Liposome polymer-modified

Yuba E, Kojima C, Sakaguchi N, Harada A, Koiwai K, Kono K (2008) Gene delivery to dendritic cells mediated by complexes of lipoplexes and pH-sensitive fusogenic polymer-modified liposomes. J Control Release 130 77-83... 

Kono, K. (2001), Thermosensitive polymer-modified liposomes, Adv. Drug Deliv. Rev., 53,307-319. 

Kono K, Henmi A, Takagishi T. Temperature-controlled interaction of thermosensitive polymer-modified cationic liposomes with negatively charged phospholipid membranes. Biochim Biophys Acta 1999 1421 183-197. 

Research on oral liposomal delivery systems has moved forward with the development of polymer-modified liposomes. For example, targeted PEGylated liposomes furnished with folic acid for oral delivery were promising, showing enhanced permeability of dextran (used as a marker) across Caco-2 cell monolayers (Anderson et al., 1999). PEG and chitosan-coated lipid nanoparticles were constmcted as oral delivery systems for salmon calcitonin (sCT). The PEG-coated nanoparticles did not alter the transepithelial electrical resistance of Caco-2 cell monolayers, while the chitosan-coated nanoparticles showed a dose-dependent increase in the permeability of dextran across the monolayers (Garcia-Fuentes et al., 2005). It demonstrated that the favourable interaction of the chitosan-coated nanoparticles with intestinal mucosa, together with their permeation enhancing characteristics, could improve the oral absorption of sCT. 

Kono K, Nakai R, Morimoto K et al. Thermosensitive polymer-modified liposomes that release contents around physiological temperature. Biochim Biophys Acta 1999 1416 239-250. 

TEMPERATURE-CONTROL OF INTERACTIONS OF THERMOSENSrriVE POLYMER MODIFIED LIPOSOMES WITH MODEL MEMBRANES AND CELLS... 

Figure I. Schematic illustration of temperature-controlled interaction of ihcrmosensitive polymer-modified liposomes with a cell. The interaction is suppressed by highly hydrated polymer chains below the LCST (left), but enhanced by dehydrated polymer chains and/or exposed bare liposome surface (right).

POLYMER-MODIFIED CATIONIC LIPOSOMES WITH MODEL MEMBRANES... 

However, its size increased significantly above 50°C, which is near the LCST ofpoly(APr). Because the diameter of the same cationic liposome did not change in the absence of the anionic liposomes in this temperature region, this result shows that the polymer-modified cationic liposomes associated with the anionic liposomes above the LCST of the polymer. Similarly, an intensive increase in diameter is seen in the case of copoly(APr-NDDAM)-modified cationic liposome above 50°C (Fig.4B). 

It has been thought that membrane fusion plays an important role in cationic liposome-mediated transfection DNA is transferred into Qdoplasm via direct fusion between cationic hposomes and plasma membrane and/or fusion between cationic liposomes and endosomal membrane after being taken up by the cell through endocytosis. Thus, the effect of temperature on fusion between the polymer-modified cationic liposomes with the anionic liposomes was examined. Liposome fusion was detected by the change in resonance energy transfer efficiency from N-(7-nitrobenz-2-oxa-l,3-diazol-4-yl)phosphatidylethanolamine (NBD-PE) to lissamine rhodamine B-sulfonyl phosphatidylethanolamine (Rh-PE) due to dilution of these fluorescent lipids in the liposomal membrane. We have already shown that the fluorescence intensity ratio of NBD to Rh (R) is useful to follow membrane fusion. ... 

Figure 5. A Time-courses of fusion of copoly(APr-NDDAM)-modified DC-Qiol/DOPE (1 I, mol/mol) liposomes with EYPC/PA (3 1, mol/mol) liposomes at 30 ( ), 50 (A), 55 ( ), and 60°C (T). B Fusion of copoly(APr-NDDAM)-modified ( ), poly(APr)-2C 2-modified (A), and unmodified ( ) DC-Chol/DOPE (1 1, mol/mol) liposomes with EYPC/PA (3 1, mol/mol) liposomes as a function of temperature. Ordinate AR) represents the inerease in R for the initial 3 min after addition of EYPC/PA liposomes. Polymer/lipid weight ratios of poly(APr)-2C 2-modified and copoly(APr-NDDAM)-modified liposomes were 0.78 and 0.65, respectively.

We examined two types of polymer-modified cationic liposomes, namely poly(APr)-2C 12-modified and copoly(APr-NDDAM)-modified cationic liposomes. While both types of liposomes exhibited similar temperature-dependence in their interactions with the anionic liposomes, the latter seems to achieve more efficient control of the interaction. As described above, in the case of the copoly (APr-NDDAM)-modified cationic liposome, the whole polymer chain might exist near surface of the liposome and, hence, this liposome has the polymer coat with higher chain density than the poly(APr)-2Ci2-modified liposome. In addition, conformational freedom of the copoly(APr-NDDAM) chain is restricted more severely than the poly(APr)-2C 2 chain, because the former is fixed hy the anchors at plural points. Such differences in density and mobility of the tethered polymer chain might result in the difference in their interactions with the anionic liposomes. 

INTERACTION OF THERMOSENSITIVE POLYMER-MODIFIED EYPC LIPOSOMES WITH CVl CELLS... 

Since the tethered polymer chain changes its character between hydrophilic and hydrophobic near the LCST of the polymer, the polymer-modified liposomes are expected to reveal its affinity to cell surfaces differently, depending on temperature. We prepared EYPC Uposomes modified with copoly(APr-NIPAM)-2C 12, which exhibits the LCST near the physiological temperature. Temperature-dependent interaction of the thermosensitive polymer-modified liposomes with CVl cells, an Afriean green monkey kidney cell line, is described. 

The influence of incubation temperature on liposome uptake by CVl cells was examined. The cells were ineubated with either the pol5mier-modified or the unmodified EYPC liposomes containing a fluorescent lipid, NBD-PE, at 37 or 42°C for 3 h. The amount of liposomal lipid associated with the cells after the incubation was evaluated from the fluorescence intensity of NBD-PE in the cells (Fig. 6). For the unmodified liposome, approximately the same amount of liposome was taken up by the cell at both temperatures, indicating that temperature does not affect the liposome uptake in this temperature region. In the case of the polymer-modified liposome, the amount of liposome taken up by the cell at 37°C was slightly lower than that of the unmodified liposome at the same temperature. However, at 42°C the amount became two times higher than that of the unmodified liposome. In... 

Figure 6. Uptake of copoly(APr-NIPAM)-2Cu-n>odified and unmodified EYPC liposomes by CVl cells after 3 h incubation in Dulbecco s modified Eagle s medium (DMEM) supplemented with 10 % fetal bovine serum (FBS) at 37 and 42°C. Polymer/lipid weight ratio of the polymer-modified liposome was 0.71.

We have reported that an efficient content release occurs from the thermosensitive polymer-modified DOPE liposomes above the polymer s LCST. " However, when phosphatidylcholines are used for the liposomal lipid, only a limited portion of the contents is released from the thermosensitive polymer-modified liposomes under the same condition. W examined retention of calcein in the copoly(APr-NIPAM)-2Ci2-modified EYPC liposomes and found that 82 and 81 % of the loaded calcein molecules were retained after 3 h incubation in DMEM with 10 % FBS at 37 and 42°C, respectively. Also, we did not observe remarkable difference in... 

Since the polymer-modified EYPC liposomes were shown to retain the hydrophilic molecule at 37 and 42 C, we investigated temperature-control of delivery of MIX to CVl cells mediated by the polymer-modified liposomes. MIX has been used for the treatment of various malignant diseases and is known to prohibit proliferation of cells by binding tightly to cytoplasmic dihydrofolate reductase. 

Figure 7. The effect of MTX-loaded liposomes on growth of CVl cells. The cells were incubated with copoly(APr-NIPAM)-2Ci2-modified ( , H ) or unmodified ( , ) EYPC liposomes encapsulating MTX in DMEM with 10 % FBS for 10, 30, or 60 min at 37°C (closed symbols) or 42°C (open symbols). The cells were washed with PBS and incubated in DMEM with 10 % FBS for 24 h at 37°C. The cell number after the 24 h incubation was shown as a function of the initial incubation time. The lower and upper arrows represent the initial number of cells and the number of cells after the 24 h incubation without the liposome treatment. Concentration of MTX and EYPC were 6.2x10 and 5.4x10 M, respectively. Polymer/lipid weight ratio of the polymer-modified liposome was 0.71.

As is seen in Figure 6, the cells took up the polymer-modified liposomes and the unmodified liposomes approximately to the same extent at 37°C. Nevertheless, MTX encapsulated in the polymer-modified liposomes was much less effective than that entrapped in the unmodified liposomes. Since MTX must be taken up in cytoplasm to be active, it might be necessary for the anionic MTX to be delivered into low-pH compartments, such as lysosome, where MTX should be protonated and thus diffuse into cytoplasm. O Brien and collaborators showed that attachment of polyethylene glycol chains to liposome surface reduces uptake of the liposome in low-pH compartments of HeLa cell. Thus, even though the same amount of liposome was taken up by CV1 cells during the incubation, a smaller fraction of the liposomes might be taken in low-pH compartments for the polymer-modified liposomes than for the unmodified liposomes. At present, the reason why the activities of these MTX-loaded liposomes are different is unclear. However, it seems that location of the liposomes in the cell affects the appearance of the MTX activity. 

The present study demonstrated that interactions of the thermosensitive polymer-modified liposomes with model membranes and cells were suppressed by the hydrated polymer chains attached to the liposome surface below the LCST. However, the hydrophilic-to-hydrophobic change of the tethered polymer chains above the LCST enhanced their interactions. As a... 

Figure 3 The principle for the evaluation of the interaction between modified or unmodified polyanionic polymer and liposomal membrane.

The modified liposomes may be separated from excess protein by gel filtration using Sephadex G-75 or by centrifugal floatation in a polymer gradient (Derksen and Scherphof, 1985). 

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