Rapid Quantification of Polyhydroxyalkanoates Accumulated in Living Cells Based on Green Fluorescence Protein-Labeled Phasins: The qPHA Method.

Polyhydroxyalkanoates (PHAs) are microbial polyesters that have the potential to replace nonbiodegradable petroplastics. A real-time in situ PHA quantification method has long been awaited to replace the traditional method, which is time- and labor-consuming. Quantification of PHA in living cells was finally developed from fluorescence intensities generated from the green fluorescence protein (GFP) fused with the Halomonas bluephagenesis phasin proteins. Phasins PhaP1 and PhaP2 were used to fuse with GFP, which reflected PHA accumulation with an R-square of over 0.9. Also, a standard correlation was established to calculate PHA contents based on the fluorescence and cell density recorded via a microplate reader with an R-square of over 0.95 when grown on various substrates. The PhaP2-GFP containing H. bluephagenesis was applied successfully to quantify PHA synthesis in a 7.5 L fermenter with high precision. Moreover, the method was found to be feasible in non-natural PHA producers such as Escherichia coli, demonstrating its broad applicability.

Synthesis and characterization of a hyperbranched grafting copolymer PEI-g-PLeu for gene and drug co-delivery.

L-Leucine (Leu) is a hydrophobic natural amino acid and can polymerize into poly-L-Leucine (PLeu) to be an excellent biocompatible material. In this paper, a hyperbranched copolymer polyethyleneimine-g-poly-L-leucine (PEI-g-PLeu) was synthesized by ring-opening polymerization with leucine NCA as monomer and PEI as initiator, which will be used as drug and gene co-delivery system for cancer therapy. To characterize the transfection efficiency in vitro, pGL3 as the reporter gene was loaded in PEI-g-PLeu to form complexes. Doxorubicin (DOX) with cis-aconitic anhydride linker (CAD) and calf thymus DNA (as model DNA) were co-loaded in PEI-g-PLeu to obtain PEI-g-PLeu/DNA/CAD nanoparticles to measure Zeta potentials and particle sizes. Lastly, CAD and modified Bc12-shRNA(as therapeutic gene) were co-loaded in PEI-g-PLeu to get PEI-g-PLeu/CAD/DNA complexes. Our finding revealed when PEI and PLeu with the molar ratio of 1:240, and PEI-g-PLeu and DNA with the mass ratio of 1:5, PEI-g-PLeu/CAD/DNA had negligible cytotoxicity with equivalent gene transfaction efficiency compared with PEI25k. As a result, PEI-g-PLeu/CAD/DNA was a promising drug and gene co-delivery system.

Pulmonary Codelivery of Doxorubicin and siRNA by pH-Sensitive Nanoparticles for Therapy of Metastatic Lung Cancer.

A pulmonary codelivery system that can simultaneously deliver doxorubicin (DOX) and Bcl2 siRNA to the lungs provides a promising local treatment strategy for lung cancers. In this study, DOX is conjugated onto polyethylenimine (PEI) by using cis-aconitic anhydride (CA, a pH-sensitive linker) to obtain PEI-CA-DOX conjugates. The PEI-CA-DOX/siRNA complex nanoparticles are formed spontaneously via electrostatic interaction between cationic PEI-CA-DOX and anionic siRNA. The drug release experiment shows that DOX releases faster at acidic pH than at pH 7.4. Moreover, PEI-CA-DOX/Bcl2 siRNA complex nanoparticles show higher cytotoxicity and apoptosis induction in B16F10 cells than those treated with either DOX or Bcl2 siRNA alone. When the codelivery systems are directly sprayed into the lungs of B16F10 melanoma-bearing mice, the PEI-CA-DOX/Bcl2 siRNA complex nanoparticles exhibit enhanced antitumor efficacy compared with the single delivery of DOX or Bcl2 siRNA. Compared with systemic delivery, most drug and siRNA show a long-term retention in the lungs via pulmonary delivery, and a considerable number of the drug and siRNA accumulate in tumor tissues of lungs, but rarely in normal lung tissues. The PEI-CA-DOX/Bcl2 siRNA complex nanoparticles are promising for the treatment of metastatic lung cancer by pulmonary delivery with low side effects on the normal tissues.

Synthesis of a biodegradable branched copolymer mPEG-b-PLGA-g-OCol and its pH-sensitive micelle.

An amphiphilic biodegradable branched copolymer, mPEG-b-PLGA-g-OCol, was synthesized by grafting copolymer (methoxy polyethylene glycol)-b-Poly (l,d-lactic-co-glycolic acid) (mPEG-b-PLGA) on oligomeric collagen (OCol), to form a branched structure with mPEG-b-PLGA as side chain and OCol as backbone. mPEG-b-PLGA and mPEG-b-PLGA-g-OCol were both amphipathic and can self-assemble into micelles in aqueous solution. The mPEG-b-PLGA-g-OCol micelles showed pH-sensitive behaviors and the particle size below 100 nm in slightly acidic environment such as tumor tissue milieu interieur to perform passive targeting. Observed by SEM, when the solution pH increased from 5 to 9, the morphology of mPEG-b-PLGA-g-OCol micelles changed from small spheres to larger ones to rings. For biodegradable mPEG-b-PLGA-g-OCol, the micelles will gradually degrade in body. Further, doxorubicin (DOX) was effectively loaded in the micelles with drug loading and encapsulation efficiency of 3.48% and 25.8%, respectively. To evaluate antineoplastic effect of DOX-laden micelles in vitro, MTT test, flow cytometry and CLSM were performed and found that DOX-laden micelles exhibited higher cellular proliferation inhibition against HeLa cells. These features indicated that the mPEG-b-PLGA-g-OCol micelles were potential drug carrier for cancer therapy.