Establishing stimulus-responsive nanosystems for the codelivery of anticancer drug and oligonucleotide is a promising strategy in cancer treatment owing to the combination of chemotherapy and gene therapy in a synergistic manner. Herein, an ATP aptamer and its cDNA sequence were first hybridized to produce the duplex, into which chemotherapeutic agent doxorubicin (DOX) interacted through the GC-rich motif of duplex, and PEI25K was then employed as a carrier to condense the DOX-loading duplex and miR-34a to construct the ternary nanocomplex PEI/DOX-Duplex/miR-34a. The nanocomplex exhibited a favorable drug release profile through the response to high concentration of ATP in the cytosol. The ATP-responsive delivery system was demonstrated to possess higher antiproliferative effect (cell viability of <40%) than the single cargo delivery, which could be attributed to the synergistic induction of cell apoptosis and cell cycle arrest from DOX and miR-34a. Furthermore, wound healing and Transwell assay elucidated the higher antimigration effect of ternary nanocomplex than DOX-Duplex or miR-34a delivery. Overall, the combinatorial delivery of DOX and miR-34a through an ATP-responsive manner could trigger the rapid release of cargoes in the cytosol and enhance the inhibition of cell proliferation and migration through the synergistic manner of these two components.Keywords: antimigration; antiproliferation; ATP response; codelivery; doxorubicin; miR-34a;

Chondroitin sulfate (CS) was modified on a polyamidoamine dendrimer (PAMAM) through Michael addition to construct a tumor-targeted carrier CS-PAMAM for miR-34a delivery. The derivative CS-PAMAM was demonstrated to achieve an efficient cellular uptake of miR-34a in a CD44-dependent endocytosis way and further facilitate the endosomal escape of miR-34a after 4 h. Through the miR-34a delivery, obvious inhibition of cell proliferation could be detected which was attributed to the enhancement of cell apoptosis and cell cycle arrest, and meanwhile the cell migration and invasion has been observed to be inhibited. Finally, the intravenous injection of CS-PAMAM/miR-34a formulation into mice bearing human lung adenocarcinoma cell A549 xenografts could efficiently inhibit the tumor growth and induce the tumor apoptosis owing to the enhanced accumulation of miR-34a in tumor tissue. Overall, CS-PAMAM is potential to be used as a tumor-targeted oligonucleotide carrier for achieving tumor gene therapy.Statement of SignificanceThe cationic dendrimer PAMAM was modified by chondroitin sulfate (CS) through Michael addition to construct a tumor-targeted carrier CS-PAMAM for miR-34a delivery. The introduction of CS could achieve an efficient cellular uptake and intracellular transfection of miR-34a in a CD44-dependent endocytosis manner. The miR-34a delivery could execute the anti-proliferation activity by simultaneously inducing cell apoptosis and cell cycle arrest, and also the anti-migration activity. The CS-PAMAM-mediated systemic delivery of miR-34a showed significant inhibition of tumor growth and induction of tumor apoptosis using a mice model of subcutaneously implanted tumors.Download high-res image (225KB)Download full-size image

The nucleobase analogue 2-amino-6-chloropurine was modified on the surface of polyamidoamine (PAMAM) to construct a derivative AP-PAMAM, and then it was used as a gene carrier for miR-23b delivery to achieve the anti-tumor effects. The carrier AP-PAMAM could condense miR-23b into stable nanoparticles with a particle size of 97.3 nm (N/P ratio of 50), which was favorable for the cellular uptake of nanoparticles. Compared with PAMAM, AP-PAMAM exhibited an obviously enhanced transfection efficiency through the transfection assay of plasmids pEGFP-N3 and pGL-3. Using the human lung adenocarcinoma cell line A549 as a model, AP-PAMAM-mediated miR-23b delivery could achieve a stronger anti-proliferative effect than PAMAM/miR-23b. The inhibition of cell proliferation was elucidated to be associated with the apoptotic induction (apoptotic ratio of 23.2%) and S phase arrest owing to the decreased expression level of cyclin D1. Meanwhile, the AP-PAMAM-mediated miR-23b delivery could suppress the cell migration and invasion of cancer cells through wound healing and Transwell migration assays. In summary, the PAMAM derivative-mediated miR-23b delivery could be a promising strategy for achieving tumor gene therapy.

Enhancing the activity and stability of enzymes and improving their reusability are critical challenges in the field of enzyme immobilization. Here we report a facile and efficient biomimetic mineralization to embed thermophilic lipase QLM in zeolite imidazolate framework-8 (ZIF-8). Systematic characterization indicated that the entrapment of lipase molecules was successfully achieved during the crystal growth of ZIF-8 with an enzyme loading of ∼72.2 ± 1.88 mg/g lipase@ZIF-8, and the enzymes could facilitate the construction of framework building blocks. Then the composite lipase@ZIF-8 was observed to possess favorable catalytic activity and stability in the ester hydrolysis, using the hydrolysis of p-nitrophenyl caprylate as a model. Finally, the composite was successfully applied in the kinetic resolution of (R,S)-2-octanol, with favorable catalytic activity and enantioselectivity during 10 cycle reactions. Thus, the biomimetic mineralization process can be potentially used as an effective technique for realizing the entrapment of biomacromolecules and constructing efficient catalysts for industrial biocatalysis.Keywords: biomimetic mineralization; ester hydrolysis; kinetic resolution; metal−organic frameworks; thermophilic lipase

Porous PLGA microparticle for the coencapsulation of doxorubicin and miR-519c was successfully constructed through the water–oil–water emulsion solvent evaporation method, using ammonium bicarbonate as a porogen. It has been characterized with high porous surface, adaptive aerodynamic diameter (<10 μm), favorable drug loading, and sustained release profile. The release supernatant exhibited a higher inhibition of cell proliferation than those from porous PLGA microparticles harboring a single component (doxorubicin or miR-519c), attributing to the enhanced induction of cell apoptosis and cell cycle arrest at S phase. Finally, the improved intracellular concentration of doxorubicin was elucidated by flow cytometry and liquid chromatography with tandem mass spectrometry, owing to the knockdown of drug transporter ABCG2 by miR-519c. Overall, the porous PLGA microparticle combining chemotherapy and gene therapy could facilitate the antitumor efficacy and reduce the side effects, and thus, it is potential to be used as a sustained release system for lung cancer treatment via pulmonary administration.Keywords: ABCG2; cell proliferation; doxorubicin; lung cancer; miR-519c; porous microparticle;

Herein, chondroitin sulfate-functionalized polyamidoamine (CS-PAMAM) was employed as a carrier in miR-34a delivery, and the inhibition of cell proliferation and migration of pancreatic cancer was systematically evaluated, using human pancreatic carcinoma cell line MiaPaCa-2 as a model. Through confocal laser scanning microscopy, an efficient cellular uptake of CS-PAMAM/miR-34a nanoparticles has been demonstrated to be executed in a CD44-dependent manner. After the successful delivery of miR-34a, the cell proliferation could be obviously inhibited due to the activation of cell apoptosis and cell cycle arrest. Meanwhile, CS-PAMAM-mediated miR-34a delivery could realize the suppression of cell migration elucidated by wound healing and transwell migration assays. Thus, the derivative CS-PAMAM could potentially be used as an effective carrier for miR-34a delivery to achieve the tumor gene therapy.

•Phenylboronic acid was conjugated to PAMAM to construct a derivative PPP.•PPP could be used as a tumor-targeted carrier for Bcl-2 siRNA delivery.•The transfection of Bcl-2 siRNA could inhibit the cell proliferation.In this study, the conjugation of phenylboronic acid (PBA) to amine-terminated polyamidoamine (PAMAM) was successfully conducted to prepare a tumor-targeted gene carrier PBA-functionalized PAMAM (PPP) for Bcl-2 siRNA delivery, using a heterobifunctional crosslinker NHS-PEG5k-Mal. The carrier possessed favorable capacity for siRNA condensation and could protect siRNA from the degradation against RNase and serum. The introduction of PBA could facilitate the cellular uptake and further transfection of Bcl-2 siRNA demonstrated by confocal laser scanning microscopy and flow cytometry. Meanwhile, PPP-mediated transfection of Bcl-2 siRNA could significantly inhibit the expression of Bcl-2 gene at both mRNA and protein levels. Furthermore, owing to the knock-down of Bcl-2, PPP/siRNA could significantly inhibit the cell proliferation by inducing the cell apoptosis, and also enhance the antitumor efficiency of doxorubicin by suppressing the resistance of tumor cells to chemotherapeutics. In conclusion, the PPP-mediated Bcl-2 siRNA delivery could potentially be an effective platform for solving the drug resistance and further achieving the combined chemotherapy and gene therapy in tumor treatment.Bcl-2 siRNA delivery mediated by phenylboronic acid-functionalized polyamidoamine.

A hybrid gene carrier, HGP, has been successfully constructed through the genipin-mediated cross-linking of thermophilic histone and PEI25K. The thermophilic histone gene GK2215 was cloned from Geobacillus kastophilus HTA426 and overexpressed in Escherichia coli BL21. The thermophilic histone was systematically characterized and then cross-linked with PEI25K by genipin to obtain HGP. Notably, HGP exhibited superior transfection efficiency due to the synergistic effects between these two components: PEI25K mainly contributed to the condensation and transfer of pDNA, while thermophilic histone could enhance the endosomal escape and further nuclear location to achieve high gene expression. Meanwhile, HGP showed much lower cytotoxicity and hemolytic activity than PEI25K due to the introduction of nontoxic thermophilic histone. In addition, a strong intrinsic red fluorescence could be obviously observed in HGP. In conclusion, the protein–polymer hybrid carrier could potentially be used as a theranostic delivery system for achieving both efficient gene therapy and in vivo imaging.

Porous PLGA microparticles were successfully prepared for achieving the co-delivery of doxorubicin and PEI25K/miR-34a, using ammonium bicarbonate as a porogen. The synergistic effect between these two components ensured the efficient anti-proliferative and anti-migration effects of porous PLGA microparticles on tumor cells.

A protein–polymer hybrid gene carrier HEP was successfully constructed based on thermophilic histone and polyethylenimine. The carrier exhibited low cytotoxicity and a high transfection efficiency due to the synergistic effects between the two components, which makes it a potential vehicle in tumor gene therapy.

•The gene carrier chondroitin sulfate-g-polyethylenimine was constructed through Michael addition.•The carrier could induce the cellular uptake of miR-34a in a CD44-mediated endocytosis manner.•The carrier-mediated miR-34a transfection could achieve the inhibition of cell proliferation and migration.Chondroitin sulfate was chemically conjugated to PEI25K through Michael addition to construct a non-viral gene carrier CS-PEI, and then the carrier was employed in miR-34a delivery to achieve the inhibition of cell proliferation and migration, using prostate tumor cell PC-3 as a model. The nanoparticle from CS-PEI and miR-34a at a mass ratio of 10 was prepared with particle size and zeta potential of 170.7 nm and +42.2 mV, respectively. Flow cytometry and fluorescence microscopy revealed that CS-PEI could efficiently induce the cellular uptake of miR-34a in a CD44-mediated endocytosis manner. Through CS-PEI-mediated miR-34a transfection, obvious cell apoptosis was observed with early apoptotic cells of 47.49%, and meanwhile the activation of caspase-3, -8 and -9, and decreased expression level of Bcl-2 were detected. Moreover, wound healing assay showed that CS-PEI/miR-34a transfection could inhibit the cell migration. Overall, CS-PEI is potentially employed as a promising tumor-targeting system for miR-34a delivery in tumor gene therapy.

•N-Isopropylacrylamide-modified polyethylenimine was employed as a p53 gene carrier.•The inhibition of cell proliferation was observed after p53 gene transfection.•The p53null cells exhibited a higher sensitivity to exogenous p53 gene expression.In this paper, N-isopropylacrylamide-modified polyethylenimine (PEN) was constructed through Michael addition and employed as a carrier to achieve the p53 gene delivery, using HeLa (p53wt) and PC-3 cells (p53null) as models. After PEN-mediated p53 transfection, expression level of p53 in HeLa and PC3 cells was up-regulated at both mRNA and protein levels. Due to the exogenous p53 expression, the inhibition of cell proliferation was observed through MTT analysis, attributing to the activation of apoptosis and cell cycle arrest. Using flow cytometric analysis, early apoptotic ratios of 54.95% and 27.06% after PEN-mediated p53 transfection were detected in PC-3 and HeLa cells, respectively, indicating that PC-3 cells were more sensitive to the exogenous p53 transfection than HeLa cells. Meanwhile, G1 phase arrest was detected in PC-3 cells while a unique G2 phase arrest was identified in HeLa cells after p53 transfection. Through Western blotting, activity analysis of caspase-3, caspase-8 and caspase-9 and mitochondrial membrane potential measurement, the apoptosis induced by PEN-mediated p53 transfection was conducted in a mitochondria-dependent apoptosis pathway. These results demonstrated that PEN could successfully mediate the p53 gene delivery and up-regulate the cellular p53 expression level, triggering a significant p53-dependent anti-proliferative effect on tumor cells.

•Porous PLGA microparticles harboring doxorubicin and PEI25K/p53 were prepared and characterized.•The microparticles exhibited an enhanced anticancer effect due to the synergistic cytotoxicity of doxorubicin and p53.•The microparticles provided a potential local drug delivery system by inhalation.In this study, porous PLGA microparticles for the co-delivery of doxorubicin and PEI25K/p53 were successfully prepared by the water–oil–water emulsion solvent evaporation method, using ammonium bicarbonate as a porogen. The porous microparticles were obtained with a mean diameter of 22.9 ± 11.8 μm as determined by laser scattering particle size analysis. The particles’ surface porous morphology and distributions of doxorubicin and p53 were systematically characterized by scanning electron microscopy, flow cytometry, fluorescence microscopy and confocal laser scanning microscopy, revealing that doxorubicin and the plasmid were successfully co-encapsulated. Encapsulation efficiencies of 88.2 ± 1.7% and 36.5 ± 7.5% were achieved for doxorubicin and the plasmid, respectively, demonstrating that the porous structure did not adversely affect payload encapsulation. Microparticles harboring both doxorubicin and PEI25K/p53 exhibited enhanced tumor growth inhibition and apoptosis induction compared to those loaded with either agent alone in A549 human lung adenocarcinoma cells. Overall, the porous PLGA microparticles provide a promising anticancer delivery system for combined chemotherapy and gene therapy, and have great potential as a tool for sustained local drug delivery by inhalation.

In the present study, block copolymers were first synthesized through a tandem ring-opening metathesis polymerization (ROMP) and conventional enzymatic ring-opening polymerization (eROP) from hydroxyl initiator. Furthermore, a novel synthesis route, single-step eROP from ester precursor was successfully developed to synthesize targeted copolymers. The as-prepared polymers were analyzed by NMR, GPC, DSC, and MALDI-TOF-MS. There was no difference in the characteristic peaks of NMR between the end products obtained from these two synthetic routes. The GPC data showed that the copolymer obtained from single-step eROP was similar to the end product obtained from the traditional multistep synthesis method. Afterward, we used model compounds to carry out the conventional eROP and the single-step eROP. Finally, through the kinetic analysis and structural analysis of the resulting product, a reasonable initiation mechanism for this single-step eROP was elucidated.

The paper explored the catalytic activity of a cell debris self-immobilized thermophilic lipase for polyester synthesis, using the ring-opening polymerization of ε-caprolactone as model. Effects of biocatalyst concentration, temperature, and reaction medium on monomer conversion and product molecular weight were systematically evaluated. The biocatalyst displayed high catalytic activity at high temperatures (70–90 °C), with 100 % monomer conversion. High monomer conversion values (>90 %) were achieved in both hydrophobic and hydrophilic solvents, and also in solvent-free system, with the exception of dichloromethane. Poly(ε-caprolactone) was obtained in 100 % monomer conversion, with a number-average molecular weight of 1,680 g/mol and a polydispersity index of 1.35 in cyclohexane at 70 °C for 72 h. Furthermore, the biocatalyst exhibited excellent operational stability, with monomer conversion values exceeding 90 % over the course of 15 batch reactions.

This paper reviews the immobilization of a thermophilic esterase, AFEST from the archaeon Archaeoglobus fulgidus, on a hydrophobic macroporous resin and its application in polyester synthesis using the ring-opening polymerization of ɛ-caprolactone as a model. Using the physical adsorption technique, the AFEST loading concentration after 24 h was 152 mg AFEST per g of support. Particle size and surface morphology of the immobilized enzyme were investigated using laser scattering analysis and scanning electron microscopy. The effects of enzyme concentration, temperature, reaction time and reaction medium on monomer conversion and product molecular weight were systematically investigated. Through the optimization of reaction parameters, poly(ɛ-caprolactone) was obtained at an almost 100% monomer conversion rate and with a low average molecular weight (< 1,100 g/mol). Finally, the immobilized enzyme exhibited good operational stability, with a monomer conversion value of more than 55% after four batch reactions.

In the past two decades, enzymatic polymerization has rapidly developed and become an important polymer synthesis technique. However, the range of polymers resulting from enzymatic polymerization could be further expanded through combination with chemical methods. This review systematically introduces recent developments in the combination of lipase-catalyzed polymerization with atom transfer radical polymerization (ATRP), kinetic resolution, reversible addition–fragmentation chain transfer (RAFT), click reaction and carbene chemistry to construct polymeric materials like block, brush, comb and graft copolymers, hyperbranched and chiral polymers. Moreover, it presents a thorough and descriptive evaluation of future trends and perspectives concerning chemoenzymatic polymerization. It is expected that combining enzymatic polymerization with multiple chemical methods will be an efficient tool for producing more highly advanced polymeric materials.