Adsorption of chitin derivatives onto liposomes: optimization of adsorption conditions.

A 2(4) factorial design is used to optimize the adsorption conditions of the hydrophilic anionic polyelectrolytes. Carboxymethylchitin (CMC) and Carboxymethyl/Glycolchitin (CO) onto liposomes at physiological ionic strength (I) and pH using phosphate buffered saline (PBS, I = 154 mM, pH = 7.4). Positive ([+]) or high surface affinity liposomes (DSPC:CHOL:DMTAP, 5:4:1), and Neutral ([N]) or low surface affinity liposomes (DSPC:CHOL, 1:1) were used as adsorption surfaces. Results of the calculations of the main effects indicate that polymer molecular weight (mwt), Surface Affinity (S), Number of Adsorption Shots (Sh), Temperature (T), and the combinations mwt x S, mwt x Sh are the most important process parameters. Results of a study conducted at T = 37 degrees C show that no loss occurs from the positive surface at the highest particle concentration, Np = 4.043 x 10(11). Finally, the extent of polymer-induced particle aggregation is decreased when the diameter of the uncoated liposomes is doubled from 0.22 to 0.45 micron. These results are as expected, given the stiffness and the dimensions of the macromolecules.

Comparison of polymerically stabilized PEG-grafted liposomes and physically adsorbed carboxymethylchitin and carboxymethyl/glycolchitin liposomes for biological applications.

The stabilities of two types of polymerically stabilized liposomes consisting of PEG-grafted (DSPC:CHOL:DSPE-PEG1900, 5:4:1) and physically adsorbed carboxymethylchitin (CMC) and carboxymethyl/glycolchitin (CO) are compared. The polyelectrolyte is adsorbed on positive (DSPC:CHOL:DMTAP, 5:4:1) and neutral (DSPC:CHOL, 1:1) liposomes at different molecular weights (Mw). In PBS buffer (c(s) = 154 mM, pH = 7.4) the theoretical stability ratios (W) calculated using the classical DLVO Theory, indicate that the CMC-coated vesicles and the negative liposomes (DSPC:CHOL:DMPG, 5:4:1) are highly stable (W >> 1) compared to the PEG-grafted (W = 0.9511) and CO-coated (W = 0.9550) liposomes. Meanwhile, experimentally determined values of W, prove that the PEG-grafted is the most stable suspension (W = 5.5). Computation of the theoretical values of W for liposome-red blood cell and liposome-macrophage indicates that the electrosterically stabilized suspensions and the negative liposomes are stable. Light scattering results show that the flocculation of liposomes in blood and plasma depends on polymer molecular weight, type of polyelectrolyte and surface charge of the uncoated liposome. Neutral liposomes coated with CMC of Mw = 1.01 x 10(5) and negative liposomes provide a more effective barrier to plasma macromolecular protein adsorption than the grafted PEG groups and are easy to resuspend in blood.

Kinetic aspects of polyelectrolyte adsorption: adsorption of chitin derivatives onto liposomes as a model system.

Carboxymethylchitin (CMC) and Carboxymethyl/Glycolchitin (CO) have been adsorbed onto liposomes at physiological ionic strength (I) and pH using phosphate buffer saline (PBS, I = 154 mM, pH = 7.4). Adsorption isotherms at different polymer weight average molecular weights (Mw), for Positive ([+]) or high surface affinity liposomes (DSPC:CHOL:DMTAP, 5:4:1), and Neutral ([N]) or low surface affinity liposomes (DSPC:CHOL, 1:1), have been obtained at T = 25 degrees C. For all CMCs, the adsorbed amount increases with polymer concentration ([P]zero) and no true plateau is observed. The CMC Mw = 4.19 x 10(5) adsorbed on a positive surface fits Langmuir kinetics with maximal coverage gamma max = 325.4 micrograms/mg (polymer/lipid), and inverse of equilibrium constant K* = 4.743 x 10(-4) mg/ml. For all isotherms the predicted amount of polymer needed to coat the entire surface based on the Radius of Gyration (Rg) is inferior to the amount adsorbed. This fact in conjunction with the dependence of gamma max on the number of adsorption shots, suggests that the adsorption is not thermodynamically but kinetically controlled.

The importance of standardization of carboxymethylchitin concentration by the dye-binding capacity of alcian blue before adsorption on liposomes.

A new method based on the measurement of the relative dye-binding capacity of Alcian Blue to carboxymethylchitin (CMC) at various molecular weights (MW) has been developed to facilitate the standardization of the initial polyelectrolyte concentration. In the absence of standardization, non-reproducible adsorption patterns are encountered during the adsorption of the MW CMC on neutral and positively charged liposomes. This method is sensitive down to a concentration of 5 mu g/ml of polymer in water. Static Light Scattering (SLS) measurements are used to obtain the weight average molecular weight (Mw) and the size of the polyelectrolyte (Rg) and overlap concentrations (c*). The Mws are then used to determine the constants K and a of the Mark-Houwink equation which are 1.65 x 10(-2) dl/g and 0.4701, respectively, evaluated at kappa = 0.154 M, pH = 7.4 and T = 25 degrees C. The critical electrolyte concentration decreases with molecular weight for Mws ranging from 5.0 x 10(4)-1.2 x 10(6). The dye-binding capacity changes with the molecular weight distribution of the polyelectrolyte demonstrating the sensitivity of this technique to polydispersity.

Purification and quantitative determination of carboxymethylchitin incorporation into submicron bilayer-lipid membrane artificial cells (liposomes) encapsulating hemoglobin.

The separation of carboxymethylchitin-coated hemoglobin-loaded liposomes (CMC-LEHb)s from the non-adsorbed CMC has been achieved by gel chromatography. This purification takes place at the physiological pH of 7.4 favoring HbO2 preservation. A comparative study between experimental techniques for the quantitative determination of the absorption of carboxymethylchitin (CMC) onto liposomes encapsulating hemoglobin (LEHb)s has been conducted. Results suggest that FT-IR spectroscopy gives a more accurate quantitative absorption index while the chitinase-based enzymatic assay should be used as a qualitative detection tool. Quantitative bilayer and surface characterization show that the RBC membrane composition has been closely simulated by that of CMC-LEHbs in terms of total lipids and carbohydrates at 87.8% (phospholipids and cholesterol) and 12.2% CMC respectively.

Purification and characterization of liposomes encapsulating hemoglobin as potential blood substitutes.

In view of the desirability to increase the survival time of the liposome-based artificial red blood cells in vivo, the variables influencing optimum hemoglobin capture and preservation for the bovine hemoglobin-loaded liposomes (LEHb) are investigated. In order to predict the in vivo response, the necessary experiments for the in vitro system characterization have been carried out. The liposomes are prepared by the Reverse Phase Evaporation technique and then purified using a Sepharose 4B column. The purified LEHbs display a unimodal size distribution in the submicron range with a volume average diameter of 0.115 microns and a particle count of 1.25* 10(15) per ml of suspension. Analysis of the lipi/Hb content of the liposomes reveals that the variations in the ratio of Hb encapsulated to lipid entrapped (Hb/L)f as a function of the initial Hb concentration ([Hb]o) is insignificant compared to the net augmentation of (Hb/L)f as a function of the increasing initial lipid to Hb loading ([L]o). Meanwhile high [Hb]o s are necessary for the preservation of oxyhemoglobin.

Preparation and surface characterization of carboxymethylchitin-incorporated submicron bilayer-lipid membrane artificial cells (liposomes) encapsulating hemoglobin.

In view of the desirability to increase the survival time of the lipsome-based artificial red blood cells in vivo, bovine hemoglobin-loaded liposomes (LEHb) are incorporated with a polyanionic polymer, a highly substituted type of carboxymethylchitin (CMC). The liposomes are prepared by a modified Reverse Phase Evaporation technique and then purified using a Sepharose 4B column. A comparative study between experimental techniques for the determination of adsorption efficiency suggests that FT-IR spectroscopy gives a more accurate quantitative adsorption index while the chitinase-based enzymatic assay should be used as a qualitative detection tool. The overall composition of the bilayer(s) can be approximated by that of the natural (RBC) membrane in terms of total lipids and carbohydrates at 87.8% (phospholipids and cholesterol) and 12.2% CMC respectively.