壳聚糖及其衍生物纳米粒的制备以及在疫苗中的应用

壳聚糖是一种高分子线性阳离子多糖。由壳聚糖及其化学改性衍生物制备的纳米粒具有生物相容性好、细胞毒性低以及可降解等特点,人们对其作为佐剂或递送系统在疫苗中的应用已开展了广泛研究。此文对壳聚糖及其衍生物纳米粒的制备方法以及在疫苗中的应用进行综述。     

壳聚糖及其衍生物作为免疫佐剂的研究进展

近年来,随着免疫学研究的不断深入,疫苗研制技术取得了很大的进步,但这些疫苗存在着给药剂量大、免疫原性弱和靶向性差等缺点,需加入免疫佐剂以提高其免疫原性和特异性免疫应答。纳米疫苗黏膜免疫递送系统具有保护抗原、简化接种程序、增强免疫效果和提高生物利用度等优点。壳聚糖及其衍生物由于具有良好的可生物降解性、生物相容性、低毒性、黏膜吸附性、免疫刺激活性、缓释和控释作用及靶向性,已成为了构建纳米疫苗黏膜免疫递送系统和疫苗免疫佐剂的优良载体。文中对疫苗递送系统、壳聚糖及其衍生物的理化性质及其作为免疫佐剂的应用、优越性和作用机制进行了综述。     

免疫佐剂与壳聚糖

随着对疫苗研究的不断深入 ,人们发现黏膜蛋白疫苗是最有希望的疫苗 ,但其作用的发挥需要佐剂的参与 ,因此寻找一种安全有效的新型免疫佐剂成为发展疫苗首先必须解决的问题。而壳聚糖具有安全无毒等特性 ,国内外许多报道说明壳聚糖具有免疫调节和佐剂效应。此文就免疫佐剂及壳聚糖的免疫调节和佐剂效应作一简要综述。     

菌影作为新型疫苗递送载体的研究进展

尽管基于重组蛋白或DNA的新型疫苗的开发以指数级上升,但是其免疫原性常弱于传统疫苗,缺少有效的递送系统是其应用的主要限制因素,因此迫切需要开发新型改良的递送系统和佐剂.菌影(bacterial ghost,BG)系统正是这类载体系统,它能将结合的抗原靶向APC,并具有佐剂活性.BG是一种无生命的革兰阴性菌包膜,无胞质内容物,但保留了细胞形态和天然表面抗原结构.由于BG具有颗粒特性并包含许多已知的免疫刺激成分,因此BG能增强抗其传递的靶抗原的免疫应答.作为一种新型的疫苗递送载体,菌影将被用于人用和兽用疫苗的生产.     

菌影作为新型疫苗递送载体的研究进展

尽管基于重组蛋白或DNA的新型疫苗的开发以指数级上升,但是其免疫原性常弱于传统疫苗,缺少有效的递送系统是其应用的主要限制因素,因此迫切需要开发新型改良的递送系统和佐剂.菌影(bacterial ghost,BG)系统正是这类载体系统,它能将结合的抗原靶向APC,并具有佐剂活性.BG是一种无生命的革兰阴性菌包膜,无胞质内容物,但保留了细胞形态和天然表面抗原结构.由于BG具有颗粒特性并包含许多已知的免疫刺激成分,因此BG能增强抗其传递的靶抗原的免疫应答.作为一种新型的疫苗递送载体,菌影将被用于人用和兽用疫苗的生产.     

壳聚糖纳米粒载体的应用研究进展

目的介绍壳聚糖纳米粒载体在药物、基因递送等方面的研究应用进展,为其在新领域的应用提供依据。方法广泛查阅中外文有关文献,整理分析归纳了其中27篇文献内容。结果壳聚糖纳米粒载体在药物和基因递送方面已经有诸多研究应用。结论壳聚糖纳米粒载体是一种有前途的非病毒递送载体,其特性和应用有待进一步探索。     

两亲性壳聚糖衍生物在药物递送中的研究进展

壳聚糖是一种来源丰富的碱性多糖,具有良好的生物相容性和生物可降解性,但是其差的溶解性限制了壳聚糖在医药领域的应用。为了提高壳聚糖的溶解性,研究者对壳聚糖进行两亲性改性,通过选择不同的亲水、疏水基团,设计合成了两亲性壳聚糖衍生物。并利用其在水溶液中的自组装性能,形成两亲性壳聚糖纳米粒,用于多种药物的递送,以达到增加药物溶解性、稳定性、降低药物毒性和提高生物利用度等目的。本文综述了两亲性壳聚糖衍生物的合成方法,以及其在药物递送系统中的应用。     

基因疫苗微粒系统黏膜免疫的研究进展

微粒系统作为一种基因疫苗的黏膜免疫递送系统,能够增强免疫效果,具有同时诱导系统免疫应答和黏膜免疫应答、产生共同黏膜免疫应答、增加病人的顺应性、降低疫苗推广成本等优点.文章综述微粒系统增强基因疫苗黏膜免疫效果的机制、常见的微粒系统、载体材料以及优化微粒系统的研究进展.     

壳聚糖及其衍生物在药物载体中的应用

摘 要壳聚糖及其衍生物具有无毒、良好的生物相容性和可降解性、黏膜黏附性和促渗性等优点,在药物载体领域具有较为广阔的研究及应用前景。本文结合国内外最新发表的相关文献,对壳聚糖及其衍生物作为相关药物载体的应用以及作用机制进行分析讨论,并对其作为抗肿瘤药物靶向载体、缓控释药物载体、眼用药物载体、基因载体和凝胶基质的应用及研究进展进行综述。     

基于脂质载体的mRNA疫苗递送系统研究进展

转录信使RNA（mRNA）具有安全有效的蛋白质表达谱,在遗传病新疗法、表达功能性蛋白和抗体、疫苗或基因编辑的开发上具有良好的应用前景。随着病原生物学与免疫学的发展,mRNA疫苗凭借其高效性与稳定性而愈发被重视。目前,有效的体内递送手段是mRNA疫苗所面临的较大挑战,脂质载体的mRNA疫苗递送系统具有靶向性强、包封率高、细胞亲和性好的特点,部分脂质载体能够使mRNA疫苗在体内以非侵入性的方式进行靶向递送。从mRNA疫苗递送的主要难点、脂质载体在mRNA疫苗递送中的研究现状以及针对新型冠状病毒（SARS-CoV-2）的mRNA脂质载体疫苗开发进展,共3个方面总结了目前基于脂质载体的mRNA疫苗递送系统研究进展,并对未来的基因疫苗递送系统研究方向进行展望。     

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.     

Chitosan for mucosal vaccination.

The striking advantage of mucosal vaccination is the production of local antibodies at the sites where pathogens enter the body. Because vaccines alone are not sufficiently taken up after mucosal administration, they need to be co-administered with penetration enhancers, adjuvants or encapsulated in particles. Chitosan easily forms microparticles and nanoparticles which encapsulate large amounts of antigens such as ovalbumin, diphtheria toxoid or tetanus toxoid. It has been shown that ovalbumin loaded chitosan microparticles are taken up by the Peyer's patches of the gut associated lymphoid tissue (GALT). This unique uptake demonstrates that chitosan particulate drug carrier systems are promising candidates for oral vaccination. Additionally, after co-administering chitosan with antigens in nasal vaccination studies, a strong enhancement of both mucosal and systemic immune responses is observed. This makes chitosan very suitable for nasal vaccine delivery. In conclusion, chitosan particles, powders and solutions are promising candidates for mucosal vaccine delivery. Mucosal vaccination not only reduces costs and increases patient compliance, but also complicates the invasion of pathogens through mucosal sites.     

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.     

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.     

Plasmid DNA loaded chitosan nanoparticles for nasal mucosal immunization against hepatitis B

This work investigates the preparation and in vivo efficacy of plasmid DNA loaded chitosan nanoparticles for nasal mucosal immunization against hepatitis B. Chitosan pDNA nanoparticles were prepared using a complex coacervation process. Prepared nanoparticles were characterized for size, shape, surface charge, plasmid loading and ability of nanoparticles to protect DNA against nuclease digestion and for their transfection efficacy. Nasal administration of nanoparticles resulted in serum anti-HBsAg titre that was less compared to that elicited by naked DNA and alum adsorbed HBsAg, but the mice were seroprotective within 2 weeks and the immunoglobulin level was above the clinically protective level. However, intramuscular administration of naked DNA and alum adsorbed HBsAg did not elicit sIgA titre in mucosal secretions that was induced by nasal immunization with chitosan nanoparticles. Similarly, cellular responses (cytokine levels) were poor in case of alum adsorbed HBsAg. Chitosan nanoparticles thus produced humoral (both systemic and mucosal) and cellular immune responses upon nasal administration. The study signifies the potential of chitosan nanoparticles as DNA vaccine carrier and adjuvant for effective immunization through non-invasive nasal route.     

Development of a novel adjuvanted nasal vaccine: C48/80 associated with chitosan nanoparticles as a path to enhance mucosal immunity

In a time in which mucosal vaccines development has been delayed by the lack of safe and effective mucosal adjuvants, the combination of adjuvants has started to be explored as a strategy to obtain potent vaccine formulations. This study describes a novel adjuvant combination as an effective approach for a nasal vaccine – the association of the mast cell activator compound 48/80 with chitosan based nanoparticles. It was hypothesized that mucoadhesive nanoparticles would promote the cellular uptake and prolong the antigen residence time on nasal cavity. Simultaneously, mast cell activation would promote a local microenvironment favorable to the development of an immune response. To test this hypothesis, two different C48/80 loaded nanoparticles (NPs) were prepared: Chitosan-C48/80 NP (Chi-C48/80 NP) and Chitosan/Alginate-C48/80 NP (Chi/Alg-C48/80 NP). The potential as a vaccine adjuvant of the two delivery systems was evaluated and directly compared. Both formulations had a mean size near 500 nm and a positive charge; however, Chi-C48/80 NP was a more effective adjuvant delivery system when compared with Chi/Alg-C48/80 NP or C48/80 alone. Chi-C48/80 NP activated mast cells at a greater extent, were better internalized by antigen presenting cells than Chi/Alg-C48/80 NP and successfully enhanced the nasal residence time of a model antigen. Superiority of Chi-C48/80 NP as adjuvant was also observed in vivo. Therefore, nasal immunization of mice with Bacillus anthracis protective antigen (PA) adsorbed on Chi-C48/80 NP elicited high levels of serum anti-PA neutralizing antibodies and a more balanced Th1/Th2 profile than C48/80 in solution or Chi/Alg-C48/80 NP. The incorporation of C48/80 within Chi NP also promoted a mucosal immunity greater than all the other adjuvanted groups tested, showing that the combination of a mast cell activator and chitosan NP could be a promising strategy for nasal immunization.     

Intranasal administration of CpG oligonucleotides induces mucosal and systemic Type 1 immune responses and adjuvant activity to porcine reproductive and respiratory syndrome killed virus vaccine in piglets in vivo

Oligonucleotides containing CpG motifs (CpG ODN) are strong adjuvants for immune responses, particularly in mice. Recently, it has been showed that CpG ODN is a promising mucosal adjuvant in mice, but data on mucosal immune responses induced by CpG ODN in piglets are scarce. We have previously demonstrated that CpG ODN is a potent adjuvant to pseudorabies attenuated virus (PRV) vaccine when administered subcutaneously (SC) in newborn piglets. Herein, we evaluated intranasal (IN) delivery of CpG ODN with porcine reproductive and respiratory syndrome (PRRS) killed virus vaccine (PRRSV) to determine its potential as a mucosal adjuvant to a commercial vaccine. CpG ODN augmented systemic (IgG in serum, Peripheral blood mononuclear cells (PBMC) proliferation) and mucosal (IgA in feces, nasal and oral secretions) immune responses against antigen. CpG ODN stimulated both T-helper type1 (Type 1) (IgG2) and Type 2 (IgA) responses when delivered intranasally. Results from this study indicate that stimulatory CpG ODN may be effective as a mucosal adjuvant with commercial vaccine in husbandry animals.     

Chitosan nanoparticles encapsulated vesicular systems for oral immunization: preparation, in-vitro and in-vivo characterization

BSA-loaded chitosan nanoparticles were prepared and encapsulated in vesicles (liposomes and niosomes) to make them acid resistant upon oral administration. Prepared systems were characterized in-vitro for shape, size, entrapment efficiency and stability in simulated gastric fluid (SGF, pH 1.2) and simulated intestinal fluid (SIF, pH 7.5). The immune stimulating activity was studied by measuring serum IgG titre and secretory IgA (sIgA) levels in mucosal secretions following oral administration of various formulations in albino rats. Significantly higher (P < 0.05) serum IgG titres were achieved following oral administration of novel nanoparticulate vesicular formulations as compared with unmodified chitosan nanoparticles. Further, high sIgA levels in mucosal secretions advocated a possible application of chitosan nanoparticle encapsulated in vesicles as an oral vaccine delivery carrier-adjuvant system.