Considerations in the developability of peptides for oral administration when formulated together with transient permeation enhancers.

This paper reviews many of the properties of a peptide that need to be considered prior to development as an oral dosage form when co-formulated with a permeation enhancer to improve oral bioavailability, including the importance and implications of peptide half-life on variability in pharmacokinetic profiles. Clinical considerations in terms of food and drug-drug interactions are also discussed. The paper further gives a brief overview how permeation enhancers overcome barriers that limit oral absorption of peptides and thereby improve their oral bioavailability, albeit bioavailabilities are still low single digit and variability is high.

Cyclodextrin-crosslinked poly(acrylic acid): Synthesis, physicochemical characterization and controlled release of diflunisal and fluconazole from hydrogels.

The aim of this work was to develop mucoadhesive hydrogels with variable drug delivery properties by crosslinking poly(acrylic acid) (PAA) with cyclodextrins (CDs). CD-PAA polymers with high CD content and good inter-batch reproducibility were synthesized by activating PAA with SOCl2, then reacting PAA chloride with CD in the presence of 4-dimethylaminopyridine at 50°C. Manipulation of the synthesis conditions affected the physicochemical character of the CD-PAA polymers and hydrogels in terms of CD content, the average number of ester bonds to an individual CD, viscosity, and the association and release of model drugs. Inclusion complexation of diflunisal (DIF) and fluconazole (FLZ) with CD-PAA hydrogels was assessed by (19)F NMR spectroscopy and association constants (Kas) for DIF were in the range 220-486M(-1) with ßCD-PAA and 1327-6055M(-1) with hydroxypropyl-ßCD-PAA. For FLZ the Ka range was 34-171M(-1) with hydroxypropyl-ßCD-PAA. The hydrogels were found to release both drugs by means of Fickian diffusion as the predominant mechanism. A slight trend toward negative correlation was found between the Ka and Higuchi kH values for DIF. These results highlight the potential of CD-PAA hydrogels to control the release of model drugs through inclusion complexation.

Cyclodextrin-crosslinked poly(acrylic acid): adhesion and controlled release of diflunisal and fluconazole from solid dosage forms.

The controlled release of diflunisal and fluconazole from tablets made of novel polymers, poly(acrylic acid) (PAA) crosslinked with either ß-cyclodextrin (ßCD) or hydroxypropyl-ßCD (HPßCD), was investigated and Carbopol 934P (Carbopol) was used as a highly crosslinked PAA for comparison. Diflunisal strongly associates with ßCD-PAA and HPßCD-PAA polymers (Ka of 486 and 6,055 M(-1) respectively); thus, it was physically mixed into the conjugates and also precomplexed to identify whether decomplexation has any influence on release kinetics. Fluconazole has poor complexing ability (Ka of 34 M(-1) with HPßCD-PAA); thus, it was only tested as a physical mixture. Swelling and adhesion studies were conducted on all tablet combinations and adhesivity of the CD-PAA polymer tablets was maintained. Diflunisal release was much slower from HPßCD-PAA tablets than from ßCD-PAA, suggesting that a higher degree of complexation retards release. The precomplexed diflunisal release was also slower than the physically mixed diflunisal of the corresponding conjugate. The release closely followed zero-order kinetics for HPßCD-PAA, but was more sigmoidal for ßCD-PAA and especially Carbopol. Conversely, poorly associating fluconazole released in almost exactly the same way across both polymers and Carbopol, indicating that the release kinetics of poorly associating drugs are not influenced by the presence of cyclodextrins. In view of the varying profiles and release rates shown with diflunisal for the different polymers, the fluconazole data support the concept that adequate complexation can indeed modulate the release kinetics of drugs.

Targeting of ICAM-1-directed immunoliposomes specifically to activated endothelial cells with low cellular uptake: use of an optimized procedure for the coupling of low concentrations of antibody to liposomes.

Targeted delivery of therapeutics to the endothelium is an important goal in the treatment of inflammatory diseases. The aim of this work was to exploit the overexpression of intercellular adhesion molecule-1 (ICAM-1) on activated endothelial cells for the targeting of anti-ICAM-1-coupled immunoliposomes with the intent for further use as drug carriers. Immunoliposomes were prepared from using an optimized method for the coupling of low concentrations of antibody to liposomes, thereby preventing the loss of antibody through the derivatization, extraction, and activation process. This is especially suitable for limiting ligand conjugates that are isolated or synthesized in small quantities, such as monoclonal antibodies (mAbs). To investigate the functionality of the resulting immunoliposomes, the specificity of binding and cellular internalization studies of liposomes, either nonconjugated or conjugated with mAbs to ICAM-1 or to irrelevant IgG to high endothelial venule (HEV) cells, were analyzed by fluorescence microplate spectroscopy at 4 and 37°C. Immunoliposomes specifically directed against ICAM-1 were shown to bind selectively and specifically to tumor necrosis factor alpha-activated endothelial cells in vitro, with minimal cellular internalization. This study provides a novel delivery system that has the potential for targeting therapeutics to inflammatory tissue.

Synthesis of cationic derivatives of Quil A and the preparation of cationic immune-stimulating complexes (ISCOMs).

The aim of the study was to prepare cationic ISCOMs using cationic derivatives of the saponin Quil A. The polyamines ethylenediamine, spermidine and spermine were conjugated with the glucuronic acid moiety of Quil A. The aqueous solubility of the derivatives increased with decreasing pH, and the pK(a) values were between 6 and 7. The CMCs of the ionised derivatives were around 0.5-1.0 mg/mL. Using the method of hydration of freeze-dried monophase systems, the interaction of each of the Quil A derivatives with phosphatidylcholine and cholesterol, at a mass ratio of 4:4:2 and a pH of 3 and 7.4, was investigated. A few ISCOM-like structures were present in the systems prepared at pH 7.4, hence the ternary system of Quil A spermine derivative, phosphatidylcholine and cholesterol was further investigated at pH 7.4 using a variety of mass ratios. A relatively high number of cationic ISCOM-like structures were observed at the mass ratio of 6:2:2. These ISCOM-like structures were less homogeneous and more irregular in shape than ISCOMs prepared from unmodified Quil A. Colloidal particles with positive zeta potential were produced and may find application in the delivery of nucleic acids or anionic proteins.

A combined approach of chemical enhancers and sonophoresis for the transdermal delivery of tizanidine hydrochloride.

The effects of chemical enhancers and sonophoresis on the transdermal permeation of tizanidine hydrochloride (TIZ) across mouse skin were investigated. Parameters including drug solubility, apparent partition coefficient (APC), drug permeation, and degradation in skin were determined. Low frequency ultrasound was also applied in the presence and absence of chemical enhancers to assess whether drug permeation improved. APC values indicated that TIZ preferentially partitions into intercellular spaces and does not form a reservoir, with the drug also exhibiting good enzymatic stability in skin. Most of the enhancers studied significantly increased the permeation rate of TIZ through full thickness mouse skin in comparison with TIZ formulated in phosphate buffer. Maximum enhancement was observed for TIZ formulated as a suspension in 50% v/v aqueous ethanol containing 5% v/v citral. Sonophoresis significantly (p < 0.05) increased the cumulative amount of TIZ permeating through the skin at 15 and 30 min in comparison to passive diffusion. A synergistic effect was noted when sonophoresis was applied in the presence of chemical enhancers. The results suggest that the formulation of TIZ with an appropriate penetration enhancer may be useful in the development of a therapeutic system to deliver TIZ across the skin for a prolonged period, i.e. 24 hr. The application of ultrasound in association with chemical enhancers, such as the combination of 5% v/v citral in 50% v/v aqueous ethanol, could further serve as a non-oral and non-invasive drug delivery modality for the immediate therapeutic effect of muscle relaxants such as TIZ.

Development of a novel method for formulating stable siRNA-loaded lipid particles for in vivo use.

PURPOSE: A simple yet novel method was developed to prepare stable PEGylated siRNA-loaded lipid particles which are suitable for in vivo use. METHODS: PEGylated siRNA-loaded lipid particles were formulated by hydration of a freeze-dried matrix. The effect of various formulation parameters on the size and homogeneity of resulting particles was studied. Particles prepared using this method were compared to those prepared using an established post-insertion procedure for the entrapment efficiency, stability, in vitro biological activity as well as in vivo biodistribution. RESULTS: Using this hydration method, a particle size of less than 200 nm can be obtained with high siRNA entrapment efficiency (>90%) and high gene-silencing efficiency. Following intravenous administration into mice, these particles achieved a similar degree of accumulation in subcutaneous tumours but displayed less liver uptake compared to the post-insertion formulations. Importantly, in contrast to post-insertion preparations, particles made by hydration method retained 100% of their gene-silencing efficiency after storage at room temperature for 1 month. CONCLUSIONS: This paper describes a simple method of formulating PEGylated siRNA-loaded lipid particles. Given the ease of preparation, long term stability and favourable characteristics for in vivo delivery, our work represents an advance in lipid formulation of siRNA for in vivo use.

Increasing entrapment of peptides within poly(alkyl cyanoacrylate) nanoparticles prepared from water-in-oil microemulsions by copolymerization.

Low molecular hydrophilic actives such as peptides are typically poorly encapsulated within poly(alkyl cyanoacrylate) nanoparticles when prepared from micellar or microemulsion templates. The aim of the present study was to investigate whether the entrapment of peptides within poly(alkyl cyanoacrylate) nanoparticles could be increased by functionalizing the peptide so that it could copolymerize with the alkyl cyanoacrylate monomer. Peptide and acryloyl functionalized peptide representing the antigenic epitope of the lymphocytic choriomeningitis virus glycoprotein (LCMV(33-41)) were synthesized using solid-phase peptide synthesis. Poly(alkyl cyanoacrylate) nanoparticles were prepared to encapsulate either peptide or functionalized peptide using both an aqueous micellar and a water-in-oil microemulsion polymerization template. Using the micellar template, nanoparticles could not be produced in the presence of acryloyl peptide. Rather an agglomerated mass formed on the stirrer. In contrast, nanoparticles could be prepared using both acryloyl and parent peptide using the water-in-oil microemulsion template. Encapsulation efficiency was more than twofold greater for functionalized peptide, being greater than 90%. Encapsulation efficiency of functionalized peptide was also observed to increase with increasing the amount of alkyl cyanoacrylate monomer used for polymerization. A biphasic release profile was observed for the nanoparticles entrapping the non-functionalized peptide with greater than 50% of peptide being released during the first 10 min and with around 90% being released at 6h. In contrast, less than 10% of the total amount of acryloyl LCMV(33-41) entrapped within the nanoparticles was detected in the release media following the initial 10 min, and no further release of peptide was observed up to the termination of the release study at 360 min. The difference in entrapment and release kinetics between the parent and functionalized peptide strongly supports the presumption that most of the acryloyl peptide actually intervened in the copolymerization with alkyl cyanoacrylate monomer and was covalently bound within the nanoparticles instead of being physically entrapped or adsorbed which appeared to be the case for the parent peptide. Thus, functionalizing a peptide so that it can copolymerize with the alkyl cyanoacrylate monomer is a strategy which can be used to increase the entrapment efficiency of peptides within poly(alkyl cyanoacrylate) nanoparticles and also maintain the peptide associated with nanoparticles so that the benefits of nanoparticulate delivery can be exploited.

A novel method for preparing immune stimulating complexes (ISCOMs) by hydration of freeze-dried lipid matrix.

The purpose of this study was to investigate the application of hydration of freeze-dried lipid monophase matrices as a novel technique to produce immune stimulating complexes (ISCOMs) encapsulating lipopeptides as potential sub-unit antigens. Size, polydispersity and morphology of the resulting colloidal particles were measured and characterized by photon correlation spectroscopy and transmission electron microscopy. The homogeneity of ISCOM preparations produced by this method was found to be influenced by the amount of matrix-forming material as well as the ratio of phospholipid:Quil A:cholesterol used for ISCOM preparation. Further, it was observed that more homogeneous ISCOM dispersions were produced if Quil A was included in the hydrating solution compared to incorporating Quil A in the lipid matrix. Entrapment of lipopeptide within ISCOMs was not affected by chain length (C12-C16) or the number of alkyl chains (1-3) and was greater than 80% when loaded at 5% w/w of total lipid. Entrapment efficiency was noted to decrease dramatically on increasing amount of lipopeptide in the ISCOMs from 5% to 10% of total lipid, decreasing to around 40%. All lipopeptide-loaded ISCOMs were observed to aggregate upon storage.

Analysis of Quil A-phospholipid mixtures using drift spectroscopy.

The aim of this study was to investigate molecular interactions between Quil A and phosphatidylcholine in the solid state using diffuse reflectance infrared Fourier-transform spectroscopy (DRIFTS). Analysis of the interactions was characterized on the different regions of phosphatidylcholine: hydrophobic chain, interfacial and headgroup regions. The spectra of the hydrocarbon region of phosphatidylcholine alone compared to that for the binary mixture of Quil A and phosphatidylcholine were similar. These findings suggest that Quil A did not cause conformational disorder of the fatty acyl chains of the phospholipid. In contrast, a shift in the wavenumber of the choline group and a broad band in this moiety indicate a modification of the phospholipid in the headgroup region due to interaction between Quil A and phosphatidylcholine. These results suggest possibly ionic interactions between the negatively charged glucuronic acid moiety of the Quil A molecule with the positively charged choline group. The findings could also be the result of conformational changes in the choline group because of the intercalation of sugar moieties in Quil A between the choline and phosphate groups due to hydrogen bonding. Shift of wavenumbers to lower values on the carbonyl group was observed suggesting hydrogen bonding between Quil A and phosphatidylcholine. The difference in degrees of wavenumber shift (choline>phosphate>carbonyl group) and observed broad bands indicated that Quil A preferentially interacted with phosphatidylcholine on the hydrophilic headgroup. Cholesterol influenced such interactions at relatively high concentration (60%, w/w).

Particulate systems as adjuvants and carriers for peptide and protein antigens.

The most common feature for antigen-delivery systems is their particulate nature. Together with a certain depot effect, it is the particulate nature that primarily dictates whether the antigen-delivery system will be successful in inducing a certain type and strength of immune response. In this article, we will summarize recent data on particulate delivery systems for peptide and protein antigens with a main focus on lipid or polymer-based particles, all of which possess high potential as both preventive and therapeutic vaccines for parenteral, nasal, and possibly oral administration.

Saponins from Quillaja saponaria Molina: isolation, characterization and ability to form immuno stimulatory complexes (ISCOMs).

ISCOMs have received much attention as vaccine adjuvants due to their immunostimulatory effects. They are colloidal particles typically comprised of phospholipids, cholesterol and Quil A, a crude mixture of saponins extracted from the bark of Quillaja saponaria Molina. We have previously shown that ISCOMs can be prepared by ether injection wherein an ether solution of phospholipids and cholesterol in a mass ratio of 5:2 is injected into a solution of Quil A at a mass ratio of 7 lipids: 3 Quil A. The aim of this study was firstly to isolate and characterise discrete fractions of Quil A and secondly to investigate which of these fractions were able to form ISCOMs by the method of ether injection. Six fractions of Quil A were isolated by semi-preparative reverse phase high performance liquid chromatography (RP-HPLC) and characterised by analytical HPLC, liquid chromatography tandem mass spectrometry (LC-MS) and the qualitative Liebermann-Burchard and Molisch tests for triterpenoids and carbohydrates respectively. ISCOMs were subsequently prepared from the isolated fractions by the method of ether injection and the resulting preparations characterized by photon correlation spectroscopy (PCS) and negative stain transmission electron microscopy (TEM). The molecular weights of the major compounds in the fractions ranged from approximately 1200 to approximately 2300 Da; all fractions tested positive for triterpenoids and saccharides and four of the fractions were identified as QS-7, QS-17, QS-18 and QS-21 by analysis (LC-MS and analytical HPLC). Injection of ether solutions of lipids into aqueous solutions of QS-17, QS-18 or QS-21 all resulted in homogeneous ISCOM dispersions. The combination of lipids and QS-7 by ether injection produced lamellae and liposomes as the prominent structures and a minor amount of ISCOMs. The remaining two hydrophilic, low molecular weight fractions of Quil A did not produce ISCOMs, instead liposomes and helical structures predominated in the samples.

Preparation of poly (alkylcyanoacrylate) nanoparticles by polymerization of water-free microemulsions.

Phase diagrams of the pseudoternary systems ethyloleate, polyoxyethylene 20 sorbitan mono-oleate/sorbitan monolaurate and propylene glycol with and without butanol as a co-surfactant were prepared. Areas containing optically isotropic, one-phase systems were identified and samples therein designated as droplet, bicontinuous or solution type microemulsions using conductivity, viscosity and self-diffusion NMR. Nanoparticles were prepared by polymerization of selected microemulsions with ethyl-2-cyanoacrylate and the morphology of the particles was investigated. Addition of monomer to all types of microemulsions led to the formation of nanoparticles, which had an average size of 244 +/- 25 nm, an average polydispersity index of 0.15 +/- 0.04 and a zeta-potential of -17 +/- 3 mV. The formation of particles from water-free microemulsions of different types is surprising, particularly considering that polymerization is expected to occur at a water-oil interface by base-catalysed polymerization. It would appear that propylene glycol is sufficiently nucleophilic to initiate the polymerization. The use of water-free microemulsions as templates for the preparation of poly (alkylcyanoacrylate) nanoparticles opens up interesting opportunities for the encapsulation of bioactives which do not have suitable properties for encapsulation on the basis of water-containing microemulsions.

Encapsulation of lipopeptides within liposomes: effect of number of lipid chains, chain length and method of liposome preparation.

The purpose of this study was to systematically investigate the effect of lipid chain length and number of lipid chains present on lipopeptides on their ability to be incorporated within liposomes. The peptide KAVYNFATM was synthesized and conjugated to lipoamino acids having acyl chain lengths of C8, C12 and C16. The C12 construct was also prepared in the monomeric, dimeric and trimeric form. Liposomes were prepared by two techniques: hydration of dried lipid films (Bangham method) and hydration of freeze-dried monophase systems. Encapsulation of lipopeptide within liposomes prepared by hydration of dried lipid films was incomplete in all cases ranging from an entrapment efficiency of 70% for monomeric lipoamino acids at a 5% (w/w) loading to less than 20% for di- and trimeric forms at loadings of 20% (w/w). The incomplete entrapment of lipopeptides within liposomes appeared to be a result of the different solubilities of the lipopeptide and the phospholipids in the solvent used for the preparation of the lipid film. In contrast, encapsulation of lipopeptide within liposomes prepared by hydration of freeze-dried monophase systems was high, even up to a loading of 20% (w/w) and was much less affected by the acyl chain length and number than when liposomes were prepared by hydration of dried lipid films. Freeze drying of monophase systems is better at maintaining a molecular dispersion of the lipopeptide within the solid phospholipid matrix compared to preparation of lipid film by evaporation, particularly if the solubility of the lipopeptide in solvents is markedly different from that of the polar lipids used for liposome preparation. Consequently, upon hydration, the lipopeptide is more efficiently intercalated within the phospholipid bilayers.

Lipid based particulate formulations for the delivery of antigen.

Particulate adjuvant systems are largely classified according to their functional characteristics, such as the nature of the typical immune response they induce, or their perceived mode of action. From a formulation science perspective, it is practical to classify antigen delivery systems according to the physical nature of the formulations. This article discusses lipid based particulate systems, grouped according to the nature of their predominant lipid constituent.

Quil A-lipid powder formulations releasing ISCOMs and related colloidal structures upon hydration.

The aim of the present study was to prepare solid Quil A-cholesterol-phospholipid formulations (as powder mixtures or compressed to pellets) by physical mixing or by freeze-drying of aqueous dispersions of these components in ratios that allow spontaneous formation of ISCOMs and other colloidal structures upon hydration. The effect of addition of excess cholesterol to the lipid mixtures on the release of a model antigen (PE-FITC-OVA) from the pellets was also investigated. Physical properties were evaluated by X-ray powder diffractometry (XPRD), differential scanning calorimetry (DSC), scanning electron microscopy (SEM), and polarized light microscopy (PLM). Characterization of aqueous colloidal dispersions was performed by negative staining transmission electron microscopy (TEM). Physically mixed powders (with or without PE-FITC-OVA) and pellets prepared from the same powders did not spontaneously form ISCOM matrices and related colloidal structures such as worm-like micelles, ring-like micelles, lipidic/layered structures and lamellae (hexagonal array of ring-like micelles) upon hydration as expected from the pseudo-ternary diagram for aqueous mixtures of Quil A, cholesterol and phospholipid. In contrast, spontaneous formation of the expected colloids was demonstrated for the freeze-dried lipid mixtures. Pellets prepared by compression of freeze-dried powders released PE-FITC-OVA slower than those prepared from physically mixed powders. TEM investigations revealed that the antigen was released in the form of colloidal particles (ISCOMs) from pellets prepared by compression of freeze-dried powders. The addition of excess cholesterol slowed down the release of antigen. The findings obtained in this study are important for the formulation of solid Quil A-containing lipid articles as controlled particulate adjuvant containing antigen delivery systems.

Incorporation of ovalbumin into ISCOMs and related colloidal particles prepared by the lipid film hydration method.

The aim of this study was to investigate the incorporation of a model antigen, fluorescently labelled ovalbumin (FITC-OVA), into various colloidal particles including immune stimulating complexes (ISCOMs), liposomes, ring and worm-like micelles, lamellae and lipidic/layered structures that are formed from various combinations of the triterpene saponin Quil A, cholesterol and phosphatidylethanolamine (PE) following hydration of PE/cholesterol lipid films with aqueous solutions of Quil A. Colloidal dispersions of these three components were also prepared by the dialysis method for comparison. FITC-OVA was conjugated with palmitic acid (P) and PE to produce P-FITC-OVA and PE-FITC-OVA, respectively. Both P-FITC-OVA and PE-FITC-OVA could be incorporated in all colloidal structures whereas FITC-OVA was incorporated only into liposomes. The incorporation of PE-FITC-OVA into all colloidal structures was significantly higher than P-FITC-OVA (P < 0.05). The degree of incorporation of protein was in the order: ring and worm-like micelles < liposomes and lipidic/layered structures < ISCOMs and lamellae. The incorporation of protein into the various particles prepared by the lipid film hydration method was similar to those for colloidal particles prepared by the dialysis method (provided both methods lead to the formation of the same colloidal structures). In the case of different colloidal structures arising due to the preparation method, differences in encapsulation efficiency were found (P < 0.05) for formulations with the same polar lipid composition. This study demonstrates that the various colloidal particles formed as a result of hydrating PE/cholesterol lipid films with different amounts of Quil A are capable of incorporating antigen, provided it is amphipathic. Some of these colloidal particles may be used as effective vaccine delivery systems.

Pseudo-ternary phase diagrams of aqueous mixtures of Quil A, cholesterol and phospholipid prepared by the lipid-film hydration method.

Pseudo-ternary phase diagrams of the polar lipids Quil A, cholesterol (Chol) and phosphatidylcholine (PC) in aqueous mixtures prepared by the lipid film hydration method (where dried lipid film of phospholipids and cholesterol are hydrated by an aqueous solution of Quil A) were investigated in terms of the types of particulate structures formed therein. Negative staining transmission electron microscopy and polarized light microscopy were used to characterize the colloidal and coarse dispersed particles present in the systems. Pseudo-ternary phase diagrams were established for lipid mixtures hydrated in water and in Tris buffer (pH 7.4). The effect of equilibration time was also studied with respect to systems hydrated in water where the samples were stored for 2 months at 4 degrees C. Depending on the mass ratio of Quil A, Chol and PC in the systems, various colloidal particles including ISCOM matrices, liposomes, ring-like micelles and worm-like micelles were observed. Other colloidal particles were also observed as minor structures in the presence of these predominant colloids including helices, layered structures and lamellae (hexagonal pattern of ring-like micelles). In terms of the conditions which appeared to promote the formation of ISCOM matrices, the area of the phase diagrams associated with systems containing these structures increased in the order: hydrated in water/short equilibration period

Liposomal delivery of antigen to human dendritic cells.

This study investigated whether formulation of antigen in mannosylated liposomes enhanced uptake and activation of dendritic cells (DC) and increased the ability of DC to induce primed T cell proliferation compared to formulation of antigen in unmodified liposomes or in solution. Immature human DC were generated from peripheral blood monocytes cultured with GM-CSF and IL-4. Uptake of antigen by DC and the degree of expression of the cell surface markers MHC class II, CD80, CD86 and the DC maturation marker CD83, was investigated by flow cytometry following incubation with liposomes or solution containing FITC-conjugated antigen. Exposure to liposomes containing FITC-ovalbumin resulted in enhanced expression of cell surface markers when compared to exposure to antigen in solution. Expression was highest following exposure to mannosylated liposomes. Mannosylated liposomes containing tetanus toxoid (TT) stimulated primed T cell proliferation more effectively than TT-neutral liposomes or TT-solution. This work suggests that mannosylated liposomes provide a versatile delivery vehicle for initiating enhanced immune responses to encapsulated peptide or protein vaccines.

A method for the incorporation of ovalbumin into immune stimulating complexes prepared by the hydration method.

This study describes the development of a method for the incorporation of fluorescently labelled ovalbumin (FITC-OVA) into immune stimulating complexes (ISCOMs) prepared by the hydration method. Conjugation of palmitic acid was performed to fluorescently labelled OVA (pFITC-OVA) or to non-labelled OVA, with subsequent conjugation of FITC to the resulting palmitified OVA (FITC-pOVA). Both pFITC-OVA and FITC-pOVA, but not FITC-OVA, could be incorporated into ISCOMs and other non-liposomal colloidal structures. The degree of incorporation of pFITC-OVA or FITC-pOVA in non-liposomal colloidal particles reaches a maximum, if ISCOMs are the predominant colloids in the system.