Amino Acid-Linked Low Molecular Weight Polyethylenimine for Improved Gene Delivery and Biocompatibility.

The construction of efficient and low toxic non-viral gene delivery vectors is of great significance for gene therapy. Herein, two novel polycations were constructed via Michael addition from low molecular weight polyethylenimine (PEI) 600 Da and amino acid-containing linkages. Lysine and histidine were introduced for the purpose of improved DNA binding and pH buffering capacity, respectively. The ester bonds afforded the polymer biodegradability, which was confirmed by the gel permeation chromatography (GPC) measurement. The polymers could well condense DNA into nanoparticles and protect DNA from degradation by nuclease. Compared with PEI 25 kDa, these polymers showed higher transfection efficiency, lower toxicity, and better serum tolerance. Study of this mechanism revealed that the polyplexes enter the cells mainly through caveolae-mediated endocytosis pathway; this, together with their biodegradability, facilitates the internalization of polyplexes and the release of DNA. The results reveal that the amino acid-linked low molecular weight PEI polymers could serve as promising candidates for non-viral gene delivery.

Cationic Heteropolymers with Various Functional Groups as Efficient and Biocompatible Nonviral Gene Vectors.

With the rise and development of gene therapy, it is of great significance to develop highly efficient and biocompatible polymeric gene carriers. In this work, a series of heteropolymers from ring-opening polymerization of diepoxide compounds and various functional primary amines were synthesized. The feed dosage of amines was adjusted to obtain the polymers with different functional group contents, and the structure-activity relationships of these polymers as nonviral gene vectors were examined in detail. Results revealed that, although the amine with the fluorinated chain seemed to be less reactive in the polymerization, the relative content of each component in the target product was consistent with the feed dosage. Compared to the "golden standard" polyethylenimine (PEI) 25 kDa, these heteropolymers showed much lower cytotoxicity and higher gene transfection efficiency, especially in serum-containing medium, and up to 78 times of efficiency than PEI was obtained. Meanwhile, they exhibited much better serum resistance than PEI, compared with the transfection efficiency in serum-free experiments, and even higher efficiency could be achieved with serum in HeLa cells. Mechanism study results suggest that the content of fluorinated chain and histamine might distinctly influence their transfection. The fluorinated chains could enhance the serum tolerance and cellular uptake efficiency (with serum), while the imidazole group in the histamine chain would improve the endosome/lysosome escape.

ROS-responsive fluorinated polycations as non-viral gene vectors.

Polycation carriers hold great potential in gene therapy. However, they usually suffer from obvious cytotoxicity and unsatisfactory transfection efficiency. In this report, a series of fluorobenzene substituted and thioacetal contained polycations (TAEA-S-xF) were prepared to explore novel alternatives for safe and efficient non-viral polymeric gene vectors. The reactive oxygen species (ROS)-responsive property of thioacetal moieties together with the fluorine effect were hope to bring the vector better performance in gene delivery process. These materials could efficiently condense DNA into nanoparticles with proper size and surface potential. The structure-activity relationship of these materials was systematically investigated, and the In vitro transfection results revealed that the amount of fluorine atoms on the linkage plays important role to ensure the transfection efficiency and serum tolerance. The ROS-responsive behavior was verified by NMR, gel electrophoresis experiment and dynamic light scattering (DLS) assay. Cytotoxicity assay results also suggest that these ROS-degradable polycations show good biocompatibility in response to higher ROS level in cancer cells. Among these fluorinated polymers, the one with the most fluorine atoms showed the best transfection efficiency, which was up to 54 times higher than polyethyleneimine (PEI) 25 kDa. Mechanism studies reveal that its better performance may come from good cellular uptake and endosome escape ability.

Low molecular weight PEI-based fluorinated polymers for efficient gene delivery.

Fluorinated biomaterials have been reported to have promising features as non-viral gene carriers. In this study, a series of fluorinated polymeric gene carriers were synthesized via Michael addition from low molecular weight polyethyleneimine (PEI) and fluorobenzoic acids (FBAs)-based linking compounds with different numbers of fluorine atoms. The structure-activity relationship (SAR) of these materials was systematically investigated. SAR studies showed that fluorine could screen the positive charge of these polymers. However, this shielding effect of fluorine would endow fluorinated polymers with good balance between DNA condensation and release. In vitro transfection results suggested that these fluorinated polymers could mediate efficient gene delivery. Flow cytometry and confocal microscopy studies demonstrated that more efficient cell uptake could be achieved by fluorinated materials with more fluorine atoms. Cytotoxicity assays showed that these fluorinated materials exhibited very low cytotoxicity even at high mass ratios. This study demonstrates that FBA-based fluorinated biopolymers have the potential for practical application.

Synthesis and Properties of Low-Molecular-Weight PEI-Based Lipopolymers for Delivery of DNA.

Rapid enzymatic degradation and fragmentation during DNA administration can result in limited gene expression, and consequently, poor efficacy. It is necessary to use novel vectors for DNA delivery. Herein, we aimed to design useful carriers for enhancing transfection efficiency (TE). These lipopolymers were prepared through Michael addition reactions from low-molecular-weight (LMW) polyethyleneimine (PEI) and linkers with three kinds of steroids. Agarose gel electrophoresis assay results displayed that the three lipopolymers could condense plasmid DNA well, and the formed polyplexes had appropriate sizes around 200⁻300 nm, and zeta potentials of about +25⁻40 mV. The results of in vitro experiments using HeLa, HEK293, and MCF-7 cells showed that these lipopolymers present higher TE than 25-kDa PEI, both in the absence and presence of 10% serum. Flow cytometry and confocal microscopy studies also demonstrated that these lipopolymer/DNA complexes present higher cellular uptake and intracellular distribution. The measurement of critical micelle concentration (CMC) revealed that these lipopolymers could form micelles, which are suited for drug delivery. All results suggest that the three materials may serve as hopeful candidates for gene and drug delivery in future in vivo applications.

Biodegradable Gene Carriers Containing Rigid Aromatic Linkage with Enhanced DNA Binding and Cell Uptake.

The linking and modification of low molecular weight cationic polymers (oligomers) has become an attracted strategy to construct non-viral gene carriers with good transfection efficiency and much reduced cytotoxicity. In this study, PEI 600 Da was linked by biodegradable bridges containing rigid aromatic rings. The introduction of aromatic rings enhanced the DNA-binding ability of the target polymers and also improved the stability of the formed polymer/DNA complexes. The biodegradable property and resulted DNA release were verified by enzyme stimulated gel electrophoresis experiment. These materials have lower molecular weights compared to PEI 25 kDa, but exhibited higher transfection efficiency, especially in the presence of serum. Flow cytometry and confocal laser scanning microscopy results indicate that the polymers with aromatic rings could induce higher cellular uptake. This strategy for the construction of non-viral gene vectors may be applied as an efficient and promising method for gene delivery.

[A histological study of three-dimensional external zygomatic suture distraction osteogenesis].

OBJECTIVE: To explore the histological change in suture of zygomatic bone for the zygomatic suture direct distraction osteogenesis. METHODS: The zygomatic bone was distracted by 3-D external distraction appliance without osteotomy. The specimens were taken 1, 3, 5 and 8 weeks after, and then examined histologically and compared with the blank contralateral side. RESULTS: There were lots of fibroblasts, osteoblasts and capillary vessels in the distracted suture tissues one week after distraction, and the fibers were observed to connect the sides of suture and arranged orderly. The surfaces of the expanded suture were irregular. Bone formation was active in the expanded side. The bone trabeculae were mature and oriented in the direction of distraction in the distracted sides at 3 weeks. A great amount of new woven bones were found in 5-week specimen. New bones were formed completely 8 weeks after the distraction. CONCLUSIONS: New bone formed rapidly in the distracted side of zygomatic bone under suture distraction osteogenesis without osteotomy.

[An experimental study of 3-dimension zygomatic suture extension for suture osteogenesis].

OBJECTIVE: To explore a methods of 3-Dimension expansion of zygomatic suture in a goat model. METHODS: Seven goats were used in this study. The 3-D extensive applicator was designed and used to extend the zygomatic suture of the goats by placing it in the zygomatic bone through an infraorbital incision. Ten days after the first operation, it was gradually extended on a speed of 0.09 cm/d for 7 days. The zygomatic movement and the osteogenesis of the suture was evaluated in two weeks. RESULTS: The zygomatic bone was extended for 0.6 cm long in average, and the osteogenesis was also shown significantly in the suture. CONCLUSION: The above mentioned technique could be a safe and effect method to be applied for the zygomatic extension.