Plain liposomes

Multilamellar liposomes composed of dipalmitoylphosphatidylcholine (DPPC) and dicetyl phosphate (DCP) were prepared by the formation of a thin lipid film and subsequent sonication, and coated with chitosan (Ch) [36], Liposomes with a size of approximately 5 pm were used in the experiment. The Ch-coated and plain liposomes were compared in terms of mucoadhesion to the rat stomach and intestinal parts. Although both the liposomes were less adhesive to the stomach, Ch-coated liposomes displayed much higher mucoadhesion to all the intestinal parts in vitro than the plain liposomes. The intestinal adhesion of the plain liposomes were minimal. Further, Ch-coated liposomes showed a great mucoadhesion to the intestine at acidic and neutral pH values. This was also confirmed by fluorescence microscopy when pyrene-loaded Ch-coated liposomes were used in the mucoadhesion test. 

The insulin solution and Ch-coated liposomes and plain liposomes containing insulin were tested for their influence on the blood glucose level after intragastric administration to normal rats. When compared with the change in the blood glucose level caused by the subcutaneous administration of insulin solution, Ch-coated liposomes had a pharmacological effect of approximately 5% (Figure 3.2). The effect of the plain liposomes and insulin solution was... 

FIGURE 3.2 Blood glucose concentration-time profiles in normal rats after oral administration of insulin-containing chitosan-coated liposomes, insulin-containing plain liposomes and insulin solution, and subcutaneous administration of insulin solution. Sol, solution Lip, liposomes Ch, chitosan s.c., subcutaneous. The results are expressed as the mean values. 

At the end of the hypoxic period, viability of the cells were assessed by the Trypan Blue dye exclusion criteria. Fig. 15 demonstrated that the viability of hypoxic cells treated with IL were not significantly different form the viability of the normoxically cultured H9C2 cardiocytes. The viability of hypoxic cardiocytes treated with plain liposomes or IgG-liposomes was significantly lower than either those of immunoliposome treated hypoxic cells or normoxica cells. Since there was no difference in viability in cardiocytes treated... 

To demonstrate that preservation of cell viability is due to fusion of IL with the cell membranes, IL were prepared with intraliposomal silver grains. The rationale was that the only way silver grains can enter the treated hypoxic cardiocytes is by fusion of the IL with the cell membrane that resulted in preservation of cardiocyte viability. If endocytosis of the IL is the process of internalization without fusion, then plain liposomes should also show internalization of the silver grains. To prepare such liposomes,silver nitrate was added in a buffer solution during liposome preparation by soni-cation. Liposomes, purified from the non-entrapped silver nitrate by dialysis were dialyzed in 0.12 M NaCl solution and exposed to light for 1 h that led to the formation of fine electron-dense precipitate of silver oxide inside liposomes. 

Fig. 3. Concanavaline-A-induced, ligand-binding assay of various liposomal formulations. Concanavalin A solution (lOOgg/mL) was added to 0.2 mL of diluted liposomes formulations and absorbance at 550 nm was measured as a function of time. The data were subtracted from the blank experiment conducted without an addition of concanavalin A. Binding of the terminal mannose residues of the mannan to the ConA causes agglutination of the complex in solution resulting in an increase in turbidity. However, plain liposomes did prevent the binding of Con A as evidenced by the lower turbidity. The accessibility of the mannan to the liposomes is critical for in vivo cell binding...

Fig. 4. Percent uptake of different iiposomai formuiations conjugated to Rtiodamine by human DCs measured through flow cytometry. Ceiis were incubated with Rhodamine-MSP-1, g-ioaded iiposomai formuiations and anaiyzed at different time intervals (0,15,30,60,120,180, and 360 min). The kinetics of uptake has been presented with mean fluorescence intensity (MFI) vs. counts by FACS analysis and percentage uptake at various time inten/als. Uptake of formulation on a per cell basis was quantified as fluorescence intensity per cell. Percentage of positive cells was determined as proportion ot cells with fluorescence intensity higher than 99% of cells of the control sample (cells incubated with unconjugated rhodamine alone). Flow cytometric analysis revealed that the percentage of rhodamine-positive DCs increased rapidly and reached a plateau after 16 h of incubation (means of three independent experiments). A steady increase in the uptake percentage (%) was recorded and a maximum cell-associated fluorescence was observed at 16 h for OPM-coated cationic liposomes. Flow cytometric analysis of DCs revealed that plain liposomes did not significantly enhance the antigen uptake by DCs compared with the uptake recorded for mannan-coated liposomes...

Treatment with CSIL enables globally ischemic hearts to return to near normal function within 15 min of reperfusion, which is consistent with the prevention of the occurrence of uncontrolled myocardial Ca overload. The absence of mitochondrial swelling and the return of function to near normal in CSIL-treated hearts are also consistent with the maintenance of Ca homeostasis. Cell membrane lesion sealing with neutral immunoliposomes may also reduce injury mediated by acid and oxidative stress. However, plain liposomes in serum-free... 

Assessment of the viability of the cells was performed after 24 h of hypoxia by Trypan-blue exclusion or by immediate further incubation of the cells with [3H]-thymidine (3HT). According to the trypan-blue dye exclusion data, almost all control HSs were nonviable. Plain liposomes provided some protection from hypoxic injury, probably, by nonspecifically sticking to cell surfaces and fortuitously sealing some of the cell membrane breaches. IPs completely prevented cell death, with a cell viability similar to that of normoxic cells. Hypoxic cells treated with nonspecific IgG liposomes demonstrated viability at the level of PL-treated cells. 

Survival of NCs as assessed by 3HT uptake, increased from 100% (the mean 3HT uptake in control NC was assigned 100%) to approx 254% after 24 h normal cell replication, whereas virtually no survival of HCs was registered after the same time period. Plain liposomes added to HC provided some protection, as evidenced by a decrease in viability after 24 h (approx 78%), of to approx 4% after 2 d of hypoxia, and essentially 0% after 3 d or more. When hypoxic cells were treated with IL, not only did IL confer protection, but it also permitted cell replication. This is evidenced by the similarity in the increase of 3HT uptake after 24 h of incubation between NCs (254%) and IL-treated HC (225%). After 48 h or more of hypoxia, replication in IL-treated HCs was decreased, but the viability of cells was maintained on the level of approx 90, 48, 15, and 7.8% after 2, 3, 4, and 5 d of hypoxia, respectively. These data show that the protective effect imposed by IL on hypoxic cells leads to a long-term preservation of cardiocyte viability, which might be especially important from a practical point of view for long-term preservation of myocardial viability. 

In an attempt to learn more about the exact mechanism of IL interaction with HCs, we performed ultrastructural studies of HCs treated with silver grain-loaded electron-dense ILs or plain liposomes (51,53,54). 

FIGURE 25.7 Fluorescence micrographs of the nasal tissue of rats excised following administration of (A) FITC-BSA solution (B) FITC-BSA encapsulated in plain liposomes or (C) FITC-BSA encapsulated in glycol chitosan coated liposomes. (From Khatri, K. et ah, Vaccine, 26, 2225, 2008. With permission.)... 

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