In vivo gene delivery into ocular tissues by eye drops of poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) polymeric micelles.

The primary objective of this study was to investigate the feasibility of using PEO-PPO-PEO non-ionic copolymeric micelles as a carrier for eye-drop gene delivery of plasmid DNA with lacZ gene in vivo. Using pyrene fluorescence probe methods, zeta potential, and dynamic light scattering test (DLS), the ability of micelle formation of these block copolymers with plasmid was studied. Gene expressions were visualized by both the quality of enzymatic color reaction using X-gal staining and by the quantification of the substrate chlorophenol red galactopyranoside (CPRG) in enucleated eyes on day 2 after gene transfer. In addition, microscopy to identify the types of cell showing uptake and expression of the transferred gene was used. We found that the block polymeric micelles were formed above 0.1% (w/v) of block copolymer with a size of 160 nm and a zeta potential of -4.4 mV. After 2 days of topically delivery three times a day, the most intense gene expression was observed on days 2 and 3. Reporter expression was detected around the iris, sclera, conjunctiva, and lateral rectus muscle of rabbit eyes and also in the intraocular tissues of nude mice upon in vivo topical application for 48 h with a DNA/polymeric micelle formulation. Furthermore, after two enhancement treatments, the transport mechanisms of the block copolymeric micelles were found through endocytosis in tissues by enhancement through the tight junction pathway. Thus, efficient and stable transfer of the functional gene could be achieved with PEO-PPO-PEO polymeric micelles through topical delivery in mice and rabbits. These in vivo experiments indicate the possible potential use of block copolymers for DNA transfer.     

Polyion complex micelles as vectors in gene therapy--pharmacokinetics and in vivo gene transfer

To establish non-viral gene delivery systems for intravenous administration, complexes of DNA and block copolymer consisting of poly-L-lysine and poly(ethylene glycol) were tested in in vivo turnover studies. The polyion complex micelles have self-assembling core-shell structures, yielding spherical nano-particles with small absolute values of zeta-potential. Southern blot analysis showed that supercoiled DNA was observed for 30 min and open circular or linear DNA was seen for 3 h after intravenous administration of PIC micelles having the charge ratios of 1:4 and PLL length of 48 mer. The PIC micelles with shorter PLL length showed lower stability in the blood stream suggesting that DNA is able to persist as an intact molecule in the blood stream using this system. Though having no ligands, PIC micelles with charge ratios of 1:2 and 1:4 transfected efficiently into HepG2 cells. Preincubation with free copolymer inhibited expression of the reporter gene, suggesting that adsorption of block copolymer to the cell surface blocked the interaction site of the PIC micelles. When the PIC micelles were injected via supramesenteric vein, expression of the gene was observed only in the liver and was sustained for 3 days. It was suggested that this gene delivery system is intrinsically efficient.     

Kallikrein protects against ischemic stroke by inhibiting apoptosis and inflammation and promoting angiogenesis and neurogenesis

Stroke-induced neurological deficits and mortality are often associated with timing of treatment after the onset of stroke. We showed that local delivery of the human tissue kallikrein gene into rat brain immediately after middle cerebral artery occlusion (MCAO) exerts neuroprotection. In this study, we investigated the effect of systemic delivery of the kallikrein gene 8 hr after MCAO. Expression of recombinant human tissue kallikrein after gene transfer was identified in the ischemic brain region and blood vessels. Intravenous injection of adenovirus encoding the kallikrein gene significantly reduced neurological deficit scores 2 and 7 days after gene transfer. Kallikrein gene transfer also reduced ischemia-reperfusion (I/R)-induced cerebral infarction and promoted the survival and migration of glial cells from penumbra to the ischemic core from 3 to 14 days after gene delivery. Kallikrein reduced I/R-induced apoptosis of neuronal cells and inhibited inflammatory cell accumulation in the ischemic brain. These effects were blocked by the kinin B2 receptor antagonist icatibant. In addition, kallikrein enhanced angiogenesis and promoted neurogenesis after I/R and the stimulatory effect of kinin on neuronal cell proliferation was confirmed in primary cultured neuronal cells. The protective effects of kallikrein, through the kinin B2 receptor, were accompanied by increased cerebral nitric oxide and Bcl-2 levels, Akt phosphorylation, and reduced NAD(P)H oxidase activity, superoxide production, Bax levels, and caspase-3 activity. These results indicate that delayed systemic administration of the kallikrein gene after onset of stroke protects against ischemic brain injury by inhibiting apoptosis and inflammation and by promoting angiogenesis and neurogenesis.     

黄芪甲苷对缺血性卒中小鼠神经功能保护作用及机制研究

目的 探讨黄芪甲苷（astragaloside-IV, AST-IV）对缺血性卒中（ischemic stroke）模型鼠神经功能保护作用及机制研究。方法 选取改良线栓法制备大脑中动脉梗死（middle cerebral artery occlusion,MCAO）小鼠模型,小鼠分成四组,每组8 只,分别为假手术组（Sham）、药物对照组（AST-IV）、MCAO 组和治疗组（AST-IV+MCAO）;采用改良神经功能损伤评分（modified neurological severity score,mNSS）实验和转棒（Rotarod）实验综合评价ASTIV的神经保护功能;采用ELISA 法检测AST-IV 对MCAO 脑组织核转录因子(NF-KB)、Toll 样受体4(TLR-4)、肿瘤坏死因子α(TNF-α)、胶质纤维酸性蛋白(glial fibrillary acidic protein ,GFAP)、血红素氧合酶-1(HO-1) 以及半胱天冬酶-1(caspase-1) 表达水平的影响;采用比色法检测AST-IV 对MCAO 脑组织丙二醛(MDA)、超氧化物歧化酶(SOD)、谷胱甘肽(GSH) 和活性氧（ROS）表达水平的影响。结果 MCAO组的mNSS评分（54.06±8.52）、脑组织ROS（54.06±8.52AFU）、MDA（2.40±0.33 nmol/mg）,NF-κB（304.94±39.93pg/mg）,TLR4（225.60±37.51pg/mg）,TNF-α（80.14±11.06pg/mg）,GFAP（181.24±19.88pg/mg）和caspase-1（1.76±0.20）表达均显著高于Sham 组（0.75±0.89,19.85±2.45AFU,1.43±0.29 nmol/mg,131.03±29.45 pg/mg,102.74±19.18 pg/mg,32.04±10.30 pg/mg,63.14±14.48pg/mg,1.00±0.09),差异具有统计学意义（F=38.572 ～ 184.478,均P<0.001）;MCAO 组在Rotarod 上的停留时间（74.63±18.73s）,脑组织SOD（2.88±0.49unin/mg）,GSH（3.49±0.64nmol/mg）和HO-1（105.93±24.45pg/ml）表达均显著低于Sham 组（186.50±48.10s,5.58±0.71unit/mg,6.32±0.82 pg/ml,203.11±28.65pg/ml）,差异具有统计学意义（F=37.586 ～ 78.889,均P<0.001）;AST-IV+MCAO组的mNSS评分（3.88±1.36）、脑组织ROS（24.64±14.26AFU）,MDA（2.00±0.21nmol/mg）,NF-κB（212.03±38.63 pg/mg）,TLR-4（134.81±25.60 pg/mg）,TNF-α（53.61±12.49pg/mg）,GFAP（101.00±15.04 pg/mg）和caspase-1（1.46±0.15）表达均显著低于MCAO 组,差异具有统计学意义（F=4.639 ～ 82.890, 均P<0.05）;AST-IV+MCAO 组在Rotarod 上的停留时间（160.75±33.33s）, 脑组织SOD（4.51±0.51unit/mg）,GSH（5.30±0.73nmol/mg）和HO-1（179.96±20.97pg/ml）均显著高于MCAO 组,差异具有统计学意义（F=27.681 ～ 42.364,均P<0.001）。结论 AST-IV 的脑保护作用可能与其抗炎、抗氧化应激、抑制焦亡进程有关。     

Polymeric micellar pH-sensitive drug delivery system for doxorubicin.

A novel polymeric micellar pH-sensitive system for delivery of doxorubicin (DOX) is described. Polymeric micelles were prepared by self-assembly of amphiphilic diblock copolymers in aqueous solutions. The copolymers consist of a biocompatible hydrophilic poly(ethylene oxide) (PEO) block and a hydrophobic block containing covalently bound anthracycline antibiotic DOX. The starting block copolymers poly(ethylene oxide)-block-poly(allyl glycidyl ether) (PEO-PAGE) with a very narrow molecular weight distribution (Mw/Mn ca. 1.05) were prepared by anionic ring opening polymerization using sodium salt of poly(ethylene oxide) monomethyl ether as macroinitiator and allyl glycidyl ether as functional monomer. The copolymers were covalently modified via reactive double bonds by the addition of methyl sulfanylacetate. The resulting ester subsequently reacted with hydrazine hydrate yielding polymer hydrazide. The hydrazide was coupled with DOX yielding pH-sensitive hydrazone bonds between the drug and carrier. The resulting conjugate containing ca. 3 wt.% DOX forms micelles with Rh(a)=104 nm in phosphate-buffered saline. After incubation in buffers at 37 degrees C DOX was released faster at pH 5.0 (close to pH in endosomes; 43% DOX released within 24 h) than at pH 7.4 (pH of blood plasma; 16% DOX released within 24 h). Cleavage of hydrazone bonds between DOX and carrier continues even after plateau in the DOX release from micelles incubated in aqueous solutions is reached.     

Intranasal Delivery of HMGB1 siRNA Confers Target Gene Knockdown and Robust Neuroprotection in the Postischemic Brain

Noninvasive intranasal drug administration has been noted to allow direct delivery of drugs to the brain. In the present study, the therapeutic efficacy of intranasal small interfering RNA (siRNA) delivery was investigated in the postischemic rat brain. Fluorescein isothiocyanate (FITC)-labeled control siRNA was delivered intranasally in normal adult rats using e-PAM-R, a biodegradable PAMAM dendrimer, as gene carrier. Florescence-tagged siRNA was found in the cytoplasm and processes of neurons and of glial cells in many brain regions, including the hypothalamus, amygdala, cerebral cortex, and striatum, in 1 hour after infusion, and the FITC-fluorescence was continuously detected for at least 12 hours. When siRNA for high mobility group box 1 (HMGB1), which functions as an endogenous danger molecule and aggravates inflammation, was delivered intranasally, the target gene was significantly depleted in many brain regions, including the prefrontal cortex and striatum. More importantly, intranasal delivery of HMGB1 siRNA markedly suppressed infarct volume in the postischemic rat brain (maximal reduction to 42.8 ± 5.6% at 48 hours after 60 minutes middle cerebral artery occlusion (MCAO)) and this protective effect was manifested by recoveries from neurological and behavioral deficits. These results indicate that the intranasal delivery of HMGB1 siRNA offers an efficient means of gene knockdown-mediated therapy in the ischemic brain.     

Poly(L-lysine)-g-poly(D,L-lactic-co-glycolic acid) micelles for low cytotoxic biodegradable gene delivery carriers.

Poly(lactic-co-glycolic acid) (PLGA)-grafted poly(L-lysine) (PLL) (PLL-g-PLGA) was synthesized to demonstrate its micelle-forming property in an aqueous solution. The micelles were used as a gene delivery carrier. The hydrodynamic diameter of PLL-g-PLGA micelles in an aqueous solution was ca. 149 nm with a narrow size distribution. Critical micelle concentration (cmc) was 9.6 mg/l. The PLL-g-PLGA micelles could be used to produce compact nanoparticulate complexes with plasmid DNA, which could efficiently protect the complexed DNA from enzymatic degradation by DNase I. The micelle/DNA complexes had highly compacted structure sized between 200-300 nm with a positive surface charge value. The PLL-g-PLGA micelles exhibited much higher transfection efficiency with lower cytotoxicity than PLL. Here, we demonstrated that biodegradable and cationic PLL-g-PLGA micelles could be used as an effective DNA condensation carrier for gene delivery system.     

Molecular design of biodegradable polymeric micelles for temperature-responsive drug release.

We designed thermo-responsive and biodegradable polymeric micelles for an ideal drug delivery system whose target sites are where external stimuli selectively release drugs from the polymeric micelles. The thermo-responsive micelles formed from block copolymers that were composed both of a hydrophobic block and a thermo-responsive block. Poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide) showing a lower critical solution temperature (LCST) around 40 degrees C was synthesized for the thermo-responsive block, while biodegradable poly(D,L-lactide), poly(epsilon-caprolactone), or poly(D,L-lactide-co-epsilon-caprolactone) was used for the hydrophobic block. By changing both the block lengths of the poly(D,L-lactide)-containing block copolymers, physical parameters such as micelle diameter and critical micelle concentration were varied. On the other hand, the choice of the hydrophobic block was revealed to be critical in relation to both on the thermo-responsive release of the incorporated anti-cancer drug, doxorubicin, and the temperature-dependent change of the hydrophobicity of the micelles' inner core. One polymeric micelle composition successfully exhibited rapid and thermo-responsive drug release while possessing a biodegradable character.     

Novel biomimetic polymersomes as polymer therapeutics for drug delivery.

Novel amphiphilic diblock copolymers, cholesterol-end-capped poly(2-methacryloyloxyethyl phosphorylcholine) (CMPC), which have poly(2-methacryloyloxyethyl phosphorylcholine) (poly(MPC)) as hydrophilic segment and cholesterol as hydrophobic segment, was specially designed as drug delivery systems. Fluorescence probe technique and transmission electron microscope (TEM) characterizations indicated that this novel amphiphilic copolymer formed micelles structure in water and the critical micelle concentration (CMC) was determined to be 1.57 x 10(-7) mol/l. A commercial obtained polymeric amphiphiles, Cholesterol end capped PEO (CPEO), which had a similar structure with CMPC, was used as a control in the cytotoxicity test. While CPEO showed obvious cytotoxicity, cytotoxicity of this novel amphiphiles was not observed as indicated by cell culture. Anti-cancer drug adriamycin (ADR) was incorporated into the micelles by oil-in-water method. The size of the drug-containing micelles was less than 200 nm, and the size distribution of the drug-containing micelles showed a narrow and monodisperse unimodal pattern. The release rate of ADR from the nanosphere was slow and the release continued over 7 days and the release rate decreased with the increase of molecular weights of the copolymer and the amount of the drug entrapped. These experimental results suggested that the nanoparticles prepared from CMPC block copolymers could be a good candidate for injectable drug delivery carrier.     

Heme oxygenase-1 contributes to imatinib resistance by promoting autophagy in chronic myeloid leukemia through disrupting the mTOR signaling pathway

Heme oxygenase-1 (HO-1) has been verified to play an important role in imatinib (IM)-resistant chronic myeloid leukemia (CML) cells, but the mechanism remains unclear. In drug resistant CML cells, HO-1 expression abnormally increased and that of autophagy-related protein LC-3I/II also increased, so we herein postulated HO-1 was associated with autophagy. HO-1 expressions in IM-sensitive/resistant K562/K562R cells were regulated through lentiviral mediation. K562 cells transfected with HO-1 resisted IM and underwent obvious autophagy. After HO-1 expression was silenced in K562R cells, autophagy was inhibited and the sensitivity to IM was increased. The findings were related with the inhibitory effects of high HO-1 expression on the mTOR signaling pathway that negatively regulated autophagy. High HO-1 expression promoted autophagy by inhibiting mTOR. Similar to the cell line results, mononuclear cells of IM-resistant CML patients became significantly sensitive to IM when HO-1 expression was inhibited. In summary, HO-1, which is involved in the development of chemoresistance in leukemia cells by regulating autophagy, may be a novel target for improving leukemia therapy.     

Hydrotropic polymer micelle system for delivery of paclitaxel.

Hydrotropic polymer micelle system has been developed for delivery of poorly water-soluble drugs such as paclitaxel. Hydrotropic polymers based on N,N-diethylnicotinamide were synthesized and used as a hydrophobic block for constructing amphiphilic block copolymers. The hydrotropic block copolymers self-assembled to form micelles in aqueous media. The size of the prepared polymer micelles was in the range of 30-50 nm, and increased to 100-120 nm after paclitaxel loading. The critical micelle concentrations (CMCs) of the block copolymers were higher by an order of magnitude than those of other typical polymer micelles, due to less hydrophobicity of the hydrotropic blocks. The drug loading capacity and physical stability of the polymer micelles were characterized and compared with those of other polymer micelles. The hydrotropic polymer micelles containing hydrotrope-rich cores showed not only higher loading capacity but also enhanced physical stability in aqueous media. They could be redissolved in aqueous media by simple vortexing and/or a mild heating. The hydrotropic polymer micelles provide an alternative approach for formulation of poorly soluble drugs.     

莱菔硫烷诱导胰岛系INS-1细胞血红素氧合酶1的表达

背景:莱菔硫烷可用于氧化应激相关疾病的治疗,血红素氧合酶是一种催化血红素降解的应激蛋白,已经成为预防氧化攻击的首选研究靶标之一。目的:观察核因子E2相关因子2激动剂莱菔硫烷对大鼠胰岛细胞系INS-1细胞血红素氧合酶1蛋白表达作用及细胞保护机制。方法:体外培养INS-1细胞,先用3μmol/L莱菔硫烷进行干预培养3h,再分别加入不同的胰岛素抵抗诱导剂葡萄糖氧化酶、地塞米松和葡萄糖胺刺激建立胰岛素抵抗细胞模型。结果与结论:3μmol/L莱菔硫烷处理INS-1细胞中血红素氧合酶1的表达增加(P<0.05),其效应在4h后达到高峰(P<0.05)。3μmol/L莱菔硫烷的预处理可逆转由胰岛素抵抗诱导剂导致的血红素氧合酶1表达下调(P<0.05)。而且在葡萄糖胺处理的INS-1细胞中,莱菔硫烷对血红素氧合酶1的表达改善与磷酸化PKB的表达上调具有正相关性(P<0.05,r=0.23)。结果证实,莱菔硫烷可能通过诱导核因子E2相关因子2介导的血红素氧合酶1表达,增强胰岛细胞抗氧化防御功能和抗损伤信号系统,从而达到拮抗胰岛素抵抗诱导剂对胰岛细胞的损伤作用。     

TAT peptide-based micelle system for potential active targeting of anti-cancer agents to acidic solid tumors.

A novel drug targeting system for acidic solid tumors has been developed based on ultra pH-sensitive polymer and cell penetrating TAT. The delivery system consisted of two components: 1) A polymeric micelle that has a hydrophobic core made of poly(l-lactic acid) (PLLA) and a hydrophilic shell consisting of polyethylene glycol (PEG) conjugated to TAT (TAT micelle), 2) an ultra pH-sensitive diblock copolymer of poly(methacryloyl sulfadimethoxine) (PSD) and PEG (PSD-b-PEG). The anionic PSD is complexed with cationic TAT of the micelles to achieve the final carrier, which could systemically shield the micelles and expose them at slightly acidic tumor pH. TAT micelles had particle sizes between 20 and 45 nm and their critical micelle concentrations were 3.5 mg/l to 5.5 mg/l. The TAT micelles, upon mixing with pH-sensitive PSD-b-PEG, showed a slight increase in particle size between pH 8.0 and 6.8 (60-90 nm), indicating complexation. As the pH was decreased (pH 6.6 to 6.0) two populations were observed, one that of normal TAT micelles (45 nm) and the other of aggregated hydrophobic PSD-b-PEG. Zeta potential measurements showed similar trend substantiating the shielding/deshielding process. Flow cytometry and confocal microscopy showed significantly higher uptake of TAT micelles at pH 6.6 compared to pH 7.4 indicating shielding at normal pH and deshielding at tumor pH. The confocal microscopy indicated that the TAT not only translocates into the cells but is also seen on the surface of the nucleus. These results strongly indicate that the above micelles would be able to target any hydrophobic drug near the nucleus.     

CNS gene transfer mediated by a novel controlled release system based on DNA complexes of degradable polycation PPE-EA: a comparison with polyethylenimine/DNA complexes

Nonviral gene delivery systems based upon polycation/plasmid DNA complexes are quickly gaining recognition as an alternative to viral gene vectors for their potential in avoiding immunogenicity and toxicity problems inherent in viral systems. We investigated in this study the feasibility of using a controlled release system based on DNA complexed with a recently developed polymeric gene carrier, polyaminoethyl propylene phosphate (PPE-EA), to achieve gene transfer in the brain. A unique feature of this gene delivery system is the biodegradability of PPE-EA, which can provide a sustained release of DNA at different rates depending on the charge ratio of the polymer to DNA. PPE-EA/DNA complexes, naked DNA, and DNA complexed with polyethylenimine (PEI), a nondegradable cationic polymer known to be an effective gene carrier, were injected intracisternally into the mouse cerebrospinal fluid. Transgene expression mediated by naked DNA was mainly detected in the brain stem, a region close to the injection site. With either PPE-EA or PEI as a carrier, higher levels of gene expression could be detected in the cerebral cortex, basal ganglia, and diencephalons. Transgene expression in the brain mediated by PPE-EA/DNA complexes at an N/P ratio of 2 persisted for at least 4 weeks, with a significant higher level than that produced by either naked plasmid DNA or PEI/DNA at the 4-week time point. Furthermore, PPE-EA displayed much lower toxicity in cultured neural cells as compared to PEI and did not cause detectable pathological changes in the central nervous system (CNS). The results established the potential of PPE-EA as a new and biocompatible gene carrier to achieve sustained gene expression in the CNS.     

Kupffer cells and neutrophils as paracrine regulators of the heme oxygenase-1 gene in hepatocytes after hemorrhagic shock

Heme oxygenase (HO) plays a pivotal role for the maintenance of liver blood flow and hepatocellular integrity after hemorrhagic shock. We investigated the role of Kupffer cells and neutrophils as paracrine modulators of hepatocellular HO-1 gene expression in a rat model of hemorrhage and resuscitation. Male Sprague-Dawley rats (n = 6-10/group) were anesthetized (pentobarbital, 50 mg/kg intraperitonal) and subjected to hemorrhagic shock (mean arterial blood pressure: 35 mmHg for 60 min) or a sham protocol. Based on the time course of HO-1 gene expression, the effect of various antioxidants, Kupffer cell blockade [gadolinium chloride (GdCl3); 10 mg/kg; 24 h prior to hemorrhage or dichloromethylene diphosphonate (Cl2MDP); 1 mg/kg; 2 days prior to hemorrhage], or neutrophil depletion (vinblastine, 0.5 mg/kg, 5 days prior to hemorrhage) on induction of the HO-1 gene was assessed at 5 h of resuscitation, i.e., the time point of maximal induction. Kupffer cell blockade and antioxidants abolished HO-1 mRNA and protein induction after hemorrhage, while neutrophil depletion failed to affect hepatocellular HO-1 gene expression. In addition, Kupffer cell blockade aggravated hepatocellular injury. N-formyl-methionine-leucyl-phenylalanin (fMLP) induced a substantial influx of neutrophils into the liver but failed to induce hepatocellular HO-1 mRNA expression. These data suggest that Kupffer cells but not neutrophils induce an adaptive hepatocellular stress response after hemorrhage and resuscitation. Oxygen-free radicals released by Kupffer cells may serve as paracrine regulators of a hepatocellular stress gene which is necessary to maintain liver blood flow and integrity under stress conditions.