End functionalized polymeric system derived from pyrrolidine provide high transfection efficiency

Chemical architecture and functionality play an important role in the physico-chemical properties of cationic polymers with applications as gene vectors. In this study, linear homopolymers of N-ethyl pyrrolidine methacrylamide (EPA), copolymers of EPA with N,N-dimethylacrylamide (DMA) and oligomers of EPA were synthesized, and the resulting structures were evaluated for their transfection efficiency as non-viral gene vectors. Specifically, polymer species with high and low molecular weights (120-2.6 kDa) and different functionalities (tertiary amines as side chains and primary amine as chain end) were prepared as non-crosslinked, linear homopolymers, copolymers and oligomers, respectively. Polymer/DNA complexes (polyplexes) formation was evaluated by agarose gel electrophoresis, showing that all systems complexed with DNA in all P/N ratios with the exception of the EPA homopolymer. Furthermore, light scattering measurements and transmission electronic microscopy (TEM) showed different size (50-450 nm) and morphology depending on the composition and concentration of the polyplex systems. Cell viability and proliferation after contact with polymer and polyplexes were studied using 3T3 fibroblasts, and the systems showed an excellent biocompatibility at 2 and 4 days. Transfection studies were performed with plasmid Gaussian luciferase kit and were found that the highest transfection efficiency in serum free was obtained with oligomers from the P/N ratio of 1/6 to 1/10. Transfection values of the functionalized oligomers with respect to the control linear poly (dimethylaminoethyl methacrylate) [poly (DMAEMA)] are very interesting in the presence of serum. Haemolysis for these polymers values below 1%, which provide attractive potential applications in gene therapy with these non-toxic readsorbable polymers.     

Synthetic modifications of the immunomodulating peptide thymopentin to confer anti-mycobacterial activity

Effective global control of tuberculosis (TB) is increasingly threatened by the convergence of multidrug-resistant TB and the human immunodeficiency virus (HIV) infection. TB/HIV coinfections exert a tremendous burden on the host's immune system, and this has prompted the clinical use of immunomodulators to enhance host defences as an alternative therapeutic strategy. In this study, we modified the clinically used synthetic immunomodulatory pentapeptide, thymopentin (TP-5, RKDVY), with six arginine residues (RR-6, RRRRRR) at the N- and C-termini to obtain the cationic peptides, RR-11 (RKDVYRRRRRR-NH₂) and RY-11 (RRRRRRRKDVY-NH₂), respectively. The arginine residues conferred anti-mycobacterial activity to TP-5 in the peptides as shown by effective minimum inhibitory concentrations of 125 mg/L and killing efficiencies of >99.99% against both rifampicin-susceptible and -resistant Mycobacterium smegmatis. The immunomodulatory action of the peptides remained unaffected as shown by their ability to stimulate TNF-α production in RAW 264.7 mouse macrophage cells. A distinct change in surface morphology after peptide treatment was observed in scanning electron micrographs, while confocal microscopy and dye leakage studies suggested bacterial membrane disruption by the modified peptides. The modified peptides were non-toxic and did not cause hemolysis of rat red blood cells up to a concentration of 2000 mg/L. Moreover, RY-11 showed synergism with rifampicin and reduced the effective concentration of rifampicin, while preventing the induction of rifampicin resistance. The synthetic peptides may have a potential application in both immunocompetent and immunocompromised TB patients.     

Adenoviral vectors coated with cationic PEG derivatives for intravaginal vaccination against HIV-1

Mucus layer coating the vaginal epithelium represents a barrier for intravaginally delivered recombined adenoviral (rAd) vectors, but it could be overcome by proper polyethylene glycol (PEG) modification. Here we synthesized two cationic PEG derivatives, amino-(EO)_n/(AGE)_m-Cyss (APCs). The polymers contained neutral linear PEG (2–5 kDa) to provide a hydrophilic surface and amine pendants to provide positive charge for coating negatively charged rAd by physical adsorption. Given proper molecular composition, the polymer (5k-APC) could coat rAd without causing aggregation, facilitating its mucus penetrating ability and enhancing gene expression both in vitro and in vivo. With HIVgag as the model antigen, the polymer-rAd complexes were administered intravaginally to elicit both systemic and mucosal immune responses. 5k-APC-rAd immunization elicited robust HIVgag-specific cellular responses and also induced higher antigen-specific serum IgG. More importantly, mice immunized with 5k-APC-rAd showed higher level of IgA in vaginal lavage fluid. These findings suggest that 5k-APC-rAd is a promising system for intravaginal immunization against infectious diseases such as HIV within the vaginal tract.     

The use of cationic MPEG-PCL-g-PEI micelles for co-delivery of Msurvivin T34A gene and doxorubicin

In our previous study, a series of triblock copolymers based on MPEG-PCL-g-PEI were successfully synthesized, and the physicochemical properties of their self-assembled micelles were also investigated. Here, a further evaluation of these micelles was carried out, including in vitro drug release behavior, body distribution as well as blood compatibility. The developed MPEG-PCL-g-PEI micelles was labeled with ⁹⁹Tc for tracing the body distribution of micelles after i.v. injection, and the results showed that the MPEG-PCL-g-PEI micelles mainly concentrated in the tumor tissue. Meanwhile, the anti-tumor activity on both B16F10 subcutaneous tumor model and lung metastasis model was tested and the results indicated that DOX-loaded micelles could significantly inhibit tumor growth as compared with free doxorubicin, which was accompanied by significantly increased apoptosis of tumor cells. By introduction of gene Msurvivin T34A in combination with chemotherapies in the treatment of lung metastasis tumor, it could greatly reduce systemic toxicity as well as improved the anti-tumor efficiency. These results demonstrated that it is possible to use cationic MPEG-PCL-g-PEI micelles for effectively co-delivering functional gene and chemotherapeutic agent, and thus improving anti-tumor effect and systemic toxicity.     

Multifunctional cationic polymer decorated and drug intercalated layered silicate (NLS) for early gastric cancer prevention

A multifunctional compound that can prevent early gastric cancer is produced by intercalating 3.20% and 1.64% of 5-FU into the interlayer of montmorillonite (MMT) and attapulgite (At), respectively. A low molecular weight cationic polymer, polyethylenimine (PEI1200), is incorporated into the surface of the 5-FU-MMT and 5-FU-At to form the multifunctional layered silicate (NLS). The chemical structure and surface morphology of the NLS are characterized and the model drug of 5-FU is intercalated into the MMT and At. The cell viability determined by the MTT assay on the BGC-823 cell lines show that over 80% of the cells are live under the experimental conditions. The PEI-5-FU-MMT and PEI-5-FU-At can carry the report gene to the BGC-823 and COS-7 cell lines efficiently. Western blotting assay shows that the pTrail protein of the BGC-823 cell lines treated with PEI-5-FU-MMT/pTrail and PEI-5-FU-At/pTrail is up-regulated, whereas the cFLIP protein is down-regulated at 48 h compared to free 5-FU, PEI1200, MMT, and At, providing evidence that the NLS can increase the sensitivity of pTrail gene and improve the effects of pTrail gene therapy. Moreover, the Helicobacter pylori (HP) bacteria are adsorbed and immobilized efficiently on the surface of the NLS according to the LIVE/DEAD^® BacLight™ Bacterial Viability Kit in the confocal fluorescence analysis. The histochemical analyses provide evidence that NLS/pTrail can prevent early gastric mucosa effectively.     

Molecular recognition and stability of Tc-UBI 29–41 based on experimental and semiempirical results

^99mTc-UBI 29–41 is an antimicrobial peptide fragment that directly radiolabeled with ^99mTc shows high in vitro and in vivo stability, rapid background clearance, minimal accumulation in non-target tissues and rapid detection of infection sites. Molecular mechanics (MM) calculation has been an essential tool in explaining experimental results associated with molecular recognition and stability. This work is an attempt to explain the ^99mTc-UBI 29–41 specificity for bacteria and to understand from a structural point of view, the experimental results indicative of a molecular recognition and stability not well favored for two other cationic peptides (^99mTc-Tat-1-Scr and ^99mTc-Tat-2-Scr ) used as control. Structures of ^99mTc-UBI, ^99mTc-Tat-1-Scr, ^99mTc-Tat-2-Scr and of the corresponding free cationic peptides were built and the optimized structures, in the best stable configurations, were calculated by a MM procedure. In order to correlate the calculated and experimental results, in vitro stability tests with cysteine challenge and stability to dilution in human serum and in saline solution, were performed for the three labeled cationic peptides. The three complexes can be represented by the general formula [Tc(V)(O)(H₂O)₂(Lys_n=1,2-Arg_n=0,1-peptide)]^10+,11+. The potential energies were 104.5, 95.6 and 90.8 kcal/mol for ^99mTc-Tat-1-Scr, ^99mTc-Tat-2-Scr and ^99mTc-UBI 29–41, respectively. Experimental and calculated results were in good agreement. It is thus possible to predict and explain that in similar solution media ^99mTc-Tat-2-Scr would be more stable than ^99mTc-Tat-1-Scr and why ^99mTc-UBI shows the highest stability. In conclusion, the in vitro specific binding to bacteria and the accumulation at infection sites in humans of ^99mTc-labeled UBI could be the result of its high thermodynamic stability, selectivity and stereospecificity.     

Reducing the renal uptake of radiolabeled antibody fragments and peptides for diagnosis and therapy: present status, future prospects and limitations

Elevated renal uptake and prolonged retention of radiolabeled antibody fragments and peptides is a problem in the therapeutic application of such agents. Over recent years, one of the focuses of research has therefore been to develop suitable methods to reduce this renal uptake, and to evaluate whether the resulting methodology will benefit therapy with antibody fragments and peptides. In these studies it has been shown that the kidney uptake of antibody fragments in animals can be reduced in a dose-dependent manner by almost one order of magnitude by the systemic administration of cationic amino acids and their derivatives, whereas the uptake in all other organs, as well as the tumor, remains unaffected. A similar reduction in renal retention is achieved for all intracellularly retained radionuclides (e.g., radiometals) or radioiodinated immunoconjugates, as well as for smaller peptides. Lysine is usually the preferred agent, and its d- and l-isomers are equally effective whether given intraperitoneally or orally. Amino sugars are effective, but their N-acetyl derivatives, lacking the positive charge, are not. Basic polypeptides are also effective, and their potency increases with increasing molecular weight (i.e., the amount of positive charges per molecule). Urine analysis of treated individuals shows the excretion of unmetabolized, intact fragments or peptides, in contrast to mostly low-molecular-weight metabolites in untreated controls. In therapy studies using radiometal-conjugated Fab fragments, the kidney is the first dose-limiting organ. Administration of cationic amino acids results in a substantial increase in the maximum tolerated dose of such Fab fragments. No biochemical or histological evidence of renal damage has been observed under these conditions. As was the case in animal studies, in pilot clinical trials the renal uptake in patients injected with Fab′ fragments and given amino acids could be decreased significantly, whereas the uptake by all other organs remained unaffected. These recent studies indicate that a variety of basic compounds are capable of inhibiting the tubular reabsorption of peptides and proteins, thus significantly lowering the renal uptake of antibody fragments or peptides in both animals and patients. On a molecular basis, the effect seems to rely essentially on the presence of a positively charged amino group. Thus, radiation nephrotoxicity of antibody fragments and peptides can be overcome successfully; this may provide new prospects for cancer therapy with radiolabeled antibody fragments and peptides.     

大豆甾醇糖苷及两亲性聚乙二醇对阳离子脂质体细胞转染与膜各向异性的影响

目的：考察不同脂质体材料对阳离子脂质体细胞转染率和脂质体膜流动性的影响。方站：合成美国FDA认可应用于人体给药的阳离子材料3β-[N-(N’，N’-二甲基胺乙基)胺基甲酰基]胆固醇(3β[N-(N’，N’-dimethy-laminoethane)carbamoyl]cholesterl，DC-chol)；以荧光素钠(fluorescein sodium，SF)为荧光标记物，二棕榈酰磷脂酰胆碱(dipalmibylphosphatidylcholine，DPPC)、二油酰磷脂酰乙醇胺(dioleoylphosphatidylethanolamine，DOPE)和胆固醇(cholesterol，ch)为辅佐膜材，分别制备DPPC／ch／DC-chol阳离子脂质体、DOPE/ch／DC-chol阳离子脂质体及大豆甾醇糖苷(soybean-derlved sterylglucoside，SG)或聚乙二醇-二硬脂酰磷脂酰乙醇胺(polyethylene glycol-dis-tearoylphosphatidylethanolamine，PEG-DSPE)表面修饰的阳离子脂质体，并制备DPPC/ch组成的中性脂质体。以HepG2 2．2．15为细胞模型观察不同脂质体处方对细胞转染率的影响；通过荧光偏振法考察不同脂质材料对脂质体膜流动性的影响。结果：DC-chol能显著增加脂质体对SF的包封率(从0．64％到的74．84％)、细胞转染率(从1．83％到32．29％)及脂质体膜的流动性。在阳离子脂质体处方中加入SG能显著提高阳离子脂质体的包封率(从52．60％到63．37％)、细胞转染率(从20．18％到47．48％)及脂质体膜的流动性；PEG-DSPE可促进阳离子脂质体的细胞转染(从20．18％到28．17％)。结论：DC-chol和SG能显著提高脂质体的细胞转染率和脂质体膜的流动性；PEG-DSPE能促进阳离子脂质体的细胞转染。     

支气管哮喘患者血清特异性IgE与ECP的关系

本文对174例支气管哮喘患者进行血清特异性IgE（Phadiatop法）和其中的46例同时作了血清嗜酸性阳离子蛋白（ECP）水平的检测。结果表明，支气管哮喘患者中有67．7％是由吸入性变应原所引起，与IgE介导的变态反应有关，血清特异性IgE水平与血清ECP小平是显著的线性正相关，说明ECP水平在一定程度上反映了吸入型（外源性）哮喘的变态反应水平，可对支气管哮喘的诊断分型提供帮助。     

Electrical charge of a protein determines penetration and localization in hyaline articular cartilage

Summary Sufficient antigen retention in joint structures is a prerequisite for sustained antigen-induced arthritis. In this in vitro study we investigated the retention of native and charge-modified bovine serum albumin (BSA) with patellar cartilage of different species (mouse, rat, rabbit, and man). Association of BSA with cartilage due to charge of the protein with that due to immune complex formation was compared. Using radiolabeled proteins we showed quantitatively that the retention of highly positively charged BSA is considerably higher (200–500 times) than retention of the anionic BSA in all cartilage species examined. Mouse, rat, and human cartilage bind more protein per mg dry weight, compared to rabbit cartilage. No clear-cut relation was found with the glycosaminoglycans (GAG) contents. Retention of native BSA by anti-BSA antibodies was low in the dense hyaline patellar cartilage in all species. Enhanced immune-complex formation was found in marginal regions of rabbit patellar cartilage, consisting of fibrous-like cartilage. Localization studies by autoradiography showed that cationic BSA penetrates deeply into the cartilage matrix. Even in a thick cartilage specimen, such as rabbit cartilage, very deep penetration into the calcified zone was demonstrated. This study indicates that binding and deep penetration of cationized protein in cartilage is not restricted to mouse specimen, as had been found previously, but is a general phenomenon.