Enhanced lymph node retention of subcutaneously injected IgG1-PEG2000-liposomes through pentameric IgM antibody-mediated vesicular aggregation

An efficient strategy for enhancing the lymph node deposition of rapidly drained liposomes from the interstitial injection site is described. Subcutaneously injected small-sized immuno-poly(ethyleneglycol)-liposomes (immuno-PEG-liposomes), containing 10 mol% mPEG₃₅₀-phospholipid and 1 mol% PEG₂₀₀₀-phospholipid in their bilayer and where IgG1 is coupled to the distal end of PEG₂₀₀₀, not only drain rapidly from the interstitial spaces into the initial lymphatic system, but also accumulate efficiently among the lymph nodes draining the region when compared with non-PEG-bearing immunoliposomes where IgG is directly coupled to the phospholipid. Liposome deposition among the draining lymph nodes, however, was further enhanced dramatically following an adjacent subcutaneous injection of a pentameric IgM against the surface attached IgG molecules (IgM:IgG, 10:1) without compromising vesicle drainage from the interstitium. This is suggested to arise either as a result of formation of large immuno-aggregates within the lymphatic vessels with subsequent transport to and trapping among the regional lymph nodes and/or following IgM binding to Fc receptors of the lymph node sinus macrophages forming a platform for subsequent trapping of drained IgG-coupled liposomes. This lymph node targeting approach may be amenable for the design and surface engineering of any rapidly drained nanoparticulate system bearing peptides and proteins that can be aggregated with a desired monoclonal pentameric IgM.     

曲妥珠单抗修饰的阿霉素免疫脂质体的制备及体外性质研究

目的:制备表面键合曲妥珠单抗(trastuzumab,TMAB)的阿霉素免疫脂质(Doxorubicin-loadedimmunoliposome,DOX-IML),并对其体外性质进行研究。方法:将磷脂酰胆碱、胆固醇、阿霉素、DSPE-MPEG2000以一定比例混合,采用薄膜超声分散法制备阿霉素脂质体,将聚乙二醇衍生物(1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[succinimidyl(polyethylene glycol)-3400]、DSPE-PEG3400-NHS)连接到TMAB;再与阿霉素脂质体连接得到DOX-IML。研究不同浓度的TMAB对DOX-IML入胞能力及细胞毒性的影响;测定免疫脂质体的包封率、载药率、粒径、电荷及稳定性等性质;动态透析法模拟体外释药特性,激光共聚焦观察免疫脂质体对AU565细胞抗体介导的入胞作用;MTT法研究DOX-IML抑制肿瘤细胞的生长。结果:成功制备了表面键合TMAB的阿霉素免疫脂质体,配体载入率分别是53%、75.5%、84%;每毫克DOX-IML中抗体的含量分别是37、83、108μg·mg-1;阿霉素的包封率为76.85%、载药量为8.03%;粒径131.8nm;表面电荷-27mV。抗体含量83μg·mg-1的DOX-IML组的细胞存活率最低,细胞内荧光强度最高,且该免疫脂质体稳定性良好,具有一定缓释作用。DOX-IML具有较强的特异性靶向作用,其入胞能力和细胞毒性均高于阿霉素脂质体。结论:DOX-IML具有较强的特异性靶向作用,其入胞能力和细胞毒性均高于阿霉素脂质体,抗体含量适中时其入胞能力和细胞毒性最强。     

Lipid contribution to the affinity of antigen association with specific antibodies conjugated to liposomes

Immunoliposomes, directed to clinically relevant cell-surface molecules with antibodies, antibody fragments or peptides, are used for site-specific diagnostic evaluation or delivery of therapeutic agents. We have developed intrinsically echogenic liposomes (ELIP) covalently linked to fibrin(ogen)-specific antibodies and Fab fragments for ultrasonic imaging of atherosclerotic plaques. In order to determine the effect of liposomal conjugation on the molecular dynamics of fibrinogen binding, we studied the thermodynamic characteristics of unconjugated and ELIP-conjugated antibody molecules. Utilizing radioimmunoassay and enzyme-linked immunosorbent assay protocols, binding affinities were derived from data obtained at three temperatures. The thermodynamic functions ΔH°, ΔG° and ΔS° were determined from van't Hoff plots and equations of state. The resultant functions indicated that both specific and nonspecific associations of antibody molecules with fibrinogen occurred through a variety of molecular interactions, including hydrophophic, ionic and hydrogen bonding mechanisms. ELIP conjugation of antibodies and Fab fragments introduced a characteristic change in both ΔH° and ΔS° of association, which corresponded to a variable contribution to binding by phospholipid gel-liquid crystal phase transitions. These observations suggest that a reciprocal energy transduction, affecting the strength of antibody-antigen binding, may be a singular characteristic of immunoliposomes, having utility for optimization and further development of the technology.     

VCAM-1 directed immunoliposomes selectively target tumor vasculature in vivo

Targeting the tumor vasculature and selectively modifying endothelial functions is an attractive anti-tumor strategy. We prepared polyethyleneglycol modified immunoliposomes (IL) directed against vascular cell adhesion molecule 1 (VCAM-1), a surface receptor over-expressed on tumor vessels, and investigated the liposomal targetability in vitro and in vivo. In vitro, anti-VCAM-1 liposomes displayed specific binding to activated endothelial cells under static conditions, as well as under simulated blood flow conditions. The in vivo targeting of IL was analysed in mice bearing human Colo 677 tumor xenografts 30 min and 24 h post i.v. injection. Whereas biodistribution studies using [³H]-labelled liposomes displayed only marginal higher tumor accumulation of VCAM-1 targeted versus unspecific ILs, fluorescence microscopy evaluation revealed that their localisations within tumors differed strongly. VCAM-1 targeted ILs accumulated in tumor vessels with increasing intensities from 30 min to 24 h, while control ILs accumulated in the tumor tissue by passive diffusion. ILs that accumulated in non-affected organs, mainly liver and spleen, primarily co-localised with macrophages. This is the first morphological evidence for selective in vivo targeting of tumor vessels using ILs. VCAM-directed ILs are candidate drug delivery systems for therapeutic anti-cancer approaches designed to alter endothelial function.     

PEG-immunoliposome

This review deals with the current status of newly developed pendant-type PEG-immunoliposomes (Type C), carrying monoclonal antibodies or their fragments (Fab') at the distal ends of the PEG chains. In terms of target binding of Type C, two different anatomical compartments are considered. They are mouse lung endothelium as a readily accessible site via the intravascular route and the implanted solid tumor as a much less accessible target site reached via extravasation. Small unilamellar liposomes (90–130 nm in diameter) were prepared from phosphatidycholine and cholesterol (2:1, m/m) containing 6 mol.% of DSPE-PEG-COOH or DPPE-PEG-Mal. For targeting to the vascular endothelial surface in the lung, 34A antibody, which is highly specific to mouse pulmonary endothelial cells, was conjugated to PEG-liposomes (34A-Type C). The degree of lung binding of 34A-Type C in BALB/c mouse was significantly higher than that of 34A-Type A, which is an ordinary type of immunoliposome (without PEG derivatives). For targeting to solid tumor tissue, 21B2 antibody (anti-human CEA) and its Fab' fragment were used. The targeting ability of Fab'-Type C was examined by using CEA-positive human gastric cancer strain MKN-45 cells inoculated into BALB/c nu/nu mice. Fab'-Type C showed low RES uptake and a long circulation time, and enhanced accumulation of the liposomes in the solid tumor was seen. The small Fab'-Type C predominantly passed through the leaky tumor endothelium by passive convective transport. These studies offer important insights into the potential of Type C liposomes for target-specific drug delivery.     

The influence of repeated injections on pharmacokinetics and biodistribution of different types of sterically stabilized immunoliposomes

Sterically stabilized immunoliposomes (IL) with diameters of about 135 nm carrying mouse IgG, either coupled directly to the liposome surface, or linked to the terminal ends of grafted poly(ethylene glycol) (PEG) chains by a recently described conjugation procedure (Cyanur-PEG-PE), were intravenously injected into rats and the elimination kinetics and biodistribution were determined and compared with control liposomes. The amounts of conjugated antibodies were about 30 μg/μmol total lipid for all IL. In naive rats, plain pegylated liposomes displayed the longest blood circulation time, whereas the terminal-coupled IL exhibited the fastest elimination. Liposomes containing the underivatized anchor molecules circulate nearly as long as plain pegylated liposomes, indicating that the fast elimination of the IL can be attributed to the presence of antibodies.A second injection of identical liposomes 14 days after the first injection had a considerable influence on the pharmacokinetic parameters of the liposomes. The circulation time of plain pegylated liposomes drastically dropped by half and their uptake by the liver increased concomitantly, indicating that the PEG, upon repeated injection, ceases to function as an efficient barrier reducing opsonization and/or immune reactions. The circulation time of conventional IL was moderately reduced upon a second injection, whereas that of the terminally coupled IL was nearly unaffected. These differences among the IL demonstrate that the pharmacokinetic behavior of IL is strongly dependent on the antibody conjugation site on the liposome. The observed effects of repeated injections were similar for liposomes of 90-nm diameter. The phenomena described may have important implications for the repeated application of IL as drug carriers.     

Expression and purification of two anti-CD19 single chain Fv fragments for targeting of liposomes to CD19-expressing cells

Antibody-targeted liposomal anticancer drugs combine the specificity of antibodies with large payloads of entrapped drugs. We previously showed that liposomal doxorubicin (DXR) targeted via anti-CD19 monoclonal antibodies (mAb) or their Fab' fragments against the B-cell antigen CD19 led to improved therapeutic effects in murine B-cell lymphoma models relative to non-targeted liposomal DXR. We now are examining the use of anti-CD19 single chain fragments of the antibody variable region (scFv) as a targeting moiety, to test the hypothesis that scFv have advantages over full-sized mAb or Fab' fragments. We expressed two different anti-CD19 scFv constructs, HD37-C and HD37-CCH in E. coli, and purified the scFvs using two different methods. The HD37-CCH construct was selected for coupling studies due to its relative stability and activity in comparison to HD37-C. When coupled to liposomes, the HD37-CCH scFv showed increased binding in vitro to CD19-positive Raji cells, compared to non-targeted liposomes. Cytotoxicity data showed that HD37-CCH scFv-targeted liposomes loaded with DXR were more cytotoxic than non-targeted liposomal DXR. Our results suggest that anti-CD19 scFv constructs should be explored further for their potential in treating B-lymphoid leukemias and lymphomas.