Entrapment of Peptidoglycans and Adamantyltripeptides into Liposomes: An HPLC Assay for Determination of Encapsulation Efficiency

Abstract

The encapsulation of different immunomodulating peptides, the peptidoglycan monomer, its semisynthetic derivatives (Adamant-1-yl)-acetyl-peptidoglycan monomer and Boc-Tyr-peptidoglycan monomer, respectively, and of two diastereoisomers of adamantyltripeptides into the large negatively charged multilamellar liposomes was investigated. The reproducible quantitative method using HPLC was established for the determination of the entrapped compounds. It was shown that the tested compounds could be efficiently incorporated into liposomes using either the film or modified film method. The results confirmed that the peptidoglycans with lipophilic substituents and particularly the adamantyltripeptides were incorporated into liposomes with higher efficiency than the peptidoglycan monomer using either of the described methods. Liposome preparations were stable at 4°C up to seven days as shown by minimal leaking of the entrapped material.     

A spin labelling study of immunomodulating peptidoglycan monomer and adamantyltripeptides entrapped into liposomes

The interaction of immunostimulating compounds, the peptidoglycan monomer (PGM) and structurally related adamantyltripeptides (AdTP1 and AdTP2), respectively, with phospholipids in liposomal bilayers were investigated by electron paramagnetic resonance spectroscopy. (1). The fatty acids bearing the nitroxide spin label at different positions along the acyl chain were used to investigate the interaction of tested compounds with negatively charged multilamellar liposomes. Electron spin resonance (ESR) spectra were studied at 290 and 310 K. The entrapment of the adamantyltripeptides affected the motional properties of all spin labelled lipids, while the entrapment of PGM had no effect. (2). Spin labelled PGM was prepared and the novel compound bearing the spin label attached via the amino group of diaminopimelic acid was chromatographically purified and chemically characterized. The rotational correlation time of the spin labelled molecule dissolved in buffer at pH 7.4 was studied as a function of temperature. The conformational change was observed above 300 K. The same effect was observed with the spin labelled PGM incorporated into liposomes. Such effect was not observed when the spin labelled PGM was studied at alkaline pH, probably due to the hydrolysis of PGM molecule. The study of possible interaction with liposomal membrane is relevant to the use of tested compounds incorporated into liposomes, as adjuvants in vivo.     

Encapsulation of immunoadjuvant [24C]peptidoglycan monomer into liposomes: Effect on metabolism and immune response in mice

¹⁴C-labeled peptidoglycan monomer was encapsulated into negatively charged, multilamellar liposomes composed of egg phosphatidylcholine, cholesterol and dicetylphosphate. Excretion and tissue distribution of the label in mice were studied after intravenous injections. Encapsulation of peptidoglycan monomer into liposomes as compared to free peptidoglycan monomer, resulted in increased retention of the label, particulary in the liver and to a lesser extent in spleen. The excretion was drastically reduced and delayed even after 4 days when cholesterol-rich (phosphatidylcholine/cholesterol, 7:5 molar ratio) liposomes were used for encapsulation of peptidoglycan monomer. Peptidoglycan monomer and liposomes, when tested separately, stimulate the immune response to sheep erythrocytes in mice. However, there was no significant additive or synergistic effect when peptidoglycan monomer was encapsulated into liposomes.     

Surface modified liposomes by mannosylated conjugates anchored via the adamantyl moiety in the lipid bilayer

The aim of the present study was to encapsulate mannosylated 1-aminoadamantane and mannosylated adamantyltripeptides, namely [(2R)-N-(adamant-1-yl)-3-(α,β-d-mannopyranosyloxy)-2-methylpropanamide and (2R)-N-[3-(α-d-mannopyranosyloxy)-2-methylpropanoyl]-d,l-(adamant-2-yl)glycyl-l-alanyl-d-isoglutamine] in liposomes. The characterization of liposomes, size and surface morphology was performed using dynamic light scattering (DLS) and atomic force microscopy (AFM). The results have revealed that the encapsulation of examined compounds changes the size and surface of liposomes. After the concanavalin A (ConA) was added to the liposome preparation, increase in liposome size and their aggregation has been observed. The enlargement of liposomes was ascribed to the specific binding of the ConA to the mannose present on the surface of the prepared liposomes. Thus, it has been shown that the adamantyl moiety from mannosylated 1-aminoadamantane and mannosylated adamantyltripeptides can be used as an anchor in the lipid bilayer for carbohydrate moiety exposed on the liposome surface.     

Effect of liposomal formulations and immunostimulating peptidoglycan monomer (PGM) on the immune reaction to ovalbumin in mice

The adjuvant activity of liposomes and immunostimulating peptidoglycan monomer (PGM) in different formulations has been studied in mice model using ovalbumin (OVA) as an antigen. PGM is a natural compound of bacterial origin with well-defined chemical structure: GlcNAc-MurNAc-L-Ala-D-isoGln-mesoDpm(epsilonNH2)-D-Ala-D-Ala. It is a non-toxic, non-pyrogenic, and water-soluble immunostimulator. The aim of this study was to investigate the influence of different liposomal formulations of OVA, with or without PGM, on the production of total IgG, as well as of IgG1 and IgG2a subclasses of OVA-specific antibodies (as indicators of Th2 and Th1 type of immune response, respectively). CBA mice were immunized s.c. with OVA mixed with liposomes, OVA with PGM mixed with liposomes, OVA encapsulated into liposomes and OVA with PGM encapsulated into liposomes. Control groups were OVA in saline, OVA with PGM in saline, and OVA in CFA/IFA adjuvant formulation. The entrapment efficacy of OVA was monitored by HPLC method. The adjuvant activity of the mixture of OVA and empty liposomes, the mixture of OVA, PGM, and liposomes and PGM encapsulated with OVA into liposomes on production of total anti-OVA IgG was demonstrated. The mixture of PGM and liposomes exhibited additive immunostimulating effect on the production of antigen-specific IgGs. The analysis of IgG subclasses revealed that encapsulation of OVA into liposomes favors the stimulation of IgG2a antibodies, indicating the switch toward the Th1 type of immune response. When encapsulated into liposomes or mixed with liposomes, PGM induced a switch from Th1 to Th2 type of immune response. It could be concluded that appropriate formulations of antigen, PGM, and liposomes differently affect the humoral immune response and direct the switch in the type of immune response (Th1/Th2).     

Reversed-phase high-performance liquid chromatographic method for the determination of peptidoglycan monomers and structurally related peptides and adamantyltripeptides

The reversed-phase HPLC method using UV detection was developed for the determination of (a) immunostimulating peptidoglycan monomers represented by the basic structure GlcNAc-MurNAc-L-Ala-D-isoGln-meso-DAP(omegaNH(2))-D-Ala-D-Ala (PGM) and two more lipophilic derivatives, Boc-Tyr-PGM and (Ada-1-yl)-CH(2)-CO-PGM, (b) two diastereomeric immunostimulating adamantyltripeptides L- and D-(adamant-2-yl)-Gly-L-Ala-D-isoGln and (c) peptides obtained by the enzyme hydrolyses of peptidoglycans and related peptides. The enzymes used, N-acetylmuramyl-L-alanine amidase and an L,D-aminopeptidase are present in mammalian sera and are involved in the metabolism of peptidoglycans and related peptides. Appropriate solvent systems were chosen with regard to structure and lipophilicity of each compound. As well, different gradient systems within the same solvent system had to be applied in order to achieve satisfactory separation and retention time. HPLC separation was developed with the aim to use this method for the study of the stability of the tested compounds, the purity during preparation and isolation and for following the enzyme hydrolyses.     

Effect of Liposomal Formulations and Immunostimulating Peptidoglycan Monomer (PGM) on the Immune Reaction to Ovalbumin in Mice

The adjuvant activity of liposomes and immunostimulating peptidoglycan monomer (PGM) in different formulations has been studied in mice model using ovalbumin (OVA) as an antigen. PGM is a natural compound of bacterial origin with well-defined chemical structure: GlcNAc-MurNAc-l-Ala-d-isoGln-mesoDpm(εNH₂)-d-Ala-d-Ala. It is a non-toxic, non-pyrogenic, and water-soluble immunostimulator. The aim of this study was to investigate the influence of different liposomal formulations of OVA, with or without PGM, on the production of total IgG, as well as of IgG1 and IgG2a subclasses of OVA-specific antibodies (as indicators of Th2 and Th1 type of immune response, respectively). CBA mice were immunized s.c. with OVA mixed with liposomes, OVA with PGM mixed with liposomes, OVA encapsulated into liposomes and OVA with PGM encapsulated into liposomes. Control groups were OVA in saline, OVA with PGM in saline, and OVA in CFA/IFA adjuvant formulation. The entrapment efficacy of OVA was monitored by HPLC method. The adjuvant activity of the mixture of OVA and empty liposomes, the mixture of OVA, PGM, and liposomes and PGM encapsulated with OVA into liposomes on production of total anti-OVA IgG was demonstrated. The mixture of PGM and liposomes exhibited additive immunostimulating effect on the production of antigen-specific IgGs. The analysis of IgG subclasses revealed that encapsulation of OVA into liposomes favors the stimulation of IgG2a antibodies, indicating the switch toward the Th1 type of immune response. When encapsulated into liposomes or mixed with liposomes, PGM induced a switch from Th1 to Th2 type of immune response. It could be concluded that appropriate formulations of antigen, PGM, and liposomes differently affect the humoral immune response and direct the switch in the type of immune response (Th1/Th2).     

Stimulating Role of Toxoids-laden Liposomes in Oral Immunization against Diphtheria and Tetanus Infections

Liposomes have been produced by injecting an ether solution of a mixture of lecithin and cholesterol into a diluted solution of prewarmed diphtheria and tetanus toxoids followed by elimination of the stream of ether vapour by vacuum.In a preliminary study, adjuvant effects of liposomes on the systemic and mucosal immune response have been studied. When a mixture of diphtheria toxoid (DT) and tetanus toxoid (TT) entrapped in liposomes were administered parenterally or orally in rabbit, a significant rise of specific antibodies against both toxoids was noticed. In monkeys receiving a mixture of DT and TT entrapped in liposomes orally, the antibody response after two and three ingestions of this product was mild but when liposomes containing toxoids were adsorbed with aluminium hydroxide in a similar experiment, a significant rise in the specific antibody response in monkey against both toxoids was recorded. Adult volunteers, similarly receiving a mixture of DT and TT, entrapped in liposomes and adsorbed with aluminium hydroxide have shown a significant rise in specific circulating antitoxins. In order to compare the efficacy of this technique of human oral immunization with the previous method, whereby a plant medicinal seed (LRS) was used as adjuvant in oral immunization of man, a second group of volunteers were simultaneously and similarly treated as suggested previously. The comparative results are discussed in the present report.     

Lectin-specific targeting of beta-glucocerebrosidase to different liver cells via glycosylated liposomes

Galactosylated and mannosylated liposomes were more efficient in transporting liposome-entrapped beta-glucocerebrosidase to liver compared to nonglycosylated liposomes. The enzyme entrapped to glycoside-bearing liposomes was found to be cleared at a much faster rate than that entrapped in liposomes having no sugar on their surface. Asialoorosomucoid and hydrolyzed mannan were found to inhibit both the clearance and the uptake of galactosylated and mannosylated liposomes, respectively, supporting involvement of lectin-sugar interaction. Further studies on the uptake of glucocerebrosidase by isolated liver cells revealed that the enzyme entrapped in mannosylated liposomes has much higher affinity for nonparenchymal cells whereas the assimilation of the entrapped enzyme into hepatocytes is clearly favored for liposomes having galactose on their surface.     

Introduction of ribonucleic acids into cells by means of liposomes.

A method of ultramicroinjection of nucleic acids into cultured cells by means of liposomes is described. Messenger RNA, ribosomal RNA and transfer RNA were entrapped in large unilamellar liposomes and subsequently the liposomes were fused with cells. The uptake of RNA by the cells was stimulated 6--8 times by our method. Possible applications of microinjection of RNA by means of liposomes are discussed.     

Liposomes are effective carriers for the ocular delivery of prophylactics

Liposomes containing acetylcholinesterase were prepared by the freeze-drying method. The multilamellar morphology of the vesicles was revealed by freeze-fracture electron microscopy and their size distribution was determined by quasi-elastic light scattering. The vesicle diameters were in the range of about 0.2-4.0 micron. The liposome preparations were tested for their ocular delivery of an entrapped cholinesterase enzyme in counteracting the miotic effect of diisopropylfluorophosphate (DFP), a prototype of a family of organophosphate poisons. The topical application of the enzyme-containing liposomes to the rabbit eye was found to confer a significant level of protection against DFP-induced miosis. In comparing the prophylactic effectiveness of different enzyme-bearing liposomes, positively charged vesicles were found to be more effective than either neutral or negatively charged vesicles. Although the precise protective mechanism is not clear, our in vitro studies indicate that DFP molecules freely associate with liposomes and tear fluid promotes the release of liposome-entrapped enzymes. Thus, it is conceivable that the enzyme-liposome complex may act somewhat like a sponge by sequestering DFP molecules which diffuse into the vesicle, and also by releasing the entrapped enzyme to combine with DFP, thereby neutralizing its in vivo toxic effect.     

Intracellular digestion of saturated and unsaturated phospholipid liposomes by mucosal cells. Possible mechanism of transport of liposomally entrapped macromolecules across the isolated vascularly perfused rabbit ileum

The mechanism of intestinal absorption of liposomally entrapped [14C]inulin and 125I-labelled poly(vinylpyrrolidone) was studied using the isolated rabbit intestinal loop with intact perfused vasculature, a system more closely resembling an in vivo system than the everted sac technique. [14C]Inulin or 125I-poly(vinylpyrrolidone) was entrapped in liposomes prepared from unsaturated egg phosphatidylcholine and soya phosphatidylcholine, and saturated distearoylphosphatidylcholine (18:0), dipalmitoylphosphatidylcholine (16:0) and dimyrostoylphosphatidylcholine (14:0). Free and liposomally entrapped macromolecules were introduced in the ileum and the transport of liposomes and entrapped macromolecules into the venous effluent was monitored by measuring the presence of the aqueous marker 125I-poly(vinylpyrrolidone) or [14C]inulin, and lipid marker [3H]cholesterol. The results show that intact liposomes are not transported across intestine into the venous effluent, but they are taken up by mucosal cells and digested intracellularly, releasing the entrapped markers 125I-poly(vinylpyrrolidone) and [14C]inulin. These markers are then transported into the venous effluent as free molecules. The absorption of liposomally entrapped [14C]inulin into the venous effluent is biphasic, first slow for 30 min (i.e., a lag period of 30 min), followed by a rapid linear increase. The duration of the lag period and the rate of absorption of the entrapped [14C]inulin are dependent on the degree of saturation and the transition temperature of the phospholipids used to prepare liposomes. The possible explanation of the lag period based on the evidence presented here is that it is the time required for the liposomes to be taken up by mucosal cells and digested intracellularly. Intracellular digestion of liposomes prepared from saturated phospholipids is more rapid than from those prepared from unsaturated phospholipids, and the greater the fatty acid chain length of the saturated phospholipids the more rapid the intracellular degradation of liposomes.     

Entrapment of plasmid DNA in liposomes.

The entrapment of plasmid DNA (pMB9) and high molecular weight DNA into large unilamellar liposomes is described. The entrapment of DNA is specific and due to encapsulation of DNA into the aqueous compartment of liposomes. The entrapped Dna, resistant to deoxyribonuclease treatment, could be reisolated from liposomes intact and, as has been shown by transformation assay, it remains biologically active. The advantages of our method and possible applications are discussed.     

Improved Cytoplasmic Delivery to Plant Protoplasts via pH-Sensitive Liposomes

We demonstrated that the liposomes composed of dioleolylphosphatidylethanolamine/cholesterol/oleic acid (4:4:2) dramatically release their contents at a pH of less than or equal to 6.0 and are capable of delivering their contents into the cytoplasm of higher plant protoplasts. This is shown by using a soluble fluorescent dye, calcein, as a liposome-entrapped marker. We found that calcein fluorescence was evenly distributed in the cytoplasm of wild carrot protoplasts after the incubation of protoplasts with liposomes in the presence of polyethylene glycol 6000. At 0.45 micro mole phospholipid per 6 × 10⁵ protoplast, for example, the percentage of protoplasts which took up liposomes was 89% which was much higher than that achieved by conventional pH-insensitive liposomes. In this study, liposomes were prepared by a detergent dialysis method which avoided sonication and organic solvents. Thus macromolecules such as proteins and nucleic acids could be entrapped in the liposomes and delivered to the cytoplasm of the protoplasts.     

Use of liposomes to aid intestinal absorption of entrapped insulin in normal and diabetic dogs

The effectiveness of liposomes in aiding intestinal absorption of entrapped insulin was studied in normal and diabetic dogs. Intraduodenal administration of free insulin (490 and 1630 U) or free insulin (88 U) plus empty liposomes to normal conscious dogs produced no change in plasma immunoreactive insulin or glucose Administration of 40–80 U insulin entrapped in liposomes composed of either phosphatidylcholine, distearoylphosphatidylcholine, or dipalmitoylphosphatidylcholine with cholesterol and dicetylphosophate ( in the ratio 10:2:1 by weight) to normal dogs produced substantial rises in peripheral plasma immunoreactive insulin after 45–60 min. However, the magnitude of these rises was neither reproducible nor dose-dependent. No fall in plasma glucose was observed. Intraduodenal administration of 50–100 U insulin entrapped in liposomes to diabetic dogs also produced rises in plasma immunoreactive insulin levels after 45–60 min but again these rises were not dose-related. However, unlike the results in normal dogs, a small fall in plasma glucose followed the plasma immunoreactive insulin rise in diabetic dogs. This glucose fall was not dose-dependent nor was it related to the magnitude of the rise in plasma immunoreactive insulin. In conclusion, it seems that administration of insulin in liposomes may allow absorption of partially degraded insulin into the circulation but the rise in plasma immunoreactive insulin observed in normal and diabetic dogs and the fall in plasma glucose in diabetic dogs are not influenced by the dose of insulin entrapped nor the lipid composition of the liposomes.     

Liposomal solubilization of new 3-hydroxy-quinolinone derivatives with promising anticancer activity: a screening method to identify maximum incorporation capacity

Four new 3-hydroxy-quinolinone derivatives with promising anticancer activity could be solubilized using liposomes as vehicle to an extent that allows their in vitro and in vivo testing without use of toxic solvent(s). A screening method to identify the maximum incorporation capacity of hydrophobic drugs within liposomes was successfully applied. The compounds and lipid(s) were dissolved in methanol, and the solvent was removed by rotary evaporation. The film was resuspended with phosphate buffer (pH 7.4), and the dispersion was sonicated to reduce vesicle size. Ultracentrifugation was used to separate liposome-associated drug from free (i.e., precipitated) drug, and the amount of drug incorporated within the liposomes was quantified using high-performance liquid chromatography. All four compounds were found to be significantly incorporated within soy phosphatidylcholine (SPC) liposomes, resulting in a 200-500-fold increase in apparent solubility. Drug-to-lipid ratios in the range of 2-5 μg/mg were obtained. Interestingly, the four quinolinone derivatives have shown different association tendencies with liposomes, probably due to the physicochemical properties of the different group bonded in position 2 of the quinolinone ring. None of the alternative lipids/lipid blends tested incorporated as much drug as SPC. Photon correlation spectroscopy analyses indicated that use of ultrasounds produced an efficient reduction in liposome size. The present approach appears suitable for incorporation capacity studies of any lipophilic drug in liposomes.     

Incorporation of a follicle stimulating hormone used for embryo transfer in cattle into multilamellar liposomes

A commercially available follicle stimulating hormone preparation (FSH-P) was successfully incorporated into multilamellar vesicles (liposomes). Multilamellar liposomes were found to contain 9.39 +/- 1.14 mg FSH-P (n=4) per 100 mg phospholipid or approximately 19.0% of the original material used to form the liposomes. A 1% solution of Triton X-100 incubated with liposomes containing FSH-P for one-half hour at 37 degrees C released 33% of the entrapped FSH-P; more than 99% of the entrapped FSH-P was released when liposomes were incubated with a 2% solution of Triton X-100. Entrapment of FSH-P increased proportionally to the mole percentage of stearylamine used in liposome formation, suggesting that FSH-P is entrapped in the aqueous interstices of the cationic liposomes. Entrapment of FSH-P in stable liposomes suggests that these multilamellar vesicles may be useful as a FSH-P delivery vehicle used for the superovulation and embryo transfer of food animals.     

Liposomal solubilization of new 3-hydroxy-quinolinone derivatives with promising anticancer activity: a screening method to identify maximum incorporation capacity

Four new 3-hydroxy-quinolinone derivatives with promising anticancer activity could be solubilized using liposomes as vehicle to an extent that allows their in vitro and in vivo testing without use of toxic solvent(s). A screening method to identify the maximum incorporation capacity of hydrophobic drugs within liposomes was successfully applied. The compounds and lipid(s) were dissolved in methanol, and the solvent was removed by rotary evaporation. The film was resuspended with phosphate buffer (pH 7.4), and the dispersion was sonicated to reduce vesicle size. Ultracentrifugation was used to separate liposome-associated drug from free (i.e., precipitated) drug, and the amount of drug incorporated within the liposomes was quantified using high-performance liquid chromatography. All four compounds were found to be significantly incorporated within soy phosphatidylcholine (SPC) liposomes, resulting in a 200–500-fold increase in apparent solubility. Drug-to-lipid ratios in the range of 2–5 µg/mg were obtained. Interestingly, the four quinolinone derivatives have shown different association tendencies with liposomes, probably due to the physicochemical properties of the different group bonded in position 2 of the quinolinone ring. None of the alternative lipids/lipid blends tested incorporated as much drug as SPC. Photon correlation spectroscopy analyses indicated that use of ultrasounds produced an efficient reduction in liposome size. The present approach appears suitable for incorporation capacity studies of any lipophilic drug in liposomes.     

Effect of diglucosamine on the entrapment of protein into liposomes

Liposomes, which had entrapped bovine serum albumin (BSA), were modified with diglucosamine by two methods. The liposome was prepared by a freeze-thawing method in the presence of the disaccharide, or the disaccharide was added to the liposome prepared in advance without it. To examine the effects of diglucosamine, the morphology, mean particle size, and zeta potential of both liposomes were compared with those of BSA-entrapping liposome prepared without the disaccharide. Diglucosamine caused no remarkable change in shape and no aggregation of the liposome. The presence of the disaccharide was confirmed on the surfaces of modified liposomes, and the entrapment of BSA into the liposomes was increased by the disaccharide. The entrapment behavior was affected by the way the disaccharide was added, and the difference in the way the BSA was entrapped was also indicated.     

Studies on osmotic stability of liposomes prepared with bacterial membrane lipids by carboxyfluorescein release.

The authors measured the osmotic stability of liposomes prepared with membrane lipids of bacteria, using the osmotic-shock release of entrapped carboxyfluorescein as an indicator. The sub-second physical changes of liposomes suspended in a solution of low osmotic pressure were examined by stopped flow spectrophotometry. The entrapped carboxyfluorescein was released when the liposomes burst on inflow of excess water. Liposomes prepared with the lipids of a stable Staphylococcus aureus L-form strain were more resistant to low osmotic pressure than those prepared from the wild strain of S. aureus, and liposomes prepared from Mycoplasma orale were even more resistant. Cardiolipin enhanced the lipid membrane stability in S. aureus and cholesterol in M. orale. The stability of lipid membranes to low osmotic pressure could be precisely determined by the present method.