PEGylation of cholecystokinin prolongs its anorectic effect in rats

The anorectic compound CCK-9 was coupled to polyethylene glycol 5 kDa, 10 kDa, 20 kDa and 30 kDa, under different reaction conditions. Conjugates were purified by HPLC and characterized by MALDI-TOF MS. A 96% PEGylation yield was obtained in buffer pH 7.5 after 6h reaction at 20 degrees C. The anorectic activity was tested in vivo in rats. A single bolus intra-peritoneal injection of non-modified CCK-9 resulted in a significant initial food intake reduction 30 min after food presentation (87% compared to paired control group). When PEG-CCK-9 conjugates modified with polymers of molecular weight up to 20 kDa were injected, lower but statistically significant initial food intake reductions were obtained (76% for PEG 10 kDa-CCK-9 conjugate compared to control group). The cumulative food intake reduction of non-modified CCK-9 is normalized within 1-2h, whereas the PEG-CCK-9 molecules showed a prolonged anorectic activity lasting for 6h for PEG 5 kDa-CCK-9; 23 h for PEG 10 kDa-CCK-9 and between 8h and 23 h for PEG 20 kDa-CCK-9. For PEG 30 kDa-CCK-9 conjugate, neither an initial nor a cumulative FI reduction was observed. PEG-CCK-9 conjugates show a significantly prolonged anorectic activity in comparison to the non-modified peptide. This effect is most evident for the PEG 10 kDa-CCK-9 conjugate.     

A poly(ethylene) glycolylated peptide for ocular delivery compacts DNA into nanoparticles for gene delivery to post‐mitotic tissues in vivo

Background

We have previously shown that a novel synthetic peptide for ocular delivery (POD) can efficiently compact DNA and deliver it to cells in vitro. This observation prompted us to develop use of POD as a nonviral vector in vivo.

Methods

POD peptide was modified using poly(ethylene) glycol (PEG‐POD) and used to compact DNA into nanoparticles that were then analysed using electron microscopy, dynamic light scattering, and fluorescent labeling. Transfection efficiency and localization were determined 48 h post‐injection into the subretinal space of the mouse eye using luciferase and LacZ, respectively. Efficiency of ocular transfection was compared to two other PEGylated peptides: PEG‐TAT and PEG‐CK30.

Results

PEG‐POD can compact DNA and form discrete nanoparticles of approximately 136 nm that can penetrate and transduce the retinal pigment epithelium (RPE) in vivo. PEG‐POD significantly increased expression of plasmid DNA by 215‐fold, PEG‐TAT by 56.52‐fold, and PEG‐CK30 by 24.73‐fold relative to DNA injected alone. In all cases β‐galactosidase was observed primarily in the RPE layer after subretinal injection. Electrophysiological analyses of PEG‐POD transduced retina indicates an absence of PEG‐POD‐mediated toxicity. PEG‐POD can protect plasmid DNA from DNaseI digestion, resulting in significant transfection of the lung after intravenous injection in mice.

Conclusions

PEG‐POD was found to significantly increase gene delivery relative to both DNA alone and other pegylated peptides. These findings highlight the use of pegylated peptides, and specifically PEG‐POD, as novel gene delivery vectors. Copyright © 2009 John Wiley & Sons, Ltd.     

PEG修饰的辣根过氧化物酶及其在非水介质中的性质

酶的化学修饰可以明显提高酶在有机相中的活力。通过氧化过氧化物酶（ＨＲＰ）的糖链后引入氨基再连接甲氧基聚乙醇（ＰＥＧ）５０００和在酶的肽链上连接ＰＥＧ５０００,发现ＨＲＰ多肽链上修饰后的酶在水相中的活力几乎没有变化,但通过氧化糖链连接ＰＥＧ的酶在水相中的活力下降近２倍。在甲苯及二氧六环含量较高的体系中,修和均呈上升趋势。特别在甲苯体系中两种修饰酶活力都比未经修饰的酶提高了近２倍。稳定性研究表明,不论     

Uptake, intracellular transport, and degradation of polyethylene glycol-modified asialofetuin in hepatocytes.

Polyethylene glycol (PEG) is attached to proteins in order to increase their half-life in the circulation and reduce their immunogenicity in vivo. For many applications involving "targeting" molecules, it is important to know how PEG modification of the molecule affects its interaction with a receptor and the subsequent internalization, intracellular transport, and lysosomal degradation. As a model system, we used asialofetuin, which binds to the galactose receptor of hepatocytes, because removal of sialic acid exposes galactose residues. We modified asialofetuin by attaching various amounts of PEG of molecular weight 1900 or 5000. The preparations were labeled with 125I so that endocytosis and degradation could be followed in suspended hepatocytes. Depending on the number of PEG molecules attached, receptor-mediated uptake was affected to varying degrees. If two-thirds of the exposed amino groups of the asialofetuin molecule were modified, the rate of uptake decreased to less than one-fourth of controls; degradation of endocytosed molecules was 12% of controls. The reduction in endocytic uptake was due to a reduced rate of formation of the receptor-ligand complex. Subcellular frationation in density gradients showed that PEG-modified asialofetuin is transported intracellularly and degraded in the same manner as the native protein, but the rate of proteolysis is reduced. This observation explains the paradoxical result of experiments with injection of modified asialofetuin into rats in vivo: even though the clearance of one preparation of PEG-asialofetuin was much slower than that of the native protein, accumulation of radioactivity in the liver from the modified protein was twice as high. The hepatocytes accounted for 85% of the hepatic accumulation of either PEG-modified or native asialofetuin in vivo.     

A new tactic for the treatment of jaundice: an injectable polymer-conjugated Bilirubin oxidase

Bilirubin oxidase (BOX) derived from Myrothecium verrucaria was modified with polyethylene glycol (PEG). When the conjugated PEG-BOX was given intravenously to rats, its plasma half-life was 20 times longer than that of native BOX. In our preliminary investigations with experimentally jaundiced rats, the plasma bilirubin level dropped to normal after only one injection, and the low bilirubin level could be maintained for 12-48 hr; native BOX had a transitory suppressive effect that lasted only a few hours. The antigenicity of PEG-BOX was greatly reduced as expected. PEG-BOX appears to have potential value for the treatment of hyperbilirubinemia observed in such diseases as fulminant hepatitis and neonatal bilirubin encephalopathy.     

Site-directed polyethylene glycol modification of trichosanthin: effects on its biological activities, pharmacokinetics, and antigenicity

Trichosanthin (TCS), a type I ribosome-inactivating protein (RIP), was modified with polyethylene glycol (PEG) in order to reduce its antigenicity and prolong its half-life. Computer modeling identified three potential antigenic sites namely Q219, K173 and S7. By site-directed mutagenesis, these sites were changed into cysteine through which PEG can be covalently attached. The resulting TCS had a PEG coupled directly above one of its potential antigenic determinants, hence masking the antigenic region and prevent binding of antibodies specific to this site. In general, mutation did not bring about significant changes in ribosome-inactivating activity, cytotoxicity, and abortifacient activity of TCS. However, the in vitro activities of PEG modified (PEGylated) TCS muteins were 3-20 folds lower and the in vivo activity 50% less than that of nTCS. Pharmacokinetics study indicated that all three PEGylated TCS muteins showed 6-fold increase in mean residence time as compared to unmodified muteins. The binding affinity of an IgE monoclonal antibody (TE1) to TCS was greatly reduced after PEG modification (PEGylation) at position Q219, suggesting that TE1 recognized an epitope very near to residue Q219. PEGylated TCS muteins induced similar IgG response but 4-16 fold lower IgE response in mice compared with nTCS.     

The Administration of an α-MSH Analogue Reduces the Serum Release of IL-1α and TNFα Induced by the Injection of a Sublethal Dose of Lipopolysaccharides in the BALB/c Mouse

The injection of α-MSH or of one of its analogues ([Nle₄-D.Phe₇] α-MSH_4–10) reduced, in vivo, the release of two cytokines (IL-1α and TNFα) involved in inflammation. The inflammatory state was induced in BALB/c mice by intraperitoneal injection of a sublethal dose of lipopolysaccharides (LPS). The assay of these cytokines by ELISA showed a reduction of 20% with α-MSH and between 30 and 60% with the α-MSH analogue. The α-MSH or the analogue was administered in one of two ways: intravenously or subcutaneously. The most efficient method seemed to be the subcutaneous one because it improved the activity 10,000 times more than the intravenous method. Moreover, the analogue induced a regression of mortality in the animals treated by the intravenous method. Our results show that α-MSH and one of its analogues inhibit IL-1α and TNFα, and can be used as anti-inflammatory molecules.     

Trafficking and activation of murine natural killer cells: differing roles for IFN-gamma and IL-2

The repeated ip injection of highly purified recombinant IFN-gamma or IL-2 resulted in a local increase in peritoneal NK activity. This increase in lytic activity was paralleled by increases in the number of peritoneal leukocytes reacting with a rat monoclonal antibody directed against the NK cell-associated surface antigen LGL-1. LGL-1 reacts specifically with the majority of murine NK cells in BALB/c and C57BL/6 mice. A single injection of IFN-gamma induced more peritoneal NK activity at 24 hr than IL-2 on a protein basis. Both cytokines induced increases in the number of LGL-1+ peritoneal cells by 24 hr after injection. Simultaneous injection of suboptimal amounts of IFN-gamma (100 U) and IL-2 (10,000 U) resulted in a significant augmentation of peritoneal NK activity over that observed with either cytokine alone. Also, the peritoneal NK activity generated in response to ip injection of high doses of IL-2 (100,000 U) could be dramatically reduced by simultaneous injection of a neutralizing monoclonal antibody to IFN-gamma. Administration of IFN-gamma 1 day prior to IL-2 resulted in a significant augmentation of the NK activity above that observed with the individual cytokines. In contrast, injection of IL-2 prior to IFN-gamma did not enhance NK activity over that observed with the individual cytokines. Both cytokines must be injected ip for the complementary effects of IFN-gamma and IL-2 on peritoneal NK activity to occur. In contrast, in vitro incubation of peritoneal leukocytes with IFN-gamma resulted in neither a significant enhancement of NK lytic activity nor an increase in the number of LGL-1+ cells. In vitro treatment of peritoneal leukocytes with IL-2 always resulted in significant augmentation of NK lytic activity in the absence of any increase in the number of LGL-1+ cells. These data are consistent with the hypothesis that the local release of IFN-gamma increases peritoneal NK activity by promoting the influx of blood-borne LGL-1+ NK cells from other sites. In contrast, low doses of IL-2 augment the lytic activity of local resident NK cells, whereas high doses of this cytokine induce both an activation of local NK cells and emigration of LGL-1+ NK cells from other sites due to the endogenous generation of IFN-gamma within the peritoneal cavity. Therefore, the local release of IFN-gamma may play an important role in regulating NK cell infiltration in vivo.     

The degradation of dynorphin A in brain tissue in vivo and in vitro

The demonstration of analgesia following in vivo administration of dynorphin A (Dyn A) has been difficult. In contrast, a number of electrophysiological and behavioral effects reported with in vivo injection of Dyn A can be produced by des-tyrosine dynorphin A (Dyn A 2-17). This suggested the extremely rapid amino terminal degradation of dynorphin A. To test this hypothesis, we examined the degradation of dynorphin A following in vivo injection into the periaqueductal gray (PAG) as well as in vitro using rat brain membranes under receptor binding conditions. In vivo, we observed the rapid amino terminal cleavage of tyrosine to yield the relatively more stable destyrosine dynorphin A. This same cleavage after tyrosine was observed in vitro. Inhibition of this aminopeptidase activity in vitro was observed by the addition of dynorphin A 2-17 or dynorphin A 7-17 but not after the addition of dynorphin A 1-13, dynorphin A 1-8, dynorphin B or alpha-neo-endorphin suggesting a specific enzyme may be responsible. The detection of the behaviorally active des-tyrosine dynorphin A following in vivo injection of dynorphin A suggests that this peptide may play an important physiological role.     

Control of hyperuricemia in subjects with refractory gout, and induction of antibody against poly(ethylene glycol) (PEG), in a phase I trial of subcutaneous PEGylated urate oxidase

PEG-modified recombinant mammalian urate oxidase (PEG-uricase) is being developed as a treatment for patients with chronic gout who are intolerant of, or refractory to, available therapy for controlling hyperuricemia. In an open-label phase I trial, single subcutaneous injections of PEG-uricase (4 to 24 mg) were administered to 13 such subjects (11 had tophaceous gout), whose plasma uric acid concentration (pUAc) was 11.3 ± 2.1 mg/dl (mean ± SD). By day seven after injection of PEG-uricase, pUAc had declined by an average of 7.9 mg/dl and had normalized in 11 subjects, whose mean pUAc decreased to 2.8 ± 2.2 mg/dl. At doses of 8, 12, and 24 mg, the mean pUAc at 21 days after injection remained no more than 6 mg/dl. In eight subjects, plasma uricase activity was still measurable at 21 days after injection (half-life 10.5 to 19.9 days). In the other five subjects, plasma uricase activity could not be detected beyond ten days after injection; this was associated with the appearance of relatively low-titer IgM and IgG antibodies against PEG-uricase. Unexpectedly, these antibodies were directed against PEG itself rather than the uricase protein. Three PEG antibody-positive subjects had injection-site reactions at 8 to 9 days after injection. Gout flares in six subjects were the only other significant adverse reactions, and PEG-uricase was otherwise well tolerated. A prolonged circulating life and the ability to normalize plasma uric acid in markedly hyperuricemic subjects suggest that PEG-uricase could be effective in depleting expanded tissue stores of uric acid in subjects with chronic or tophaceous gout. The development of anti-PEG antibodies, which may limit efficacy in some patients, is contrary to the general assumption that PEG is non-immunogenic. PEG immunogenicity deserves further investigation, because it has potential implications for other PEGylated therapeutic agents in clinical use.     

Modification of batroxobin with activated polyethylene glycol: reduction of binding ability towards anti-batroxobin antibody and retention of defibrinogenation activity in circulation of preimmunized dogs

Amino groups of batroxobin (Bothrops atrox thrombic protease) were modified with 2,4-bis(O-methoxypolyethylene glycol)-6-chloro-s-triazine (activated PEG2). The modified batroxobin had the reduced binding ability towards anti-batroxobin antibody but retained its enzymic activity in vitro and in vivo. Administration of modified batroxobin in which 29% of the total amino groups in the molecule had been modified, to beagle dogs preimmunized with native batroxobin gave rise to a marked reduction of the fibrinogen level in plasma, accompanied with an increased level of fibrinogen (fibrin) degradation products, FDP. On the other hand, no reduction of fibrinogen level was observed when native batroxobin instead of modified batroxobin was injected to immunized dogs.     

Control of hyperuricemia in subjects with refractory gout, and induction of antibody against poly(ethylene glycol) (PEG), in a phase I trial of subcutaneous PEGylated urate oxidase

PEG-modified recombinant mammalian urate oxidase (PEG-uricase) is being developed as a treatment for patients with chronic gout who are intolerant of, or refractory to, available therapy for controlling hyperuricemia. In an open-label phase I trial, single subcutaneous injections of PEG-uricase (4 to 24 mg) were administered to 13 such subjects (11 had tophaceous gout), whose plasma uric acid concentration (pUAc) was 11.3 +/- 2.1 mg/dl (mean +/- SD). By day seven after injection of PEG-uricase, pUAc had declined by an average of 7.9 mg/dl and had normalized in 11 subjects, whose mean pUAc decreased to 2.8 +/- 2.2 mg/dl. At doses of 8, 12, and 24 mg, the mean pUAc at 21 days after injection remained no more than 6 mg/dl. In eight subjects, plasma uricase activity was still measurable at 21 days after injection (half-life 10.5 to 19.9 days). In the other five subjects, plasma uricase activity could not be detected beyond ten days after injection; this was associated with the appearance of relatively low-titer IgM and IgG antibodies against PEG-uricase. Unexpectedly, these antibodies were directed against PEG itself rather than the uricase protein. Three PEG antibody-positive subjects had injection-site reactions at 8 to 9 days after injection. Gout flares in six subjects were the only other significant adverse reactions, and PEG-uricase was otherwise well tolerated. A prolonged circulating life and the ability to normalize plasma uric acid in markedly hyperuricemic subjects suggest that PEG-uricase could be effective in depleting expanded tissue stores of uric acid in subjects with chronic or tophaceous gout. The development of anti-PEG antibodies, which may limit efficacy in some patients, is contrary to the general assumption that PEG is non-immunogenic. PEG immunogenicity deserves further investigation, because it has potential implications for other PEGylated therapeutic agents in clinical use.     

Large and prolonged in vivo response of pancreatic secretion to phenylethylamide and phenylethylester derivatives of Boc-[Nle-Nle]CCK(26–33) in the rat

Supramaximal doses of cholecystokinin (CCK) induce in vitro submaximal biological responses (i.e., smaller by 50% than the response to a maximal dose of CCK), desensitization and residual stimulation, and in vivo secretory inhibition and edematous pancreatitis. It has been reported previously that supramaximal doses of Boc-[Nle²⁸-Nle³¹]CCK(27–32)/-phenylethylester (JMV180) do not produce these effects. The aim of this study was to analyze the in vivo response of pancreatic secretion of the rat to a wide dose range of Boc-[Nle²⁸-Nle³¹]CCK(26–33) (JMV118), an analog of CCK8 with the same activity spectrum as CCK8, to JMV180 and to Boc-[Nle²⁸-Nle³¹]CCK(27–32)-phenylethylamide (JMV170). The three peptides were administered as intravenous infusions and as bolus intravenous injections. In the case of infusions, the same maximal effect was observed with all three peptides. It was obtained with 22.5 pmol/kg · min of JMV118; JMV180 and JMV170 were about 700 times less potent. In the case of bolus injections, the maximal response to JMV118 was observed with 450 pmol/kg, and the response peaked 10–15 min after the injection. Higher doses of JMV118 induced a secretory peak that was smaller and delayed relative to the moment of injection. JMV180 and JMV170 were about 500 times less potent: the maximal response was observed with 218700 pmol/kg and peaked 10–15 min after the injection. Larger doses of JMV180 and JMV170 produced neither supramaximal inhibition nor a delayed peak response, but induced a sustained stimulation of pancreatic secretion that could last more than 3 h after the injection. These data indicate that single large doses of JMV180 and JMV170 can produce a large and long-lasting stimulation of pancreatic secretion in vivo, a goal that cannot be reached with JMV118 or CCK8.     

Behavioral recovery from traumatic brain injury after membrane reconstruction using polyethylene glycol

Polyethylene glycol (PEG; 2000 MW, 30% by volume) has been shown to mechanically repair damaged cellular membranes and reduce secondary axotomy after traumatic brain and spinal cord injury (TBI and SCI respectively). This repair is achieved following spontaneous reassembly of cell membranes made possible by the action of targeted hydrophilic polymers which first seal the compromised portion of the plasmalemma, and secondarily, allow the lipidic core of the compromised membranes to resolve into each other. Here we compared PEG-treated to untreated rats using a computer-managed open-field behavioral test subsequent to a standardized brain injury. Animals were evaluated after a 2-, 4-, and 6-hour delay in treatment after TBI. Treated animals receive a single subcutaneous injection of PEG. When treated within 2 hours of the injury, injured PEG-treated rats showed statistically significant improvement in their exploratory behavior recorded in the activity box when compared to untreated but brain-injured controls. A delay of 4 hours reduced this level of achievement, but a statistically significant improvement due to PEG injection was still clearly evident in most outcome measures compared at the various evaluation times. A further delay of 2 more hours, however, eradicated the beneficial effects of PEG injection as revealed using this behavioral assessment. Thus, there appears to be a critical window of time in which PEG administration after TBI can provide neuroprotection resulting in an enhanced functional recovery. As is often seen in clinically applied acute treatments for trauma, the earlier the intervention can be applied, the better the outcome.     

A sensitive,specific radioisotope assay for l-glutamine-d-fructose-6-phosphate aminotransferase

A sensitive and specific radioassay for l-glutamine-d-fructose-6-phosphate aminotransferase (EC 5.3.1.19) activity is presented. Picomoles of product are measurable, and the assay can be applied to systems having limited quantities of available protein, particularly in extracts of either cell or organ cultures. The assay is at least 10,000 times more sensitive under K₁ concentrations of fructose 6-phosphate than the modified Elson-Morgan colorimetric assay and 20 times more sensitive under saturating conditions of fructose 6-phosphate. As little as 0.5 μg of cell-extract protein will yield measurable product. In contrast, 280 μg of crudeextract protein from colon is required with the modified Elson-Morgan colorimetric assay.     

Polyethylene glycol conjugation enhances the nitrite reductase activity of native and cross-linked hemoglobin

Although stabilized hemoglobins have been evaluated as oxygen-carrying replacements for red cells in transfusions, in vivo evaluations have noted that these materials are associated with vasoactivity, a serious complication. Scavenging of endogenous nitric oxide by the deoxyheme sites of the stabilized proteins is one likely source of vasoactivity. Recent reports indicate that modification of cell-free hemoglobin derivatives with multiple chains of polyethylene glycol (PEG) suppresses vasoactivity. Gladwin and co-workers observed that the nitrite reductase activity of hemoglobin serves as a major endogenous source of nitric oxide. If PEG conjugation leads to enhanced nitrite reductase activity, this could compensate for scavenged endogenous nitric oxide. To test this possibility, the rates of conversion of nitrite ion to nitric oxide by altered hemoglobins with and without PEG were measured at 25 degrees C. Fumaryl (alpha99-alpha99) cross-linked hemoglobin reacts with nitrite with a bimolecular rate constant of 0.52 M (-1) s (-1), which is comparable to that associated with native hemoglobin (0.25 M (-1) s (-1)). Addition of PEG chains to the cross-linked hemoglobin at beta-Cys93 (alphaalpha-Hb-PEG5K 2) results in a material that produces nitric oxide much more rapidly ( k = 1.41 M (-1) s (-1)). R-State-stabilized hemoglobins with multiple PEG chains (Hb-PEG5K 2 and Hb-PEG5K 6) react 10 times faster with nitrite to produce nitric oxide than does native hemoglobin ( k = 2.5 and 2.4 M (-1) s (-1), respectively). These results, showing enhanced production of nitric oxide resulting from an increased proportion of the protein residing in the R-state, are consistent with the decrease in vasoactivity associated with PEG conjugation.     

Phospholipid synthesis in rat liver endoplasmic reticulum after the administration of phenobarbitone and 20-methylcholanthrene

1. Phenobarbitone injection did not affect the concentration of phospholipids in the liver endoplasmic reticulum, but it increased the rate of incorporation of [(32)P]orthophosphate into the phospholipids. 20-Methylcholanthrene caused a transient increase in total phospholipid but a decrease in the turnover rate of the phospholipids. 2. Incorporation of [(32)P]orthophosphate into phosphatidylcholine, compared with that into phosphatidylethanolamine, was increased by phenobarbitone injection but decreased by 20-methylcholanthrene injection. 3. The activity of S-adenosylmethionine-phosphatidylethanolamine methyltransferase increased 12h after phenobarbitone injection, when incorporation of [(32)P]orthophosphate into phosphatidylcholine was a maximum, but at other times, and after 20-methylcholanthrene injection, the activity of the enzyme did not correlate with the rate of phosphatidylcholine synthesis. 4. [(14)C]Glycerol was incorporated more rapidly into phosphatidylcholine than into phosphatidylethanolamine, whereas [(32)P]orthophosphate and [(14)C]ethanolamine were incorporated more rapidly into phosphatidylethanolamine than into phosphatidylcholine. 5. Incorporation of [(32)P]orthophosphate into phosphatidylethanolamine of liver slices incubated in vitro was much more rapid than into phosphatidylcholine, and incorporation into phosphatidylcholine was markedly stimulated by addition of methionine to the medium. Changes in the incorporation of [(32)P]orthophosphate into phospholipids observed in vivo after injection of phenobarbitone or methylcholanthrene could not be reproduced in slices incubated in vitro. 6. It is concluded that phenobarbitone injection causes an increased rate of turnover of total phospholipids in the endoplasmic reticulum and an increased conversion of phosphatidylethanolamine into phosphatidylcholine, whereas 20-methylcholanthrene injection depresses both the turnover rate of total phospholipids and the formation of phosphatidylcholine.     

Regulation of epithelial cell morphology and functions approaching to more in vivo-like by modifying polyethylene glycol on polysulfone membranes

Cytocompatibility is critically important in design of biomaterials for application in tissue engineering. However, the currently well-accepted "cytocompatible" biomaterials are those which promote cells to sustain good attachment/spreading. The cells on such materials usually lack the self-assembled cell morphology and high cell functions as in vivo. In our view, biomaterials that can promote the ability of cells to self-assemble and demonstrate cell-specific functions would be cytocompatible. This paper examined the interaction of polyethylene glycol (PEG) modified polysulfone (PSf) membranes with four epithelial cell types (primary liver cells, a liver tumor cell line, and two renal tubular cell lines). Our results show that PSf membranes modified with proper PEG promoted the aggregation of both liver and renal cells, but the liver cells more easily formed aggregates than the renal tubular cells. The culture on PEG-modified PSf membranes also enhanced cell-specific functions. In particular, the cells cultured on F127 membranes with the proper PEG content mimicked the in vivo ultrastructure of liver cells or renal tubules cells and displayed the highest cell functions. Gene expression data for adhesion proteins suggest that the PEG modification impaired cell-membrane interactions and increased cell-cell interactions, thus facilitating cell self-assembly. In conclusion, PEG-modified membrane could be a cytocompatible material which regulates the morphology and functions of epithelial cells in mimicking cell performance in vivo.     

PEG-g-poly(GdDTPA-co-L-cystine): effect of PEG chain length on in vivo contrast enhancement in MRI

Biodegradable macromolecular Gd(III) complexes, Gd-DTPA cystine copolymers (GDCP), were grafted with PEG of different sizes to modify the physicochemical properties and in vivo MRI contrast enhancement of the agents and to study the effect of PEG chain length on these properties. Three new PEG-grafted biodegradable macromolecular gadolinium(III) complexes were synthesized and characterized as blood pool MRI contrast agents. One of three different lengths of MPEG-NH(2) (MW = 550, 1000, and 2000) was grafted to the backbone of GDCP to yield PEG(n)()-g-poly(GdDTPA-co-l-cystine), PEG(n)()-GDCP. The PEG chain length did not dramatically alter the T(1) relaxivity, r(1), of the modified agents. The MRI enhancement profile of PEG(n)()-GDCP with different PEG sizes was significantly different in mice with respect to both signal intensity and clearance profiles. PEG(2000)-GDCP showed more prominent enhancement in the blood pool for a longer period of time than either PEG(1000)-GDCP or PEG(550)-GDCP. In the kidney, PEG(2000)-GDCP had less enhancement at 2 min than PEG(1000)-GDCP, but both PEG(550)-GDCP and PEG(1000)-GDCP showed a more pronounced signal decay thereafter. The three agents behaved similarly in the liver, as compared to that in the heart. All three agents showed little enhancement in the muscle. Chemical grafting with PEG of different chain lengths is an effective approach to modify the physiochemistry and in vivo contrast enhancement dynamics of the biodegradable macromolecular contrast agents. The novel agents are promising for further clinical development for cardiovascular and cancer MR imaging.     

Experimental study on the treatment of intracerebral glioma xenograft with human cytokine-induced killer cells

Objective. To investigate the phenotype changes and proliferation activities of cytokine-induced killer cells (CIKs) and lymphokine-activated killer cells (LAKs) from healthy donor, and the cytotoxicities of CIKs and LAKs to human in vitro glioma cell lines U251 and U87. Therapy of CIK intratumoral injection was evaluated in nude mouse models. Methods. CIK cells were induced from peripheral blood mononuclear cells (PBMC) of healthy donors with multiple cytokines. Phenotype analysis of CIKs and LAKs was performed with flow cytometer (FCM). The specific cytotoxicities of CIKs and LAKs against cell line U251 and U87 were determined by LDH method. After intracerebral injection of CIKs, the distribution of CIKs and the inflammatory reaction of their surrounding brain tissue were observed through continuous pathological sections. In vivo anti-tumor activity of CIKs was evaluated in athymic nude mice with intracerebral xenotransplanted U251 glioma by MRI. Results. Amount of CIKs was increased (49.83+/-2.04) times and double positive cells, CD3(+)/CD56(+) cells, were increased from (3.36+/-1.85%) to (44.07+/-14.14%) with elevated absolute amount over 1000 times after 2 week culture. In vitro experiments demonstrated that compared with LAK, CIKs possessed more obvious cytotoxic activity to U251 and U87. In vivo experiments showed that there was no severe inflammatory reaction in brain tissue. CIKs can markedly inhibit intracranial xenotransplanted glioma growth by intracranial injection (P<0.01). Conclusion. CIKs are a kind of highly effective immune cells which have a strong suppressive effect on growth for in vitro and in vivo glioma. Local injection of CIKs does not produce severe damage to normal brain tissue and is likely to be used in clinical adoptive immunotherapy of intracerebral glioma.