In vivo uptake of chitosan microparticles by murine Peyer's patches: visualization studies using confocal laser scanning microscopy and immunohistochemistry

Although oral vaccination has numerous advantages over parenteral injection, degradation of the vaccine and low uptake by the gut associated lymphoid tissue (GALT) still complicate the development of efficient oral vaccines. However, previous studies in our laboratory demonstrated that chitosan microparticles can have suitable size, charge, loading and release characteristics for oral vaccination using ovalbumin as model vaccine. In this study, two different approaches were used to investigate the in vivo uptake of chitosan microparticles by murine Peyer's patches. Firstly, a confocal laser scanning microscopy (CLSM) study was performed to visualize the uptake of fluorescent-labeled chitosan microparticles in the Peyer's patches after intragastrical feeding. Subsequently, the intestinal epithelial uptake of ovalbumin loaded chitosan microparticles was visualized using immunohistochemical staining of ovalbumin. Because the microparticles are biodegradable, this entrapped ovalbumin will be released after intracellular digestion in the Peyer's patches. CLSM visualization demonstrated that chitosan microparticles enhance the uptake of fluorescent-labeled ovalbumin by the epithelium of the Peyer's patches. No ovalbumin uptake by the intestinal epithelium was observed when the protein was administered without microparticles. Moreover, immunohistochemical visualization studies revealed that ovalbumin could only be transported into the Peyer's patches after association to chitosan microparticles. Since uptake by Peyer's patches is an essential step in oral vaccination, these in vivo experiments demonstrate that chitosan microparticles are very promising vaccine delivery systems.     

Chitosan-based delivery systems for mucosal vaccines

Introduction: Mucosal vaccine development faces several challenges and opportunities. Critical issues for effective mucosal vaccination include the antigen-retention period that enables interaction with the lymphatic system, choice of adjuvant that is nontoxic and induces the required immune response and possibly an ability to mimic mucosal pathogens. Chitosan-based delivery systems are reviewed here as they address these issues and hence represent the most promising candidates for the delivery of mucosal vaccines. Areas covered: A comprehensive literature search was conducted, to locate relevant studies published within the last 5 years. Mucosal delivery via nasal and oral routes is evaluated with respect to chitosan type, dosage forms, co-adjuvanting with novel adjuvants and modulation of the immune system. Expert opinion: It is concluded that chitosan derivatives offer advantageous opportunities such as nanoparticle and surface charge manipulation that facilitate vaccine targeting. Nevertheless, these technologies represent a longer-term goal. By contrast, chitosan (unmodified form) with or without a co-adjuvant has significant toxicology and human data to support safe mucosal administration, and thus has the potential for earlier product introduction into the market.     

Chitosan and its derivatives in mucosal drug and vaccine delivery.

Numerous studies have demonstrated that chitosan and their derivatives (N-trimethyl chitosan, mono-N-carboxymethyl chitosan) are effective and safe absorption enhancers to improve mucosal (nasal, peroral) delivery of hydrophylic macromolecules such as peptide and protein drugs and heparins. This absorption enhancing effect of chitosans is caused by opening of the intercellular tight junctions, thereby favouring the paracellular transport of macromolecular drugs. Chitosan nano- and microparticles are also suitable for controlled drug release. Association of vaccines to some of these particulate systems has shown to enhance the antigen uptake by mucosal lymphoid tissues, thereby inducing strong systemtic and mucosal immune responses against the antigens. The aspecific adjuvant activity of chitosans seems to be dependent on the degree of deacetylation and the type of formulation. From the studies reviewed it is concluded that chitosan and chitosan derivatives are promising polymeric excipients for mucosal drug and vaccine delivery.     

Nanoparticles for nasal vaccination

The great interest in mucosal vaccine delivery arises from the fact that mucosal surfaces represent the major site of entry for many pathogens. Among other mucosal sites, nasal delivery is especially attractive for immunization, as the nasal epithelium is characterized by relatively high permeability, low enzymatic activity and by the presence of an important number of immunocompetent cells. In addition to these advantageous characteristics, the nasal route could offer simplified and more cost-effective protocols for vaccination with improved patient compliance.The use of nanocarriers provides a suitable way for the nasal delivery of antigenic molecules. Besides improved protection and facilitated transport of the antigen, nanoparticulate delivery systems could also provide more effective antigen recognition by immune cells. These represent key factors in the optimal processing and presentation of the antigen, and therefore in the subsequent development of a suitable immune response. In this sense, the design of optimized vaccine nanocarriers offers a promising way for nasal mucosal vaccination.     

Chitosan-based delivery systems for mucosal vaccines

Introduction: Mucosal vaccine development faces several challenges and opportunities. Critical issues for effective mucosal vaccination include the antigen-retention period that enables interaction with the lymphatic system, choice of adjuvant that is nontoxic and induces the required immune response and possibly an ability to mimic mucosal pathogens. Chitosan-based delivery systems are reviewed here as they address these issues and hence represent the most promising candidates for the delivery of mucosal vaccines.

Areas covered: A comprehensive literature search was conducted, to locate relevant studies published within the last 5 years. Mucosal delivery via nasal and oral routes is evaluated with respect to chitosan type, dosage forms, co-adjuvanting with novel adjuvants and modulation of the immune system.

Expert opinion: It is concluded that chitosan derivatives offer advantageous opportunities such as nanoparticle and surface charge manipulation that facilitate vaccine targeting. Nevertheless, these technologies represent a longer-term goal. By contrast, chitosan (unmodified form) with or without a co-adjuvant has significant toxicology and human data to support safe mucosal administration, and thus has the potential for earlier product introduction into the market.     

Rational design of nasal vaccines

Nasal vaccination is a promising alternative to classical parental vaccination, as it is non-invasive and, in principle, capable of eliciting strong systemic and local immune responses. However, the protective efficacy of nasally administered antigens is often impaired because of delivery problems: free antigens are readily cleared from the nasal cavity, poorly absorbed by nasal epithelial cells and generally have low intrinsic immunogenicity. In this review paper, we describe the main physiological hurdles to nasal vaccine delivery, survey the progress made in technological approaches to overcome these hurdles and discuss emerging opportunities for improving nasal vaccines. According to current insights, encapsulation of the antigen into bioadhesive (nano)particles is a promising approach towards successful nasal vaccine delivery. These antigen-loaded particles can be tailor made by supplying them with targeting ligands, adjuvants or endosomal escape mediators to form the desired vaccine that provides long-lasting protective immunity.     

Polymeric lamellar substrate particles for intranasal vaccination

In recent years, several strategies have been under investigation to achieve safe and effective immunisation, in terms of new antigens, adjuvants and routes of vaccination. The latter include mucosal sites such as oral, rectal, vaginal and nasal. Biodegradable microparticles produced from polymers such as poly(D,L-lactide) (PLA) and poly(D,L-lactide-co-glycolide) (PLGA) containing encapsulated vaccine antigens have been extensively studied for immunisation. These microparticles allow controlled release of vaccines with the aim to develop as single dose vaccines. However there are concerns regarding the integrity and immunogenicity of the antigen during the encapsulation process when the antigen is exposed to organic solvents, high shear stresses and the exposure of antigen to low pH which is caused by polymer degradation. Polymeric lamellar substrate particles (PLSP) produced by simple precipitation of PLA, form a novel polymeric system for the adsorption of antigens. This procedure avoids pH changes, exposure to organic solvents and hence allows the integrity of the antigen to be retained. The aim of this article is to discuss the factors affecting the characteristics of PLSP and adsorption of antigens onto PLSP and consider their potential as adjuvants for the nasal delivery of protein, peptide or viral vaccines.     

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.     

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.     

Uptake of Ovalbumin-conjugated Starch Microparticles by Pig Respiratory Nasal Mucosa In Vitro

The uptake of ovalbumin-conjugated starch microparticles (OVA-MP) was studied after application to porcine respiratory nasal mucosa in vitro. Nasal mucosa from freshly slaughtered pigs was mounted in horizontal Ussing chambers, which permit monitoring of the viability of the tissue exposed to microparticles and ensure that the microparticles are deposited on the mucosa. The antigen-conjugated starch microparticles have previously been shown to produce strong mucosal, cellular and systemic immune responses to conjugated model antigens following oral administration. Intranasal administration of vaccines for mucosal immunisation is an interesting alternative to oral administration, since nasal delivery systems generally require lower doses of antigen and the site of application is better suited for protection against air-borne antigens. Most of a nasally administered dose is deposited on the surface of the respiratory area of the nasal mucosa. It is therefore important to examine whether the microparticles are taken up in this area and, if so, by which cell type.

Confocal laser scanning microscopy and transmission electron microscopy (TEM) of the nasal tissue both showed intracellular OVA-MP in non-ciliated epithelial cells after 45 min' incubation. The morphology of the cells in the TEM preparations did not support the presence of either M cells (specialised antigen sampling cells) or adjacent lymphocytes. Anticytokeratin-18 (Ac18) was used as a potential M cell marker. However, there was no indication of Ac18 binding to M cells, but it did bind to mucus-producing cells in the respiratory nasal mucosa. In conclusion, OVA-MP were taken up intracellularly by non-ciliated epithelial cells in the nasal respiratory mucosa of pigs, in vitro.     

疫苗口服接种及其微粒传输系统

为说明疫苗口服接种产生黏膜免疫的生理学基础，突出微粒作为口服疫苗载体的研究意义，本文分析了肠系淋巴组织的抗原呈递及黏膜免疫反应特点，并结合肠道吸收屏障，进一步讨论微粒载体经肠道的摄取和转运，阐述疫苗微粒口服接种的研究概况。参与免疫调节的M-细胞和派伊尔集合淋巴结是口服疫苗产生免疫应答的重要部位，采用微粒作为疫苗转运载体，可克服肠道屏障的影响，赋予了口服疫苗以新的内涵，特别是凝集素化微粒在提高疫苗转运及免疫接种效率方面的作用。可见经肠黏膜免疫系统进行的疫苗口服接种，通过微粒载体介导，将实现定位触发和效应放大，具有潜在的研究和应用价值。     

Development of chitosan–pullulan composite nanoparticles for nasal delivery of vaccines: in vivo studies

Abstract

Here, we aimed at developing chitosan/pullulan composite nanoparticles and testing their potential as novel systems for the nasal delivery of diphtheria toxoid (DT). All the chitosan derivatives [N-trimethyl (TMC), chloride and glutamate] and carboxymethyl pullulan (CMP) were synthesised and antigen-loaded composites were prepared by polyion complexation of chitosan and pullulan derivatives (particle size: 239–405?nm; surface charge: +18 and +27?mV). Their immunological effects after intranasal administration to mice were compared to intramuscular route. Composite nanoparticles induced higher levels of IgG responses than particles formed with chitosan derivative and antigen. Nasally administered TMC–pullulan composites showed higher DT serum IgG titre when compared with the other composites. Co-encapsulation of CpG ODN within TMC-CMP-DT nanoparticles resulted in a balanced Th₁/Th₂ response. TMC/pullulan composite nanoparticles also induced highest cytokine levels compared to those of chitosan salts. These findings demonstrated that TMC-CMP-DT composite nanoparticles are promising delivery system for nasal vaccination.     

Bioadhesive delivery systems for mucosal vaccine delivery

Mucosal vaccine delivery potentially induces mucosal as well as systemic immune responses and may have advantages particularly for optimal protection against pathogens that infect the host through mucosal surfaces. However, the delivery of antigens through mucosal membranes remains a major challenge due to unfavorable physiological conditions (pH and enzymes) and significant biological barriers, which restrict the uptake of antigens. To improve mucosal vaccine delivery, the use of bioadhesive delivery systems offers numerous advantages, including protection from degradation, increasing concentration of antigen in the vicinity of mucosal tissue for better absorption, extending their residence time, and/or targeting them to sites of antigen uptake. Although some bioadhesives have direct immune stimulating properties, it appears most likely that successful mucosal vaccination will require the addition of vaccine adjuvants for optimal immune responses, particularly if they are to be used in an unprimed population. Thus, complex vaccine formulations and delivery strategies have to be carefully designed to appropriately stimulate immune response for the target pathogen. In addition, careful consideration is needed to define the “best” route for mucosal immunization for each individual pathogen.     

Uptake of ovalbumin-conjugated starch microparticles by pig respiratory nasal mucosa in vitro

The uptake of ovalbumin-conjugated starch microparticles (OVA-MP) was studied after application to porcine respiratory nasal mucosa in vitro. Nasal mucosa from freshly slaughtered pigs was mounted in horizontal Ussing chambers, which permit monitoring of the viability of the tissue exposed to microparticles and ensure that the microparticles are deposited on the mucosa. The antigen-conjugated starch microparticles have previously been shown to produce strong mucosal, cellular and systemic immune responses to conjugated model antigens following oral administration. Intranasal administration of vaccines for mucosal immunisation is an interesting alternative to oral administration, since nasal delivery systems generally require lower doses of antigen and the site of application is better suited for protection against air-borne antigens. Most of a nasally administered dose is deposited on the surface of the respiratory area of the nasal mucosa. It is therefore important to examine whether the microparticles are taken up in this area and, if so, by which cell type. Confocal laser scanning microscopy and transmission electron microscopy (TEM) of the nasal tissue both showed intracellular OVA-MP in non-ciliated epithelial cells after 45 min' incubation. The morphology of the cells in the TEM preparations did not support the presence of either M cells (specialised antigen sampling cells) or adjacent lymphocytes. Anticytokeratin-18 (Ac18) was used as a potential M cell marker. However, there was no indication of Ac18 binding to M cells, but it did bind to mucus-producing cells in the respiratory nasal mucosa. In conclusion, OVA-MP were taken up intracellularly by non-ciliated epithelial cells in the nasal respiratory mucosa of pigs, in vitro.     

Intranasal administration of peptide antigens of HIV with mucosal adjuvant CpG ODN coentrapped in microparticles enhances the mucosal and systemic immune responses

The mucosal immune system acts as a first line of defense against infection caused by luminal pathogens. Because HIV is transmitted primarily via mucosal associated tissues, particularly with sexual transmission, understanding antiviral immunity present at these sites is important. As most of the peptide antigens show poor immunogenicity when immunized alone but after incorporating the same peptide antigens along with adjuvant CpG ODN in microparticles has shown enhanced immunogenicity in the murine model. In the present study we have investigated the immunomodulatory effects of two adjuvants, CpG 1826 and CpG 2006 (Class B, Also known as type K) to the four peptide antigens of HIV such as envelope glycoproteins gp41 Leucine Zipper, gp41 fusion domain and gp120-C2 as well as regulatory protein (Nef) in microparticles, exploring nasal route with single immunization schedule. Peptide (s) alone in the microparticles elicited low peptide specific IgG and IgA peak titres in the sera, whereas the inclusion of CpG ODN with peptides in microparticles significantly enhanced peptide specific IgG and IgA peak titres and such responses were sustained for longer durations. Similarly higher SIgA response was achieved in the mucosal washes with CpG encapsulated in microparticles. Such presence of SIgA in washes was further correlated with the presence of secretory component (SC) in the respective washes. Both adjuvants induced excellent peptide specific IgG and IgA immune responses. Thus the overall study highlighted the importance of CpG ODNs as a mucosal adjuvant for weaker peptide antigens and thus can explore for developing peptide based vaccine against HIV.     

Protective immune responses to meningococcal C conjugate vaccine after intranasal immunization of mice with the LTK63 mutant plus chitosan or trimethyl chitosan chloride as novel delivery platform

Chitosan and its derivative N-trimethyl chitosan chloride (TMC), given as microparticles or powder suspensions, and the non-toxic mucosal adjuvant LTK63, were evaluated for intranasal immunization with the group C meningococcal conjugated vaccine (CRM-MenC). Mice immunized intranasally with CRM-MenC formulated with chitosan or TMC and the LTK63 mutant, showed high titers of serum and mucosal antibodies specific for the MenC polysaccharide. Neither significant differences were observed between microparticle formulations and powder suspensions nor when LTK63 was pre-associated to the delivery system or not. The bactericidal activity measured in serum of mice immunized intranasally with the conjugated vaccine formulated with the delivery systems and the LT mutant was superior to the activity in serum of mice immunized sub-cutaneously. Importantly, intranasal but not parenteral immunization, induced bactericidal antibodies at the nasal level, when formulated with both delivery system and adjuvant.     

Protective immune responses to meningococcal C conjugate vaccine after intranasal immunization of mice with the LTK63 mutant plus chitosan or trimethyl chitosan chloride as novel delivery platform

Chitosan and its derivative N-trimethyl chitosan chloride (TMC), given as microparticles or powder suspensions, and the non-toxic mucosal adjuvant LTK63, were evaluated for intranasal immunization with the group C meningococcal conjugated vaccine (CRM-MenC). Mice immunized intranasally with CRM-MenC formulated with chitosan or TMC and the LTK63 mutant, showed high titers of serum and mucosal antibodies specific for the MenC polysaccharide. Neither significant differences were observed between microparticle formulations and powder suspensions nor when LTK63 was pre-associated to the delivery system or not. The bactericidal activity measured in serum of mice immunized intranasally with the conjugated vaccine formulated with the delivery systems and the LT mutant was superior to the activity in serum of mice immunized sub-cutaneously. Importantly, intranasal but not parenteral immunization, induced bactericidal antibodies at the nasal level, when formulated with both delivery system and adjuvant.     

壳聚糖衍生物在口鼻黏膜疫苗中的应用

壳聚糖是一种有效的黏膜疫苗佐剂和递送载体,但因其水溶性差,应用受到一定限制.通过对壳聚糖进行不同的化学修饰可得到各类壳聚糖衍生物,这些衍生物不仅溶解性较好,而且保持了壳聚糖良好的生物相容性、生物降解性、免疫刺激活性等优势,为黏膜疫苗,尤其是经口、鼻途径递送的疫苗提供了新型候选佐剂和递送载体.此文对修饰壳聚糖的主要方法以及其衍生物在口鼻黏膜疫苗中的应用做一综述.     

Brain uptake of an anti-ischemic agent by nasal administration of microparticles

N(6)-cyclopentyladenosine (CPA) has neuronal anti-ischemic properties, but it is not absorbed into the brain from the bloodstream, where it shows poor stability and induces side effects. Microparticulate drug delivery systems designed for CPA nasal administration and constituted by mannitol or chitosan, were prepared by spray-drying and characterized. Mannitol-lecithin microparticles showed high CPA dissolution rate, whereas chitosan microparticles controlled its release rate. In vitro mucoadhesion studies indicated that CPA-loaded chitosan microparticles had higher mucoadhesive properties compared to mannitol particles. Ex vivo studies on sheep nasal mucosa showed that mannitol microparticles promoted CPA permeation across the mucosa, whereas chitosan microparticles controlled CPA permeation rate in comparison with CPA raw material. In vivo studies were carried out on rats. No CPA was detected in rat cerebrospinal fluid (CSF) and brain sections after intravenous administration. In contrast, after nasal administration of loaded microparticles CPA was found in the CSF at concentrations ranging from high nM to microM values and up to two order of magnitude higher than those obtained at systemic level. CPA was also found in the olfactory bulb at concentrations around 0.1 ng/mg of tissue. These results can open new perspectives for the employment of CPA against brain damages following stroke.     

Effect of vitamin E TPGS on immune response to nasally delivered diphtheria toxoid loaded poly(caprolactone) microparticles

The nasal mucosa has many advantages as a potential site for drug and vaccine delivery. The present study has sought to exploit this route of delivery using microparticles composed of D-alpha-tocopheryl polyethylene glycol 1000 succinate (TPGS) as a matrix material blended with poly(caprolactone) for nasal immunisation with diphtheria toxoid. Particles were prepared by a double emulsion method, followed by spray drying and the effect of TPGS on size, zeta potential, loading and release of antigen was assessed. Particles composed of TPGS-PCL blends were spherical, smooth and monodisperse, displaying increasing yields after spray drying with increasing concentrations of TPGS. The immune response to diphtheria toxoid loaded PCL-TPGS microspheres after nasal administration was shown to be higher than that achieved using PCL microspheres alone. We conclude that TPGS shows significant potential as a novel adjuvant either alone or in combination with an appropriate delivery system.