Rational Construction of a Mitochondrial Targeting, Fluorescent Self-Reporting Drug-Delivery Platform for Combined Enhancement of Endogenous ROS Responsiveness.

To maximize the utilization and response to the high oxidative stress environment of tumor sites while avoiding the dilemma of enhancing reactive oxygen species (ROS) response in a single way, mitochondrial targeting combined with fluorescent self-reporting polymeric nanocarriers (1K-TPP and 2K-TPP) with grafted structures were synthesized via a chemoenzymatic method in a high yield to simultaneously enhance the drug delivery of endogenous ROS responses. 1K-TPP and 2K-TPP loaded doxorubicin (DOX) at a high content over 12% and formed homogeneous spherical micelles. In vitro, both of them showed promising high sensitivity (detection limit below 200 nM H2O2), fast response, and ratiometric fluorescent self-reporting properties (fluorescent enhancement more than 200 times) to ROS and excellent stability under physiological conditions, while achieving a rapid release of the DOX in response to 1 mM H2O2. Cell co-localization experiments exhibited that they had favorable mitochondrial targeting, and mitochondrial isolation experiments also confirmed that the TPP-modified 1K-TPP selectively accumulated nearly three times in mitochondria than that in total cells. The internalization of 1K-TPP and 2K-TPP into cancer cells was greatly improved by nearly 200% compared to that of unmodified control (1K-OH and 2K-OH) and also explored a unique energy-dependent endocytosis. Furthermore, stimulation of endogenous ROS enhanced the green fluorescence intensity (up to 51.4%) of the linked probe so as to destroy the internal structure of the nanocarriers, achieving self-reporting of the drug's intracellular release and tracking of the intracellular location of nanocarriers. The cytotoxicity of DOX-loaded 1K-TPP and 2K-TPP in tumor cells with a higher ROS content showed statistical superiority to that of 1K-OH and 2K-OH, benefiting from the extremely good endogenous ROS response sensitivity leading to the differential selective release of drugs. These results demonstrate the potential of 1K-TPP and 2K-TPP, especially for 1K-TPP, as mitochondria-targeted, fluorescent self-reporting nanocarriers for combined enhancement of endogenous ROS responsiveness.

Novozym 435-Catalyzed Synthesis of Well-Defined Hyperbranched Aliphatic Poly(ß-thioether ester).

A series of new hyperbranched aliphatic poly(ß-thioether ester)s were prepared by the enzymatic ring-opening polycondensation of 1,4-oxathiepan-7-one (OTO) and AB2/ABB' comonomer with acid-labile ß-thiopropionate groups. Two kinds of comonomers, methyl 3-((3-hydroxy-2-(hydroxymethyl)propyl)thio)propanoate (HHTP) and methyl 3-((2,3-dihydroxypropyl)thio)propanoate (DHTP), with different primary alcohols and secondary alcohols, were synthesized by thiol-ene click chemistry and thiol-ene Michael addition, respectively. Immobilized lipase B from Candida antarctica (CALB), Novozym 435, was used as the catalyst. The random copolymers were characterized by 1H-NMR, 13C-NMR, GPC, TGA, and DSC. All branched copolyesters had high molecular weights over 15,000 Da with narrow polydispersities in the range of 1.75-2.01 and were amorphous polymers. Their degradation properties under acidic conditions were also studied in vitro. The polymeric nanoparticles of hyperbranched poly(ß-thioether ester)s were successfully obtained and showed good oxidation-responsive properties, indicating their potential for biomedical applications.

Preparation of fluorophore-tagged polymeric drug delivery vehicles with multiple biological stimuli-triggered drug release.

In the field of drug delivery, the controlled release of drugs is continuously one of the highly prioritized research domains. Stimuli-responsive polymers are being investigated as drug delivery vehicles that modulate pharmaceutical effect via tumor specific mechanisms. In this work, a biocompatible graft copolymer (denoted as PSNC-g-mPEG/TPE) was constructed, which comprised a triple-responsive polycarbonate backbone coupled with fluorescent TPE and hydrophilic methoxypoly(ethylene glycol) (mPEG) segments. This multifunctional amphiphilic copolymer was able to self-assemble in aqueous solutions and acted as a drug delivery vehicle that releases cargo in response to multiple biological stimuli (ROS, pH and enzymes). And the results of confocal laser scanning microscopy (CLSM) suggested that these micelles could be rapidly internalized by cells and achieve more effective drug release in cancer cells. Furthermore, the cytotoxicity assays proved the safety of this material. It is anticipated that this strategy has enormous potential in constructing novel anticancer therapeutics.

GSH/pH dual-responsive biodegradable camptothecin polymeric prodrugs combined with doxorubicin for synergistic anticancer efficiency.

Dual stimuli-responsive camptothecin polymeric prodrugs (CPT Prodrugs) with grafted structures were designed via chemoenzymatic methods and combined with doxorubicin (DOX) for synergistic drug delivery to improve anticancer efficiency. The CPT Prodrugs loaded DOX with a high efficiency through the cooperative contribution of several interaction forces. The produced amphiphilic polymeric prodrugs loaded with DOX, referred to as DOX@CPT Prodrugs, formed homogeneous spherical micelles of appropriate sizes (sub-50 nm). The DOX@CPT Prodrug micelles showed excellent stability in release experiments under in vitro physiological conditions and maintained over 80% drug loading after 4 weeks when stored at 4 °C. Under weakly acidic pH and reduced glutathione (GSH) conditions, the DOX@CPT Prodrugs with high disulfide and tertiary amine content achieved synergistic release of the two loaded drugs and biodegraded into low-molecular-weight compounds. The cell experiments confirmed that the internalization of DOX@CPT Prodrugs into cancer cells was greatly improved by nearly 30% compared with that of free drugs. Furthermore, the synergistic drug delivery system exhibited superior anticancer efficiency with highly improved cell selectivity ratios (up to 127.0%) and greatly enhanced synergistic effects (up to 23.9%) benefiting from good long-term stability, better internalization by cells and sensitive pH and GSH dual-responsivity. In addition, the DOX@CPT Prodrugs with suitable sizes and good water solubility also exhibited a greater penetrability in the case of simulating solid tumors than the free drugs. These results demonstrate the potential of DOX@CPT Prodrugs as biodegradable, dual-responsive combination therapy nanocarriers for synergistic anticancer treatment.

Chemoenzymatic synthesis of dual-responsive graft copolymers for drug delivery: long-term stability, high loading and cell selectivity.

A series of amphiphilic graft copolymers, poly(N-propargyldiethanolamine 4,4'-dithiodibutyionate)-graft-monomethoxy poly(ethylene glycol) (PPD-g-mPEG), were designed via a chemoenzymatic method for pH and reduced glutathione (GSH) dual-responsive drug delivery. The effects of percent grafting and molecular weights of mPEG on critical micelle concentration (CMC) values, size of micelles, drug loading and dual-response were tested. The graft copolymers could easily form homogeneous spherical micelles with appropriate sizes and zeta-potentials. The micelles of PPD-g-mPEG copolymers loaded doxorubicin (DOX) in high efficiency, and showed excellent stability under physiological conditions and synergetic dual-response to weakly acidic pH and GSH. In vitro experiments confirmed that the DOX-loaded micelles could be internalized into cancer cells efficiently and release DOX over time. Furthermore, cell cytotoxicity assays indicated that the graft copolymers were non-cytotoxic to both cancerous and normal cells while the DOX-loaded micelles greatly improved the selectivity ratios between HeLa cells and HL-7702 cells. DOX-loaded micelles also avoided hemolysis of red blood cells (RBCs) effectively compared with commercialized doxorubicin hydrochloride. All these demonstrated the potential of PPD-g-mPEG as a model to create more functional dual-responsive nanocarriers for controlled drug delivery.

Amphiphilic polymers formed from ring-opening polymerization: a strategy for the enhancement of gene delivery.

Cationic liposomes and polymers are both important candidates for use as non-viral gene vectors. However, both of them have special shortcomings and application limits. This work is devoted to the combination of advantages of liposomes and polymers. The ring-opening polymerization strategy was used for the preparation of amphiphilic polymers from cyclen-based cationic small lipids. The non-hydrophobic polymer and the corresponding lipids were also prepared for performing structure-activity relationship studies. Gel electrophoresis results reveal that both the lipopolymers and liposomes could effectively condense DNA into nanoparticles and protect DNA from degradation. Compared to polymers, the DNA binding ability of liposomes is more affected by hydrophobic tails. Under the same dosage, the synthetic polymers have stronger DNA binding ability than the liposomes. In vitro transfection experiments show that the polymers could give better transfection efficiency, which was much higher than those of the corresponding liposomes and non-hydrophobic polymer. The oleyl moiety is suitable for lipidic vectors, but things were different for polymers. Under optimized conditions, up to 14.2 times higher transfection efficiency than that for 25 kDa bPEI could be obtained. More importantly, the lipopolymers showed much better serum tolerance, which was further confirmed by protein adsorption, gel electrophoresis, flow cytometry, and CLSM assays. The results indicate that ring-opening polymerization is a promising strategy for the enhancement of the gene delivery efficiency and biocompatibility of cationic lipids.

Ring-opening polymerization for hyperbranched polycationic gene delivery vectors with excellent serum tolerance.

In order to improve the transfection efficiency (TE) and biocompatibility, we synthesized a series of hyperbranched cationic polymers by ring-opening polymerization between diepoxide and several polyamines. These materials can condense plasmid DNA efficiently into nanoparticles that have much lower cytotoxicity than those derived from bPEI. In vitro transfection experiments showed that polymers prepared from branched or cyclic polyamine (P1 and P5) exhibited TE several times higher than 25KDa bPEI. More significantly, serum seemed to have no negative effect on P1-P5 mediated transfection. On the contrary, the TE of P1 improved, even when the serum concentration reached 70%. Several assays demonstrated the excellent serum tolerance of such polycationic vectors: bovine serum albumin (BSA) adsorption assay revealed considerably lower protein adsorption of P1-P5 than PEI; P1 showed better DNA protection ability from degradation by DNase I than PEI; flow cytometry results suggested that any concentration of serum may not decrease the cellular uptake of P1/DNA polyplex; and confocal laser scanning microscopy also found that serum has little effect on the transfection. By using specific cellular uptake inhibitors, we found that the polyplexes enter the cells mainly via caveolae and microtubule-mediated pathways. We believe that this ring-opening polymerization may be an effective synthetic approach toward gene delivery materials with high biological activity.

Low molecular weight PEI-appended polyesters as non-viral gene delivery vectors.

Routine clinical implementation of human gene therapy requires safe and efficient gene delivery methods. Linear biodegradable polyesters with carbon-carbon double bonds are prepared from unsaturated diacids and diols. Subsequent appending of low molecular weight PEI by Michael addition gives target cationic polymers efficiently. Agarose gel retardation and fluorescence quenching assays show that these materials have good DNA binding ability and can completely retard plasmid DNA at weight ratio of 0.8. The formed polyplexes have appropriate sizes around 275 nm and zeta-potential values about +20-35 mV. The cytotoxicities of these polymers assayed by MTT are much lower than that of 25 kDa PEI. In vitro transfection toward 7402, HEK293 and U-2OS cells show that polymer P1 may give dramatically higher transfection efficiency (TE) than 25 kDa PEI, especially in U-2OS cells, suggesting that such polymer might be promising non-viral gene vectors.

Lipase-catalyzed synthesis of azido-functionalized aliphatic polyesters towards acid-degradable amphiphilic graft copolymers.

A series of novel aliphatic polyesters with azido functional groups were synthesized via the direct lipase-catalyzed polycondensation of dialkyl diester, diol and 2-azido-1,3-propanediol (azido glycerol) using immobilized lipase B from Candida antarctica (CALB). The effects of polymerization conditions including reaction time, temperature, enzyme amount, substrates and monomer feed ratio on the molecular weights of the products were studied. The polyesters with pendant azido groups were characterized by (1)H NMR, (13)C NMR, 2D NMR, FTIR, GPC and DSC. Alkyne end-functionalized poly(ethylene glycol) containing a cleavable acetal group was then grafted onto the polyester backbone by copper-catalyzed azide-alkyne cycloaddition (CuAAC, click chemistry). Using fluorescence spectroscopy, dynamic light scattering (DLS) and transmission electron microscopy (TEM), these amphiphilic graft copolymers were found to readily self-assemble into nanosized micelles in aqueous solution with critical micelle concentrations between 0.70 and 1.97 mg L(-1), and micelle sizes from 20-70 nm. The degradation of these polymers under acidic conditions was investigated by GPC and (1)H NMR spectroscopy. Cell cytotoxicity tests indicated that the micelles had no apparent cytotoxicity to Bel-7402 cells, suggesting their potential as carriers for controlled drug delivery.

Effects of additives on lipase immobilization in microemulsion-based organogels.

An inexpensive, facile, and environmentally benign method was developed to improve the activity and stability of Candida rugosa lipase (triacylglycerol acylhydrolase) immobilized on microemulsion-based organogels (CRL MBGs) via the addition of additives during immobilization. The additives used were polyethylene glycol (PEG) or polysaccharides. This study is the first report on the effect of additives in CRL MBGs. Among the tested additives, PEG produced the most improvement in the immobilized CRL, enhancing its stability in organic solvents (specifically polar solvents). The results of circular dichroism and fluorescence spectra experiments indicated that exposure of the acidic CRL to electronegative additives in the buffer, such as polyethylenimine and the electropositive surfactant cetyltrimethylammonium bromide, may change the lipase secondary structure, ultimately causing enzyme inactivation. However, sodium bis(2-ethylhexyl)sulfosuccinate and PEG 2000 had minimal effects on the secondary structure of CRL. The CRL MBGs containing PEG 2000 demonstrated remarkable retention of their catalytic activity during the recycling test. No significant changes in enzymatic activity were observed, even after nine runs, and 90% of the original yield was maintained after 15 cycles.

Trypsin-catalyzed tandem reaction: one-pot synthesis of 3,4-dihydropyrimidin-2(1H)-ones by in situ formed acetaldehyde.

A simple, mild, one-pot tandem method catalyzed by trypsin was developed for the synthesis of 3,4-dihydropyrimidin-2(1H)-ones by the Biginelli reaction of urea, ß-dicarbonyl compounds, and in situ-formed acetaldehyde. Trypsin was found to display dual promiscuous functions to catalyze transesterification and the Biginelli reaction in sequence.