Long-living liposomes as potential drug carriers

Neutral, unilamellar liposomes (vesicles) composed of a dialkyl analog of phosphatidylcholine and cholesterol, and containing ¹⁴C-maltose as entrapped marker, were administered intravenously to mice. After one and two days, radioactivity in blood and liver remained 3–4 times higher than after administration of liposomes of egg (diester) phosphatidylcholine and cholesterol. It appears that the vesicles were taken into liver cells by endocytosis, and that phospholipases are involved in the capture as well as in the breakdown of conventional liposomes. Liposomes that are semi-resistant to catabolic enzymes may become useful in the manipulation of drug delivery.     

Lung mechanics and blood gases in pregnant guinea pigs

Lung resistance and compliance were measured in early pregnant, late pregnant and non pregnant guinea pigs. Lung resistance was increased, lung compliance was decreased in pregnancy. A new finding was the increase of chest wall compliance in the course of pregnancy. In order to obtain a sufficient gas exchange during artificial ventilation, pregnant animals have to be ventilated with a higher frequency or a higher pressure than non pregnant controls.     

Study of quercetin-loaded liposomes as potential drug carriers: in vitro evaluation of human complement activation

Liposomes have been employed as potential drug carriers. However, after their in vivo administration, they can be destabilized by proteins of complement system, contributing to the clearance of vesicles from blood circulation. Antioxidant flavonoids such as quercetin have been reported to be beneficial to human health, but their low water solubility and bioavailability limit their enteric administration. Therefore, the development of appropriate flavonoid-carriers could be of great importance to drug therapy. The aim of the present study was to evaluate the activation of human complement system proteins by liposomes composed of soya phosphatidylcholine (SPC) and cholesterol (CHOL) or cholesteryl ethyl ether (CHOL-OET) loaded with quercetin or not. The consumption of complement, via classical (CP) and alternative (AP) pathways, by different vesicles was evaluated using a hemolytic assay and quantitative determination of iC3b and natural antibodies deposited on empty liposomal surfaces by ELISA. The main results showed that empty liposomes composed of large amounts of CHOL consumed more complement components than the others for both CP and AP. Furthermore, replacement of CHOL with CHOL-OET reduced complement consumption via both CP and AP. Incorporation of quercetin did not change CP and AP consumption. Deposition of iC3b, IgG and IgM in vesicles composed of SPC:CHOL-OET at a molar ratio of 1.5:1 was lower compared to the others. Taken together, these observations suggest that liposomes composed of SPC:CHOL-OET at a molar ratio of 1.5:1 are the most appropriate among the vesicles studied herein to be used as a drug carrier system in further investigations.     

Small liposomes are better than large liposomes for specific drug delivery in vitro

We have compared drug transfer into target cells in vitro from liposomes of different sizes. Liposomes of mean diameter 800 Å, 2000 Å or 4000 Å, containing the folate analogue, methotrexate, and the fluorophore, carboxyfluorescein, were covalently coupled to Staphylococcus aureus protein A. Cells of the murine k haplotype were preincubated with an anti-H-2K^k monoclonal antibody. Excess antibody was removed and then cells were incubated with liposomes. The number of cell-bound liposomes was determined by fluorimetry. The drug effect was assayed by the methotrexate-mediated inhibition of radiolabeled deoxyuridine uptake. The drug effect was more important in the case of the 800 Å vesicles than for the larger liposomes, despite the fact that the quantity of drug bound to cells was several-fold greater for large liposomes than for small ones. Since fusion is excluded by the non-proportionality of drug binding and drug effect, the predominant manner of liposome entry seems to be endocytosis. At least for these in vitro studies, the endocytosis by target cells of small liposomes seems to be more efficient than that of large liposomes.     

"Smart" drug carriers: PEGylated TATp-modified pH-sensitive liposomes

To engineer drug carriers capable of spontaneous accumulation in tumors and ischemic areas via the enhanced permeability and retention (EPR) effect and further penetration and drug delivery inside tumor or ischemic cells via the action of the cell-penetrating peptide (CPP), we have prepared liposomes simultaneously bearing on their surface CPP (TAT peptide, TATp) moieties and protective PEG chains. PEG chains were incorporated into the liposome membrane via the PEG-attached phosphatidylethanolamine (PE) residue with PEG and PE being conjugated with the lowered pH-degradable hydrazone bond (PEG-HZ-PE). Under normal conditions, liposome-grafted PEG "shielded" liposome-attached TATp moieties since the PEG spacer for TATp attachment (PEG(1000)) was shorter than protective PEG(2000). PEGylated liposomes are expected to accumulate in targets via the EPR effect, but inside the "acidified" tumor or ischemic tissues lose their PEG coating due to the lowered pH-induced hydrolysis of HZ and penetrate inside cells via the now-exposed TATp moieties. This concept is shown here to work in cell cultures in vitro as well as in ischemic cardiac tissues in the Langendorff perfused rat heart model and in tumors in experimental mice in vivo.     

Use of liposomes as carriers for radiation synovectomy

Using [^99mTc]pertechnetate as an aqueous space marker, the permeability of liposomes composed of seven different mixtures of distearoylphosphatidylcholine (DSPC) and sphingomyelin (SM) was determined. Liposomes containing 20–33% SM were the least permeable in the presence of rheumatoid synovial fluid. Following injection of ^99mTc-containing liposomes into the knee joints of rabbits, retention of ^99mTc in the knee was more than 200 times greater than following injection of nonencapsulated [^99mTc]pertechnetate. The knee clearance biologic half time of ^99mTe with DSPC/SM (4:1) liposomes was 64 h. Most of the activity that had leaked from the knee was not found in extra-articular tissues, suggesting rapid excretion. When DSPC/SM (4:1) liposomes were labeled with ¹¹¹In(oxine), a knee clearance biologic half time of greater than 1200 h was observed.     

Liposomes as drug carriers.

The use of liposomes as drug carrying vehicles are summarized. Introduction of smaller doses, increased permeability, reduced toxicological and allergic effects and altered immunological responses are the advantages of the method. Experimental techniques for the preparation of drug containing liposomes and for their deliveries are surveyed. Proposed mechanisms for the incorporation of liposomes in cells are discussed. Examples for $\text{in vivo}$ applications and for functional liposomes capable of “homing-in” to given targets are presented.     

Characterization of diacetylenic liposomes as carriers for oral vaccines

In order to evaluate liposomes as vehicle for oral vaccines the characterization and stability of polymerized and non-polymerized liposomes were examined. Mixtures of 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3 phosphocholine) (DC8,9PC) with saturated 1,2-dimiristoyl-sn-glycero-3-phosphocholine in molar ratio 1:1 were used. Saturated and non-saturated lipids were combined to give a chemically modified membrane by UV polymerization derived from DC8,9PC. Characterization was carried out by electronic microscopy, differential scanning calorimetry (DSC) and by hydrophobicity factor (HF). The stability towards the digestive tract (including saliva): acidic solutions, bile and pancreatin are compared to buffer pH 7.4, measuring the release of Glucose-6-phosphate or bovine plasma albumin entrapment. The polymerized liposomes showed further augmentation of the HF and the size. DSC showed phase separation and lower Tt if compared to data obtained for DC8,9PC. The HF, as main factor is discussed in relation to in vitro stability, suggesting that polymerized and non-polymerized liposomes would serve effectively as an oral delivery vehicle.     

Antigen-nonspecific and specific suppression of lymphokine production in desensitized guinea pigs

Doubly immunized guinea pigs may be desensitized with respect to delayed hypersensitivity reactions against both antigens (anergy) by injection of large doses of either one. This anergic response therefore has both a specific and nonspecific component. The specific component of desensitization persists longer than the nonspecific one. In the present study, we have explored the mechanism of both antigen-specific and antigen-nonspecific suppression during the later stages of desensitization. Guinea pigs immunized with two antigens, DNP-KLH and DNP-EA, were desensitized with DNP-EA. The lymph node cells obtained from the animals 1 day after desensitization were unable to produce MIF in the presence of either antigen. The cells obtained 3, 5, and 7 days after desensitization were able to generate MIF when stimulated with the non-specific antigen (DNP-KLH), but not with specific antigen (DNP-EA). It was shown that both T- and non-T-cell fractions obtained 1 day after desensitization had the capacity to antigen-nonspecifically suppress MIF production. In contrast, if the cells were obtained 3 or 5 days after desensitization, T cells could inhibit only the antigen-specific production of MIF, while non-T cells were still capable of suppressing antigen-specific and nonspecific MIF production. Interestingly, when these two populations were mixed back again, it was now only suppressive to the specific antigen-induced MIF production. This latter observation indicates that nonspecific suppressor non-T cells may themselves be regulated by suppressor T cells. Furthermore, antigen-specific suppressor T cells were shown to produce soluble factor(s) which inhibited the production of MIF.     

Active specific immunotherapy of guinea pigs with visceral tumor implants

Summary Guinea pigs, each with established, 7-day-old, syngeneic visceral micrometastases of line 10 tumor implanted intravenously, were immunized by intradermal inoculations into several sites of a mixture of irradiated line 10 cells and an emulsion containing heat-killed BCG or Mycobacterium phlei bacilli. This treatment led to survival of 72 of 80 treated animals (90%). Therapeutic effectiveness depended on the dose of mycobacteria and on that of irradiated tumor cells. Animals treated by intradermal injection of mycobacteria attached to oil droplets alone or with irradiated tumor cells alone, all died with multiple foci of pulmonary tumor.     

Liposomes as drug carriers in leishmaniasis and malaria

Experimental studies suggest that liposomes could substantially improve the performance of anti-leishmanial drugs in the chemotherapy of visceral leishmaniasis. In this article, Carl Alving discusses the potential for overcoming resistance to antimonial drugs; for 'buffering' the toxicity of drugs, and for drug delivery under conditions where hospitalization is impossible or inconvenient. Liposomes can also be used experimentally to reduce the toxicity and increase the efficacy of parenterally-administered primaquine in the treatment of sporozoite-induced murine malaria.     

Characterization and in vitro evaluation of starch based hydrogels as carriers for colon specific drug delivery systems

A series of starch/methacrylic acid (MAAc) copolymer hydrogels of different compositions were synthesized using γ-rays induced polymerization and crosslinking. The effects of the preparation conditions such as the feed solution concentration, feed solution composition and irradiation dose on the gelation process of the synthesized copolymer were investigated. The swelling behavior of the starch/methacrylic acid (MAAc) copolymer hydrogels was characterized by studying the effect of the hydrogel composition on the time- and pH-dependent swelling. Swelling kinetics showed that the synthesized hydrogels possessed Fickian diffusion behavior at pH 1 and non-Fickian diffusion at pH 7 which recommend them as good candidate for colon specific drug delivery systems. The synthesized hydrogels were loaded with ketoprofen as a model drug to investigate the release behavior of the synthesized hydrogels. The results showed the ability of the hydrogels to keep the loaded drug at pH 1 and release it at pH 7. The data also showed that the release rate can be controlled by controlling the preparation conditions such as comonomer concentration and composition and irradiation dose.     

阳离子脂质体作为基因治疗药物载体在小鼠体内的毒性研究

目的:探讨阳离子脂质体作为基因治疗药物载体在小鼠体内的毒性。方法:采用流体动力学法,经尾静脉给小鼠注射不同剂量的阳离子脂质体,对照组注射等量5%GLU液。于注射前、注射后第1天和第3天,经眶静脉采血,用自动血球计数仪测定外周抗凝血中的白细胞(WBC)、淋巴细胞(LY%)、中性粒细胞(GR%)。用自动生化分析仪测定血浆中的清蛋白(Alb)、谷丙转氨酶(ALT)、尿素氮(BUN)、肌酐(CR)。同时做脏器组织切片HE染色,观察肝、肾脏组织结构的变化。结果:与对照组比较,12μl/g剂量以上组的WBC、BUN、CR均明显升高(P<0.05),ALT、Alb无显著性差异(P>0.05)。12μl/g剂量以下组无明显变化(P均>0.05)。与注射前比较,注射后第1天,WBC、BUN、CR显著升高(P<0.05),而注射后第3天除CR外均基本恢复正常(P>0.05)。而ALT、Alb无显著性差异(P>0.05)。肝肾脏组织切片HE染色,各荆量组间组织结构变化无差异。结论:阳离子脂质体对小鼠外周血相和肾脏功能的毒性作用呈剂量和时间依赖性,故使用其作为核酸药物载体用于基因治疗研究时,剂量应小于12ul/g体重,给药时间应隔1天以上。     

Antigen-presenting liposomes are effective in treatment of recurrent herpes simplex virus genitalis in guinea pigs.

The therapeutic and immunologic effects of a liposome preparation containing both a macrophage activator, muramyl-tripeptide-phosphatidylethanolamine, and a recombinant antigen, glycoprotein D of herpes simplex virus type 1, have been investigated. This preparation was tested in vitro for the ability to stimulate peripheral blood lymphocytes and in vivo for the control of recurrent herpes genitalis in guinea pigs. Our results show that the liposome-antigen-adjuvant preparation is capable of enhancing antigen-specific lymphocyte stimulation, which may be related to the observed 75% suppression of the frequency and severity of reactivation of recurrent herpes simplex virus type 2 genitalis compared with that of placebo controls.