Microparticles for intranasal immunization

Of the several routes available for mucosal immunization, the nasal route is particularly attractive because of ease of administration and the induction of potent immune responses, particularly in the respiratory and genitourinary tracts. However, adjuvants and delivery systems are required to enhance immune responses following nasal immunization. This review focuses on the use of microparticles as adjuvants and delivery systems for protein and DNA vaccines for nasal immunization. In particular we discuss our own work on poly(lactide co-glycolide) (PLG) microparticles with entrapped protein or adsorbed DNA as a vaccine delivery system. The possible mechanisms involved in the enhancement of immune responses through the use of DNA adsorbed onto PLG microparticles are also discussed.     

Polylactide-co-glycolide microparticles with surface adsorbed antigens as vaccine delivery systems

Several groups have shown that vaccine antigens can be encapsulated within polymeric microparticles and can serve as potent antigen delivery systems. We have recently shown that an alternative approach involving charged polylactide co-glycolide (PLG) microparticles with surface adsorbed antigen(s) can also be used to deliver antigen into antigen presenting cell (APC). We have described the preparation of cationic and anionic PLG microparticles which have been used to adsorb a variety of agents, which include plasmid DNA, recombinant proteins and adjuvant active oligonucleotides. These PLG microparticles were prepared using a w/o/w solvent evaporation process in the presence of the anionic surfactants, including DSS (dioctyl sodium sulfosuccinate) or cationic surfactants, including CTAB (hexadecyl trimethyl ammonium bromide). Antigen binding to the charged PLG microparticles was influenced by several factors including electrostatic and hydrophobic interactions. These microparticle based formulations resulted in the induction of significantly enhanced immune responses in comparison to alum. The surface adsorbed microparticle formulation offers an alternative and novel way of delivering antigens in a vaccine formulation.     

Anionic microparticles are a potent delivery system for recombinant antigens from Neisseria meningitidis serotype B

The adsorption behavior of model proteins onto anionic poly(lactide-co-glycolide) (PLG) microparticles was evaluated. PLG microparticles were prepared by a w/o/w solvent evaporation process in the presence of the anionic surfactant dioctyl sodium sulfosuccinate (DSS). The effect of surfactant concentration and adsorption conditions on the adsorption efficiency and release rates in vitro was also studied. Subsequently, the microparticle formulation was tested to evaluate the efficacy of anionic microparticles as delivery systems for recombinant antigens from Neisseria meningitides type B (Men B), with and without CpG adjuvant. Protein (antigen) binding to anionic PLG microparticles was influenced by both electrostatic interaction and by other mechanisms, including hydrophobic attraction. The Men B antigens adsorbed efficiently onto anionic PLG microparticles and, following immunization in mice, induced potent enzyme-linked immunosorbent assay (ELISA) and serum bactericidal activity in comparison to alum-adsorbed formulations. These Men B antigens represent an attractive approach for vaccine development.     

Recent developments in vaccine delivery systems

New generation vaccines, particularly those based on recombinant proteins and DNA, are likely to be less reactogenic than traditional vaccines, but are also less immunogenic. Therefore, there is an urgent need for the development of new and improved vaccine adjuvants. Adjuvants can be broadly separated into two classes, based on their principal mechanisms of action; vaccine delivery systems and 'immunostimulatory adjuvants'. Vaccine delivery systems are generally particulate e.g. emulsions, microparticles, iscoms and liposomes, and mainly function to target associated antigens into antigen presenting cells (APC), including macrophages and dendritic cells. This review will focus on recent developments in vaccine delivery systems. Immunostimulatory adjuvants are predominantly derived from pathogens and often represent pathogen associated molecular patterns (PAMP) e.g. LPS, MPL, CpG DNA, which activate cells of the innate immune system. Once activated, cells of innate immunity drive and focus the acquired immune response. In some studies, delivery systems and immunostimulatory agents have been combined for more effective delivery of the immunostimulatory adjuvant into APC. A rational approach to the development of new and more effective vaccine adjuvants will require much further work to better define the mechanisms of action of existing adjuvants. The discovery of more potent adjuvants may allow the development of vaccines against infectious agents such as HIV which do not naturally elicit protective immunity. New adjuvants and delivery system combinations may also allow vaccines to be delivered mucosally.     

The potency of the adjuvant, CpG oligos, is enhanced by encapsulation in PLG microparticles

The objective of this work was to evaluate the potency of the CpG containing oligonucleotide encapsulated within poly(lactide-co-glycolide), and coadministered with antigen adsorbed to poly(lactide-co-glycolide) microparticles (PLG particles). The formulations evaluated include, CpG added in soluble form, CpG adsorbed, and CpG encapsulated. The antigen from Neisseria meningitidis serotype B (Men B) was used in these studies. The immunogenicity of these formulations was evaluated in mice. Poly(lactide-co-glycolide) microparticles were synthesized by a w/o/w emulsification method in the presence of a charged surfactant for the formulations. Neisseria meningitidis B protein was adsorbed to the PLG microparticles, with binding efficiency and initial release measured. CpG was either added in the soluble or adsorbed or encapsulated form based on the type of formulation. The binding efficiency, loading, integrity and initial release of CpG and the antigen were measured from all the formulations. The formulations were then tested in mice for their ability to elicit antibodies, bactericidal activity and T cell responses. Encapsulating CpG within PLG microparticles induced statistically significant higher antibody, bactericidal activity and T cell responses when compared to the traditional method of delivering CpG in the soluble form.     

A comparison of anionic nanoparticles and microparticles as vaccine delivery systems

The objective of this work was to conduct an in vivo comparison of nanoparticles and microparticles as vaccine delivery systems. Poly (lactide-co-glycolide) (PLG) polymers were used to create nanoparticles size 110 nm and microparticles of size 800-900 nm. Protein antigens were then adsorbed to these particles. The efficacy of these delivery systems was tested with two protein antigens. A recombinant antigen from Neisseria meningitides type B (MenB) was administered intramuscularly (i.m.) or intraperitonealy (i.p.). An antigen from HIV-1, env glycoprotein gp140 was administered intranasally (i.n.) followed by an i.m. boost. From three studies, there were no differences between the nanoparticles and micro-particles formulations. Both particles led to comparable immune responses in mice. The immune responses for MenB (serum bactericidal activity and antibody titers) were equivalent to the control of aluminum hydroxide. For the gp140, the LTK63 was necessary for high titers. Both nanoparticles and microparticles are promising delivery systems.     

A comparative study of the antigen-specific immune response induced by co-delivery of CpG ODN and antigen using fusion molecules or biodegradable microparticles

CpG ODN are toll-like receptor 9 (TLR9) agonists that can enhance antigen presentation by antigen presenting cells (APCs) such as dendritic cells (DCs). The most potent antigen-specific responses are seen when CpG ODN and the antigen are co-localized in the same APC. CpG ODN-antigen fusion molecules and biodegradable microparticles entrapping CpG ODN and antigen can ensure both components are delivered to the same APC. In this study, we compared the efficacy of the CpG-ODN fusion molecules against biodegradable microparticles entrapping antigen and CpG ODN. Microparticles were prepared using a double emulsion solvent evaporation methodology. CpG ODN-OVA fusion molecules were prepared by mixing maleimide-activated protein with thiolated CpG ODN. Both CpG ODN-OVA fusion molecules and microparticles co-entrapping CpG ODN and OVA generated stronger IgG2a and interferon-gamma (IFN-gamma) responses than delivery of soluble CpG ODN and OVA. The microparticles generated stronger IgG2a and IFN-gamma immune responses than did CpG ODN-antigen fusion molecules.     

Evaluation of nano- and microparticle uptake by the gastrointestinal tract

Numerous papers over the last two decades have demonstrated that particle uptake by the gastrointestinal tract is a reality. In addition, polymeric nano- and microparticles have proved to be useful delivery systems to enhance oral bioavailability of poorly absorbed drugs or to induce mucosal immune response. However, despite the amount of data available, no set criteria are available for the design of a good particulate carrier for oral delivery of peptides or antigens. This is partly due to the publication of conflicting and confusing data. The source of discrepancy is actually multiparametric (e.g., methodology, mode of evaluation, animal species) and is still not fully understood. The purpose of this review is to discuss the advantages and the limitations of the methodologies and the models used to evaluate gastrointestinal uptake of nano- and microparticles.     

Innovative strategies for co-delivering antigens and CpG oligonucleotides

Cytosine-phosphorothioate-guanine oligodeoxynucleotides (CpG ODN) is a recent class of immunostimulatory adjuvants that includes unmethylated CpG dinucleotide sequences similar to those commonly found in bacterial DNA. CpG ODN specifically triggers toll like receptor 9 (TLR9), which is found within phagoendosomes of antigen presenting cells (APCs) such as dendritic cells (DCs). CpG ODN triggers activation and maturation of DCs and helps to increase expression of antigens. CpG ODN can be used to induce polarized Th1 type immune responses. Several studies have shown that antigens and CpG ODN must be co-localized in the same APC to generate the most potent therapeutic antigen-specific immune responses. Delivery vehicles can be utilized to ensure co-delivery of antigens and CpG ODN to the same APCs and to significantly increase uptake by APCs. These strategies can result in antigen-specific immune responses that are 5 to 500-fold greater than administration of antigen alone. In this review, we discuss several recent and innovative strategies to co-delivering antigens and CpG ODN adjuvants to APCs. These approaches include the utilization of conjugate molecules, multi-component nanorods, liposomes, biodegradable microparticles, pulsatile release chips and cell-microparticle hybrids.     

Adsorption of a Novel Recombinant Glycoprotein from HIV (Env gp120dV2 SF162) to Anionic PLG Microparticles Retains the Structural Integrity of the Protein,Whereas Encapsulation in PLG Microparticles Does Not

No HeadingPurpose. To evaluate the delivery of a novel HIV-1 antigen (gp120dV2 SF162) by surface adsorption or encapsulation within polylactide-co-glycolide microparticles and to compare both the formulations for their ability to preserve functional activity as measured by binding to soluble CD4.Methods. Poly(lactide-co-glycolide) microparticles were synthesized by a water-in-oil-in-water (w/o/w) emulsification method in the presence of the anionic surfactant dioctylsulfosuccinate (DSS) or polyvinyl alcohol. The HIV envelope glyocoprotein was adsorbed and encapsulated in the PLG particles. Binding efficiency and burst release measured to determine adsorption characteristics. The ability to bind CD4 was assayed to measure the functional integrity of gp120dV2 following different formulation processes.Results. Protein (antigen) binding to PLG microparticles was influenced by both electrostatic interaction and other mechanisms such as hydrophobic attraction and structural accommodation of the polymer and biomolecule. The functional activity as measured by the ability of gp120dV2 to bind CD4 was maintained by adsorption onto anionic microparticles but drastically reduced by encapsulation.Conclusions. The antigen on the adsorbed PLG formulation maintained its binding ability to soluble CD4 in comparison to encapsulation, demonstrating the feasibility of using these novel anionic microparticles as a potential vaccine delivery system.     

Lipid-based delivery of CpG oligonucleotides enhances immunotherapeutic efficacy

There has been significant interest in the potential of cytosine-guanine (CpG) containing oligodeoxynucleotides (ODN) as an immunotherapy for malignant, infectious and allergic diseases. While human trials have yielded promising results, clinical use of free CpG ODN still faces several challenges which limit their effectiveness. These include suboptimal in vivo stability, toxicity, unfavorable pharmacokinetic/biodistribution characteristics, lack of specificity for target cells and the requirement for intracellular uptake. To overcome these challenges, optimized lipid-based delivery systems have been developed to protect the CpG ODN payload, modify their circulation/distribution so as to enhance immune cell targeting and facilitate intracellular uptake. Ultimately, lipid-mediated delivery has the capacity to increase the immunopotency of CpG ODN and enhance their prophylactic or therapeutic efficacy in a range of diseases. Lipid-encapsulation provides a feasible strategy to optimize the immunostimulatory activity and immunotherapeutic efficacy of CpG ODN, thereby allowing their full clinical potential to be realized.     

Inactive Vibrio cholerae whole-cell vaccine-loaded biodegradable microparticles: in vitro release and oral vaccination

An approach is proposed using Vibrio cholerae (VC)-loaded microparticles as oral vaccine delivery systems for improved vaccine bioavailability and increased therapeutic efficacy. The VC-loaded microparticles were prepared with 50:50 poly(DL-lactide-co-glycolide) (PLG), 75:25 poly(DL-lactide-co-glycolide) and poly(lactide acid) (PLA)/PEG blend copolymers by the solvent evaporation method. VC was successfully entrapped in three types of microparticles with loading efficiencies and loading levels as follows: 50:50 PLG systems: 97.8% and 55.4 +/- 6.9 micro g/mg; 75:25 PLG systems: 89.2% and 46.5 +/- 4.4 micro g/mg; PLA/PEG-blended systems: 82.6% and 53.7 +/- 5.8 micro g/mg. The different distributions of VC in the core region and on the surface were as follows: 50:50 PLG systems 25.7 +/- 1.9 and 6.2 +/- 0.9 micro g/mg; 75:25 PLG systems: 25.8 +/- 2.2 and 3.6 +/- 0.4 micro g/mg; PLA/PEG-blended systems: 32.4 +/- 2.1 and 5.2 +/- 1.0 micro g/mg, respectively. In vitro active release of VC was affected mainly by matrix type and VC-loaded location in microparticles. The therapeutic immunogenic potential of VC loaded with 50:50 PLG, 75:25 PLG and PLA/PEG-blended microparticles was evaluated in adult mice by oral immunization. Significantly higher antibody responses and serum immunoglobin Ig G, IgA and IgM responses were obtained when sera from both VC-loaded 75:25 PLG and PLA/PEG-blended microparticles immunized mice were titrated against VC. The most immunogenicity in evoking serum IgG, IgA and IgM responses was immunized by VC-loaded PLA/PEG-blended microparticles, and with VC challenge in mice, the survival rate (91.7%).     

The Preparation and Characterization of Poly(lactide-co-glycolide) Microparticles. II. The Entrapment of a Model Protein Using a (Water-in-Oil)-in-Water Emulsion Solvent Evaporation Technique

Poly(lactide-co-glycolide) (PLG) microparticles with entrapped antigens have recently been investigated as controlled-release vaccines. This paper describes the preparation of PLG microparticles with an entrapped model antigen, ovalbumin (OVA), using a (water-in-oil)-in-water emulsion solvent evaporation technique. In a series of experiments, the effects of process parameters on particle size and OVA entrapment were investigated. It was found that smooth, spherical microparticles 1–2 µm in diameter containing up to 10% (w/w) OVA could be produced using a small volume of external aqueous phase containing a high concentration of emulsion stabilizer and a 1:5 antigen:polymer ratio. PAGE analysis, isoelectric focusing, and Western blotting of OVA released from the microparticles in vitro confirmed that the molecular weight and antigenicity of the protein remained largely unaltered by the entrapment procedure.     

Inactive Vibrio cholerae whole-cell vaccine-loaded biodegradable microparticles: in vitro release and oral vaccination

An approach is proposed using Vibrio cholerae (VC)-loaded microparticles as oral vaccine delivery systems for improved vaccine bioavailability and increased therapeutic efficacy. The VC-loaded microparticles were prepared with 50:50 poly(DL-lactide-co-glycolide) (PLG), 75:25 poly(DL-lactide-co-glycolide) and poly(lactide acid) (PLA)/PEG blend copolymers by the solvent evaporation method. VC was successfully entrapped in three types of microparticles with loading efficiencies and loading levels as follows: 50:50 PLG systems: 97.8% and 55.4 ± 6.9 µg/mg; 75:25 PLG systems: 89.2% and 46.5 ± 4.4?µg/mg; PLA/PEG-blended systems: 82.6% and 53.7 ± 5.8?µg/mg. The different distributions of VC in the core region and on the surface were as follows: 50:50 PLG systems 25.7 ± 1.9 and 6.2 ± 0.9?µg/mg; 75:25 PLG systems: 25.8 ± 2.2 and 3.6 ± 0.4?µg/mg; PLA/PEG-blended systems: 32.4 ± 2.1 and 5.2 ± 1.0?µg/mg, respectively. In vitro active release of VC was affected mainly by matrix type and VC-loaded location in microparticles. The therapeutic immunogenic potential of VC loaded with 50:50 PLG, 75:25 PLG and PLA/PEG-blended microparticles was evaluated in adult mice by oral immunization. Significantly higher antibody responses and serum immunoglobin Ig G, IgA and IgM responses were obtained when sera from both VC-loaded 75:25 PLG and PLA/PEG-blended microparticles immunized mice were titrated against VC. The most immunogenicity in evoking serum IgG, IgA and IgM responses was immunized by VC-loaded PLA/PEG-blended microparticles, and with VC challenge in mice, the survival rate (91.7%).     

Recent Advances in Vaccine Adjuvants

New generation vaccines, particularly those based on recombinant proteins and DNA, are likely to be less reactogenic than traditional vaccines but are also less immunogenic. Therefore, there is an urgent need for the development of new and improved vaccine adjuvants. Adjuvants can be broadly separated into two classes based on their principal mechanisms of action: vaccine delivery systems and immunostimulatory adjuvants. Vaccine-delivery systems generally are particulate (e.g., emulsions, microparticles, iscoms, and liposomes)and function mainly to target associated antigens into antigen-resenting cells. In contrast, immunostimulatory adjuvants are derived predominantly from pathogens and often represent pathogen-ssociated molecular patterns (e.g., lipopolysaccaride, monophosphoryl lipid A, CpG DNA), which activate cells of the innate immune system. Recent progress in innate immunity is beginning to yield insight into the initiation of immune responses and the ways in which immunostimulatory adjuvants may enhance this process. The discovery of more potent adjuvants may allow the development of prophylactic and therapeutic vaccines against cancers and chronic infectious diseases. In addition, new adjuvants may also allow vaccines to be delivered mucosally.     

Characterization of antigens adsorbed to anionic PLG microparticles by XPS and TOF-SIMS

The chemical composition of the surface of anionic PLG microparticles before and after adsorption of vaccine antigens was measured using X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (TOF-SIMS). The interfacial distributions of components will reflect underlying interactions that govern properties such as adsorption, release, and stability of proteins in microparticle vaccine delivery systems. Poly(lactide-co-glycolide) microparticles were prepared by a w/o/w emulsification method in the presence of the anionic surfactant dioctyl sodium sulfosuccinate (DSS). Ovalbumin, lysozyme, a recombinant HIV envelope glyocoprotein and a Neisseria meningitidis B protein were adsorbed to the PLG microparticles, with XPS and time-of-flight secondary mass used to analyze elemental and molecular distributions of components of the surface of lyophilized products. Protein (antigen) binding to PLG microparticles was measured directly by distinct elemental and molecular spectroscopic signatures consistent with amino acids and excipient species. The surface sensitive composition of proteins also included counter ions that support the importance of electrostatic interactions being crucial in the mechanism of adsorptions. The protein binding capacity was consistent with the available surface area and the interpretation of previous electron and atomic force microscope images strengthened by the quantification possible by XPS and the qualitative identification possible with TOF-SIMS. Protein antigens were detected and quantified on the surface of anionic PLG microparticles with varying degrees of efficiency under different adsorption conditions such as surfactant level, pH, and ionic strength. Observable changes in elemental and molecular composition suggest an efficient electrostatic interaction creating a composite surface layer that mediates antigen binding and release.     

Transport of Chitosan Microparticles for Mucosal Vaccine Delivery in a Human Intestinal M-cell Model

Uptake of particulate antigen carrier systems by specialized M-cells of the gut-associated lymphoid tissue is still a limiting step in inducing efficient immune responses after oral vaccination. Although transport of soluble drugs over the epithelial barrier of the gut is extensively studied in vitro by using the Caco-2 cell model, this was for long time not possible for particles due to the absence of M-cells. By co-culturing Caco-2 cells with cultured human B-lymphocytes (Raji-cells), cells which are morphologically and functionally similar to M-cells can be induced. This human M-cells model makes it possible to study the uptake of microparticles for oral vaccine delivery. In this way, chitosan microparticles, which have demonstrated to target the Peyer's patches efficiently in vivo, could be tested in vitro. The development of this M-cells model facilitates the optimization of the microparticles in order to target them even more efficiently to the M-cells in the gut. In this study, the integrity of the human M-cell model was investigated by determining the transepithelial electrical resistance (TEER), 14 C-mannitol transport and morphology using scanning electron microscopy. The uptake of particles was investigated by measuring transport of both fluorescently labeled microspheres (Fluospheres ®) and chitosan microparticles using flowcytometry. No discontinuities or abnormalities could be found in the co-culture. Scanning electron microscopy showed that morphologically different cells were present in the human M-cell model. Both commercially available Fluospheres ® (size 0.2 μ m) and chitosan microparticles (size 1.7 μ m) for oral vaccine delivery were transported at a significantly higher amount by the human M-cell model compared to the transport by the Caco-2 cell monoculture. Since chitosan microparticles were proven to be taken up by Peyer's patches in mice as well, this human M-cell model is able to predict the M-cell uptake of microparticles for oral vaccine delivery. This M-cell model is a new tool, which can be used to scan, develop and optimize microparticles for oral vaccine delivery. Since the M-cell uptake can now be studied in vitro, the targeting of these cells can be studied more efficiently and can now be done in cells from human origin.     

Biodegradable microparticles as a delivery system for the allergens of Dermatophagoides pteronyssinus (house dust mite): I. Preparation and characterization of microparticles

The encapsulation of allergens into biodegradable microparticles may offer the potential for novel forms of hyposensitization therapy. The use of microparticles for hyposensitization therapy may offer the potential advantages of a reduced number of doses, through controlled release of allergens, and the possibility of oral delivery. Nevertheless, a possible concern is the effect of the microencapsulation process on the integrity and activity of the entrapped allergens. Therefore, in the current studies, an allergen, Dermatophagoides pteronyssinus (D.Pt.), was entrapped in poly(lactide-co-glycolide) (PLG) microparticles and several established techniques were used to investigate the integrity of the entrapped allergen. Isoelectric focusing indicated that all the protein components of the allergen were successfully entrapped in the microparticles. A radioallergosorbent test (RAST) inhibition assay indicated that there was a minor reduction in the binding of specific IgE to the entrapped allergen, but this was thought unlikely to affect the ability of the allergen to act as a hyposensitizing agent. Finally, the IgG binding activity of the major allergenic component of D.Pt. (Der P1) was shown to be unchanged following microencapsulation. Hence, the current studies indicated that the allergen D.Pt., was not adversely affected following encapsulation into PLG microparticles. Therefore, microparticles may have potential as delivery systems for hyposensitization therapy.     

Biodegradable Microparticles Loaded with Doxorubicin and CpG ODN for In Situ Immunization Against Cancer

In situ immunization is based on the concept that it is possible to break immune tolerance by inducing tumor cell death in situ in a manner that provides antigen-presenting cells such as dendritic cells (DCs) with a wide selection of tumor antigens that can then be presented to the immune system and result in a therapeutic anticancer immune response. We designed a comprehensive approach to in situ immunization using poly(lactic-co-glycolic acid) (PLGA)-biodegradable microparticles (MPs) loaded with doxorubicin (Dox) and CpG oligodeoxynucleotides (CpG) that deliver Dox (chemotherapy) and CpG (immunotherapy) in a sustained-release fashion when injected intratumorally. Dox induces immunogenic tumor cell death while CpG enhances tumor antigen presentation by DCs. PLGA MPs allow their safe co-delivery while evading the vesicant action of Dox. In vitro, we show that Dox/CpG MPs can kill B and T lymphoma cells and are less toxic to DCs. In vivo, Dox/CpG MPs combined with antibody therapy to enhance and maintain the T cell response generated systemic immune responses that suppressed injected and distant tumors in a murine B lymphoma model, leading to tumor-free mice. The combination regimen was also effective at reducing T cell lymphoma and melanoma tumor burdens. In conclusion, Dox/CpG MPs represent an efficient and safe tool for in situ immunization that could provide a promising component of immunotherapy for patients with a variety of types of cancer.KEY WORDS: cancer vaccine, CpG, doxorubicin, in situ immunization, microparticles, PLGA     

Transport of chitosan microparticles for mucosal vaccine delivery in a human intestinal M-cell model

Uptake of particulate antigen carrier systems by specialized M-cells of the gut-associated lymphoid tissue is still a limiting step in inducing efficient immune responses after oral vaccination. Although transport of soluble drugs over the epithelial barrier of the gut is extensively studied in vitro by using the Caco-2 cell model, this was for long time not possible for particles due to the absence of M-cells. By co-culturing Caco-2 cells with cultured human B-lymphocytes (Raji-cells), cells which are morphologically and functionally similar to M-cells can be induced. This human M-cells model makes it possible to study the uptake of microparticles for oral vaccine delivery. In this way, chitosan microparticles, which have demonstrated to target the Peyer's patches efficiently in vivo, could be tested in vitro. The development of this M-cells model facilitates the optimization of the microparticles in order to target them even more efficiently to the M-cells in the gut. In this study, the integrity of the human M-cell model was investigated by determining the transepithelial electrical resistance (TEER), 14C-mannitol transport and morphology using scanning electron microscopy. The uptake of particles was investigated by measuring transport of both fluorescently labeled microspheres (Fluospheres) and chitosan microparticles using flowcytometry. No discontinuities or abnormalities could be found in the co-culture. Scanning electron microscopy showed that morphologically different cells were present in the human M-cell model. Both commercially available Fluospheres (size 0.2 microm) and chitosan microparticles (size 1.7 microm) for oral vaccine delivery were transported at a significantly higher amount by the human M-cell model compared to the transport by the Caco-2 cell monoculture. Since chitosan microparticles were proven to be taken up by Peyer's patches in mice as well, this human M-cell model is able to predict the M-cell uptake of microparticles for oral vaccine delivery. This M-cell model is a new tool, which can be used to scan, develop and optimize microparticles for oral vaccine delivery. Since the M-cell uptake can now be studied in vitro, the targeting of these cells can be studied more efficiently and can now be done in cells from human origin.