A novel polymer-lipid hybrid nanoparticle for efficient nonviral gene delivery.

AIM: To develop a novel non-viral vector with high transfection efficiency and low cytotoxicity. METHODS: Poly (ethylene glycol)-distearoylphosphatidylethanolamine (PEG-DSPE) was incorporated into polymer-lipid hybrid nanoparticles (PLN) to construct a PEG-DSPE modified long circulating PLN (L-PLN). The L-PLN was prepared by the emulsifying-solvent evaporation method, L-PLN and L-PLN/DNA complexes were characterized. Both HEK293 and MDA-MB-231 cells transfected by L-PLN/DNA complexes were observed under a fluorescence microscope. The transfection efficiency of the complexes to HEK293 cells was further evaluated by flow cytometry. RESULTS: The GFP fluorescence intensity in HEK293 cells transfected by the L-PLN/DNA complexes (N/P=10) was about 37.2%, which was higher than those transfected by PLN alone or commercial Lipofectamine 2000. The L-PLN exhibited minimal toxicity at a low N/P ratio compared with other vectors. CONCLUSION: L-PLN as a novel gene delivery system, has higher transfection efficiency and acceptable cytotoxicity compared to the corresponding PLN, which is beneficial for the development of non-viral gene transfer vectors and may offer an alternative strategy for the future gene therapy.

[Influences of two different endolymphatic infusion ways to cochlear morphology and function in guinea pigs].

OBJECTIVE: To explore the influence of two different endolymphatic infusion ways on cochlear morphology and function. METHODS: Forty healthy pigment guinea pigs (250 - 350 g) with normal Prey's reflex were divided into A and B group with 20 animals respectively. The right ears served as treated ears and the left ones as control ones. In group A, 5 microl of adenovirus 5-enhanced green fluorescence protein (Ad5-EGFP) suspension was infused into the scala media through an opened cochleostomy on the lateral wall of the scala media (LWS). In group B, the same volume of Ad5-EGFP suspension was infused into the scala media through punctured round window membrane and the basilar membrane (RBM). Cochlear morphology was examined under scan electric microscope and phalloidin staining was used to observe the hair cells in the infused ears after the animals were sacrificed. Auditory brainstem thresholds of the ears of all the animals were measured before and after treatment. RESULTS: All the animals recovered well after operation. The holes on the lateral wall of the scala media and punctures on the round window membrane were healed completely. EGFP labeling appeared in the organ of Corti and lining wall of the stria vascularis indicated that adenovirus suspension was injected into the scala media using LWS (succeed in 14 animals accounted for 70%) and RBM (in 8 animals accounted for 40%) ways. Viruses were inoculated into the scala media with only locally inflammation reaction. In group A the hearing threshold decreased significantly in the treated ears compared with the control ears after the operation [(33.1+/-10.3) dB, (9.4+/-3.9) dB, F=46.34, P=0.0005]. However, in group B there was no significantly different between the treated ears and the control ears after the operation [(2.5+/-3.8) dB, (2.5+/-3.8) dB, F=0.00, P=1.000]. Phalloidin staining indicated that in group A the extension of hair cells loss was bigger than in group B. In some animals of two groups, EGFP labeling appeared in the extra-lymphatic system indicating that some of the injected suspension leaked out of the scala media. CONCLUSIONS: Ad5-EGFP could be infused into the scala media through LWS or RBM and adenovirus could infect the lining cells of scala media and supporting cells in the basal membrane successfully without causing immunoreaction in the whole cochlea. LWS caused more hair cell loss and hearing loss than RBM. However, the cochlear morphology could be recovered completely after surgery. The positive inoculation rate was relatively higher that through LWS than that through RBM.

[Cultivation, identification and ultrastructural observation of the proliferative cells from the newborn rat cochlea].

OBJECTIVE: To explore whether there could be proliferative cells in the cochlea of the newborn rat or not and what kinds of cells should be differentiated from the proliferative cells while to study the effect of the growth factors on the proliferative cells and the ultrastructure of the proliferative cells. METHODS: The Corti's organ were dissected from the cochlea of newborn SD rats and cultured. The proliferative condition of cells was tested by infusing the 5-bromo-2-deoxyuridine (BrdU) into the culture medium. And the variety of the spheres and differentiated cells were identified by immunohistochemistry. Corti's organ from forty-eight surface preparations was randomly divided into 4 groups: control group; epidermal growth factor (EGF) group; basic fibroblast growth factor (bFGF) group and EGF + bFGF group, with each group including 12 Corti's organ, and then the number of cell spheres of each Corti's organ was counted. The data was statistically analysed with ANOVE. Finally, the proliferative cells were observed under scanning electron microscope and transmission electron microscope. RESULTS: (1) The cell spheres can be observed in the cell culture of the Corti's organ. In present experiment, 90.1% of cells in spheres were labeled by BrdU, while nestin of spheres, the marker of hair cells--myosin 7A, espin, and phalloidin of the differentiated cells were positive. The marker of neuron-microfilament-M was also positive, and some differentiated cells were labeled by myosin 7A and BrdU, espin and BrdU, NF-M and BrdU at the same time. (2) The average number (x +/- s) of spheres from single Corti's organ was: 45.3 +/- 23.00 in control group, 86.2 +/- 34.1 in EGF group, 96.5 +/- 33.6 in bFGF group and 131.2 +/- 47.00 in EGF + bFGF group. There were significant differences between other groups respectively (P < 0.05) but there was no significant differences between EGF group and bFGF group (P > 0.05). (3) Scanning electron microscopy and transmission electron microscopy showed that cells of the spheres were round and had the same size and many short and thin microvilli on the surface of these cells. The cytoplasm were rich of organellae such as endoplasmic reticulum, mitochondrion, and cytoskeleton such as microfilament, microtube, et al. Tight junction, desmosomes and gap junctions between two adjacent cells were seen. CONCLUSIONS: The proliferative cells are observed in the cochlea of the newborn rats and proliferative cells could differentiated into hair cells with bundles-like structure and neuron. Both EGF and bFGF possess the promoting effects for proliferation on the proliferative cells while the proliferative cells have characters of earlier immature cells.

[Apoptosis of olfactory receptor neurons induced by bulbectomy].

OBJECTIVE: To study whether apoptosis plays a role in controlling the number of olfactory receptor neurons, so as to reveal the specialty and mystery of neurogenesis. METHODS: Using terminal deoxynucleotidyl transferase-mediated dUTP-fluorescein nick end labeling (TUNEL) and transmission electron microscopy to detect apoptosis in olfactory mucosa of normal adult rats and damaged olfactory mucosa of 16, 32, 48 hours and 3, 7, 30 days after bulbectomy. RESULTS: In normal olfactory epithelium, a subpopulation of immature neurons, as well as mature neurons, showed internucleosomal DNA-fragmentation. The number of TUNEL-labeled neurons increased dramatically 32 hours after removal of olfactory bulb. Then it declined quickly and remained at low level. Ultrastructural data of olfactory mucosa showed that the feature of apoptotic neurons was chromatin condensation and cell shrinkage. Besides, some dying cells were characterized by the formation of numerous autophagic vacuoles, and few had some of the features of necrosis but without obvious mitochondrial swelling. CONCLUSIONS: Apoptosis might play a role in turnover of the olfactory epithelium and regeneration in adult rats. There might be other two types of neural death through different mechanism.