsive Polymer-Drug Conjugates with a Phenylboronic Ester Linker.

Polymer-drug conjugates have attracted great interest as one category of various promising nanomedicines due to the advantages of high drug-loading capacity, negligible burst release, and improved pharmacokinetics as compared with the small molecular weight drugs or the polymeric delivery systems with physically encapsulated drugs. Herein, a new type of oxidation-responsive polymer-drug conjugates composed of a poly(ethylene glycol) (PEG) block and a hydrophobic polyacrylate block to which Naproxen is attached through a phenylboronic ester linker is reported. The amphiphilic block copolymers are synthesized through the reversible addition-fragmentation chain transfer polymerization of the Naproxen-containing acrylic monomer using a PEG chain transfer agent. In neutral aqueous buffer, the conjugates formed nanoparticles with diameters of ≈150-300 nm depending on the length of the hydrophobic segment. The dynamic covalent bond of the phenylboronic ester is stabilized due to the hydrophobic microenvironment inside the nanoparticles. Upon exposure to H2 O2 , the phenylboronic ester is oxidized rapidly into the phenol derivative which underwent a 1,6-elimination reaction, releasing the intact Naproxen. The rate of drug release is influenced by the concentration of H2 O2 and the hydrophobic block length. This type of oxidation-responsive polymer-drug conjugate is feasible for other drugs containing hydroxyl group or amino group.