凝集素修饰纳米粒经鼻入脑的递药特性研究
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摘要
血脑屏障(BBB)是存在于血液和脑组织间的一层屏障系统，由极化的脑毛细血管内皮细胞通过复杂的细胞间紧密连接构成，它对于维系脑内环境的相对恒定十分重要，同时也是药物进入脑组织发挥治疗作用的主要屏障。随着生物技术、神经科学的迅速发展，已经发现许多具有中枢治疗活性的多肽蛋白药物，但由于BBB的存在，很大程度上限制了这些药物的脑内转运和治疗作用的发挥。因此，研究药物，尤其是多肽蛋白类药物的脑内递释技术，具有很高的研究价值和临床意义。
鼻脑通路的存在为多肽蛋白药物入脑提供了可能，但多肽蛋白药物的透膜吸收能力差，给药易受鼻腔中酶的降解和鼻纤毛的清除，因此经鼻入脑的药量仍然很低，无法达到有效的临床治疗效果。纳米粒递药系统能够有效减少多肽蛋白类药物的降解，而且有望通过载体的跨膜转运增加药物的吸收。然而，普通纳米粒鼻腔给药仍存在三大问题：①纳米粒的粒径远大于细胞间隙，不易通过细胞间紧密连接被吸收，又无法有效诱导细胞内吞，因而透过鼻粘膜吸收的能力不很理想；②由于受到鼻纤毛的清除，纳米粒在鼻腔中通常仅能滞留15～20 min，因而往往尚未被完全摄取即被清除；③普通纳米粒在鼻粘膜上的吸收缺乏部位选择性：其可能通过呼吸部粘膜吸收增加药物进入血液循环，也可能通过嗅粘膜吸收增加药物入脑，因而脑内递药效率较差。
为了解决上述问题，本课题构建了一种新型的经鼻给药脑内递药系统—凝集素修饰纳米粒载药系统。以纳米粒作为多肽蛋白药物的载体，可保护其体内稳定性；而纳米粒表面修饰的麦胚凝集素(WGA)或荆豆凝集素(UEAI)可分别与鼻腔嗅粘膜上选择性高表达的糖基受体N-乙酰氨基葡萄糖或L-岩藻糖特异结合，延长载药系统在鼻粘膜，尤其是嗅粘膜上的滞留时间，介导鼻粘膜特别是嗅粘膜对载药系统的吸收，选择性地递送更多的药物入脑，提高对中枢疾病的预防和治疗效果，降低全身性的毒副作用。该递药系统的构建模式国内外未见报道，具有较高的创新性。
本文第一部分为凝集素修饰纳米粒的构建和表征。将一定比例的甲氧基聚乙二醇—聚乳酸(MPEG-PLA)和马来酰亚胺聚乙二醇—聚乳酸(Maleimide-PEG-PLA)经复乳／溶媒蒸发法制备得到纳米粒(NP)，通过表面的马来酰亚胺基与巯基化凝集素共价连接得到凝集素修饰纳米粒。所得纳米粒平均粒径100 nm以下，Zeta电位-20 mV。免疫电镜观察证实纳米粒表面连接有凝集素；红细胞凝集实验证实连接于纳米粒表面的凝集素仍具特异性糖基结合活性；以纳米粒的粒径、表面凝集素密度和蛋白连接效率为指标，筛选优化制备条件：MPEG-PLA：Maleimide-PEG-PLA质量比为9：1；巯基化试剂2-IT：蛋白摩尔比为60：1；Maleimide-PEG-PLA：蛋白摩尔比为3：1；反应时间为8-10h。
为了评价凝集素修饰纳米粒经鼻给药后的脑内递药特性，第二部分采用香豆素-6为荧光探针，观察并测定载香豆素-6的凝集素修饰纳米粒经鼻给药后脑组织中的荧光探针强度，并对WGA和UEAI修饰纳米粒鼻腔给药后的脑内靶向递送效果进行评价。首先制备载香豆素-6的凝集素修饰纳米粒，通过体外泄露实验和红细胞凝集试验证实香豆素-6是一种较理想的荧光探针，可用于示踪纳米粒在大鼠体内的转运情况。定性观察发现，鼻腔给予NP和WGA修饰纳米粒(WGA-NP)15 min即可在嗅球、大脑组织切片中观察到纳米粒绿色荧光，说明纳米粒鼻腔给药后可迅速转运入脑，纳米粒经WGA修饰，其入脑量有所提高。定量测定结果显示，WGA和UEAI修饰显著增加包载于纳米粒中的香豆素-6经鼻给药后的入脑量：WGA-NP和UEAI修饰纳米粒(UEA-NP)组脑组织中香豆素-6平均峰浓度分别为NP组的2.26(P＜0.05)和1.33倍，药时曲线下面积分别为NP组的1.97(P＜0.05)和1.70倍(P＜0.05)。3种纳米粒的脑靶向效率依次为：UEA-NP＞WGA-NP＞NP。
第三部分将脑内递药能力较强的WGA-NP用于包载多肽药物—血管活性肠肽(vasoactive intestinal peptide，VIP)，研究其用于多肽蛋白药物脑内递送的可行性。采用复乳／溶媒蒸发法制备载VIP的PEG-PLA纳米粒(VIP-NP)，该纳米粒与巯基化WGA共价连接制得WGA修饰纳米粒(WGA-VIP-NP)，粒径100 nm左右，Zeta电位-20 mV。VIP-NP的包封率70％以上，载药量可达1.4％。在血浆中12 h累积释放约35％，在鼻洗液中8 h释放17％，突释效应不大，显示一定的缓释特性。VIP-NP在血浆、鼻洗液中的稳定性均显著优于游离VIP溶液，为其体内应用提供基础。采用~(125)I标记VIP，HPLC偶联γ计数器检测脑组织中的完整~(125)I-VIP，结果发现VIP溶液剂鼻腔给药、皮下注射后15 min可在脑中检测到完整VIP，随着时间的延长，完整VIP所占比例减小，鼻腔给药后1 h，皮下注射后2 h，即在脑组织检测不到完整VIP。而鼻腔给予~(125)I-VIP-NP和WGA-~(125)I-VIP-NP，直到给药后12 h，仍可在脑中检测到完整VIP，说明采用纳米粒包封VIP，提高其稳定性，大大延长其体内作用时间。VIP-NP鼻腔给药后脑内完整VIP的AUC为VIP溶液鼻腔给药的3.5-4.7倍(P＜0.05)；VIP-NP经WGA修饰后鼻腔给药，入脑量显著提高，脑内完整VIP的AUC为VIP溶液鼻腔给药的5.6-7.7倍(P＜0.05)。药效学研究采用AF64A侧脑室注射构建大鼠中枢胆碱能神经元损毁Alzheimer症动物模型，通过Morris水迷宫实验评价VIP制剂对大鼠空间记忆障碍的改善作用，测定大鼠海马乙酰胆碱酯酶活力评价VIP制剂对中枢胆碱能神经元损毁的保护效果，结果发现VIP-NP鼻腔给药后，能够改善AF64A侧脑室注射所致大鼠空间记忆障碍，提高大鼠海马乙酰胆碱酯酶含量，对中枢胆碱能系统具有一定的保护作用。VIP-NP经WGA修饰后，在剂量减半的情况下仍可产生明显的记忆改善作用，显著提高模型大鼠海马乙酰胆碱酯酶含量，说明WGA-NP是更有效的递药系统，为具有脑内治疗活性药物，特别是稳定性差、难以通过血脑屏障的多肽蛋白药物脑内递送提供了有效的载体，具有很高的应用价值。
第四部分对凝集素修饰纳米粒经鼻入脑转运机理进行研究。首先，以香豆素-6为荧光探针，探讨凝集素修饰纳米粒经鼻入脑转运通路。通过比较WGA-NP和NP不同时间在鼻粘膜不同部位分布情况发现：WGA-NP较NP具有更强的透粘膜吸收能力；在靠近嗅球的筛板处观察到WGA-NP的分布量高于NP，说明WGA-NP更易被摄取进入嗅球。形态学观察结合免疫荧光标记区分嗅粘膜和呼吸部粘膜发现，相对于NP，WGA-NP和UEA-NP均呈现出在嗅粘膜上选择性分布的特点。NP、WGA-NP和UEA-NP主要通过细胞内途径吸收。免疫荧光标记嗅粘膜及粘膜下神经束，初步确定WGA-NP经嗅粘膜上皮细胞或腺体吸收进入粘膜下层，经神经束或神经束周围结缔组织转运入脑。其次，以光稳定性好的CdSe量子点(QDs)作为荧光探针，采用大鼠在体鼻腔灌流技术对WGA-NP鼻粘膜吸收机理进行探讨。结果显示，WGA的特异糖基N-乙酰氨基葡萄糖可显著抑制鼻腔对WGA-QDs-NP的摄取，说明WGA通过与鼻粘膜上的特异糖基结合，增加递药系统在鼻腔的滞留进而介导其吸收。能量代谢抑制剂叠氮钠显著抑制鼻粘膜对WGA-QDs-NP的摄取，提示WGA-QDs-NP以能量依赖方式吸收。包被凹陷内吞抑制剂PhAsO和chlorpromazine显著抑制鼻腔对WGA-QDs-NP的摄取，表明WGA-QDs-NP内吞主要通过细胞膜上的clathrin蛋白构成的包被凹陷介导；而细胞膜穴样内馅caveolae的内吞抑制剂filipin对摄取无显著影响，说明caveolae途径可能并非主要的内吞途径。高尔基体破坏剂BFA抑制鼻腔对WGA-QDs-NP的摄取，说明高尔基体参与WGA-QDs-NP在细胞内转运；溶酶体抑制剂Monensin抑制鼻腔对WGA-QDs-NP的摄取，提示至少部分WGA-QDs-NP在胞内转运过程中经过溶酶体。该机理研究有助于阐明凝集素修饰纳米粒的细胞吸收途径，为后续的递药系统设计提供思路。
为了评价WGA-NP鼻腔应用的生理适应性，第五部分考察了该递药系统对鼻纤毛和鼻粘膜形态及细胞分化情况的影响。采用两种模型评价WGA-NP对鼻粘膜纤毛的影响情况，结果显示：WGA-NP对蟾蜍上颚粘膜纤毛运动持续时间影响较小，与生理盐水组对照，纤毛持续运动时间百分率达90％以上；对大鼠鼻粘膜纤毛形态影响小，持续给药6天后，大鼠鼻粘膜纤毛形态与生理盐水阴性对照组相似，纤毛浓密，未出现明显脱落，说明WGA-NP对鼻纤毛毒性小。鼻粘膜细胞形态观察、嗅粘膜厚度测量和神经特异性烯醇化酶免疫组化观察发现，大鼠鼻腔持续给予WGA-NP，对鼻粘膜形态和细胞分化情况无显著影响，给药7天后，WGA-NP组大鼠鼻粘膜完整，结构清楚，嗅粘膜厚度不变，粘膜上纤毛整齐浓密，粘膜下腺体、血管、神经束清晰可见，与阴性对照组相似，说明WGA-NP鼻腔给药不会引起鼻粘膜形态和细胞分化情况的急性改变，证实该系统具有较高的应用安全性。
Drug delivery into the brain is made difficult by the presence of the blood-brain barrier(BBB), which is formed by tight junctions within the capillary endothelium of thevertebrate brain. The development of neuroscience has facilitated the discoveries ofpeptides and proteins with considerable potential in the treatment of central nervoussystem (CNS) diseases such as Alzheimer's disease and Parkinson's disease. However, asignificant challenge to their clinical administration is their ideal delivery to the CNScrossing the BBB.
Intranasal drug delivery system offers a non-invasive alternative for the delivery oftherapeutics, effectively bypassing the blood-brain barrier. Indeed, the past few yearshave witnessed a sharp increase in the amount of research on the nasal pathway for theCNS drug delivery. However, the total amount of drugs accessing the brain has beenreported to be low, especially in the form of peptides and proteins, which were highlysusceptible to the unfavorable environment of the nasal cavity. The encapsulation ofthese drugs into nanoparticles might be a promising approach, since the colloidalformulations have been shown to protect the drugs from the degrading milieu in the nasalcavity and facilitate their transport across the mucosal barriers. Nevertheless, the amountof nanoparticles accessing the brain is still limited because of the following reasons: firstof all, the penetration of the nanometer-size particles through tight junctions betweencells was negligible and the amount of unmodified nanoparticles endocytosed by thenasal epithelium was limited; secondly, the resident time of nanoparticles in nasal cavityis short because of mucociliary clearance (particles cleared within the nose every 15 to 20min), which is not available for the complete absorption of the formulation; finally,unmodified nanoparticles distributed in the nasal cavity without selectivity, resulting inpoor brain targeting efficiency of the formulation.
To address these problems, novel lectin-modified nanoparticles were constructed.Wheat germ agglutinin (WGA), specifically binding to N-acetyl-D-glucosamine and sialic acid, both of which were abundantly observed in the nasal cavity especially in theolfactory mucosa, and ulex europeus agglutinin I (UEA I), specifically binding toL-fucose, which was largely located in the olfactory epithelium, were selected aspromising targeting ligands. The incorporation of peptides in the nanoparticles mightimprove their stability in vivo while the conjugation of lectin at the nanoparticles surfacemight induce strong mucoadhesion for a longer duration, or a close contact of thenanoparticles with the mucosal cells especially in the olfactory mucosa so as to produce astronger penetration. Such lectin-modified nanoparticles might serve as potential carriersfor brain delivery of peptides and proteins with enhanced brain-targeting efficiency andminimized adverse effects in the peripheral tissues.
In the first part, lectin-conjugated nanoparticles were prepared by incorporatingmaleimide into one end of the PLA-PEG copolymer and taking advantage of its thiolgroup-binding reactivity to conjugate with the lectins thiolated with 2-iminothialane. Themean size of the resulted nanoparticles was about 100 nm and the zeta potential was-20mV. The coupling of WGA with PEG-PLA nanoparticles (NP) was confirmed by theexistence of gold-labeled WGA-NP under TEM. The retention of biorecognitive activityof WGA after the covalent coupling procedure was confirmed by haemagglutination tests.The preparation protocol was optimized based on the following endpoints: lectin densityat the particle surface, particle size and conjugation efficiency, through which the optimalratio of maleimide-PEG-PLA to MePEG-PLA around 1: 9, lectin: 2-iminothiolane 1: 60,thiolated lectin: maleimide 1: 3 and the conjugation time of 8-10 h were obtained.
In order to evaluate the capacity of the lectin-conjugated nanoparticles for drugdelivery into the CNS, in the second part, a lipophilic fluorescent probe with highsensitivity, coumarin-6, was incorporated into the nanoparticles, and the concentrationsof the fluorescent marker in blood and brain tissues following intranasal administration ofWGA-NP were determined and compared with those after nasal delivery of NP. It wassuggested by the in vitro release study and the haemagglutination test that coumarin-6was an appropriate fluorescent probe and the fluorescence signal detected or observedwas mainly attributed to the coumarin-6 encapsulated into the nanoparticles. Strongergreen fluorescent signals were observed on both olfactory bulb and cerebrum slicesfifteen minutes after intranasal administration of WGA-NP than that of NP. In vivopharmacokinetie results in rats suggested that the WGA and UEA I modification at the nanoparticles surface facilitated the absorption of the associated coumarin-6 into thebrain following intranasal administration with significant increase in the peakconcentration (about 2.26 and 1.33 times, respectively) and the area under theconcentration-time curve (about 2 and 1.7 times, respectively) in different brain tissuescompared with that of coumarin-6 incorporated in NP. The brain drug-targetingefficiency of the nanoparticles was in the following order: UEA-NP＞WGA-NP＞NP.
In the third part, WGA-NP was used to deliver vasoactive intestinal peptide (VIP) intothe CNS via nasal administration. VIP was efficiently incorporated into PEG-PLAnanoparticles followed by surface modification with WGA, producing nanoparticles withparticle size of 100 nm, zeta potential of-20 mV, encapsulation efficiency of more than70％and drug loading capacity of 1.4％. In vitro release of VIP from VIP-NP showed aslight burst at the beginning and a long sustained release of about 35％aider incubationin plasma for 12 h and 17％in nasal wash for 8 h. It was also showed that the stability ofentrapped VIP in the plasma and nasal wash was significantly improved compared withthat of VIP. In the pharmacokinetic study, VIP was radio-labeled with Na ~(125)I and intact~(125)I-VIP in the CNS was determined with HPLC coupled with aγ-counter. The amount ofintact VIP detected in the brain tissues suggested that the incorporation of VIP into thenanoparticles significantly increased the stability of VIP in vivo and the area under theconcentration-time curve of intact ~(125)I-VIP in mice brain was significantly enlarged by3.5～4.7 folds and 5.6～7.7 folds following intranasal administration of ~(125)I-VIP carried byNP and WGA-NP, respectively, compared with that after intranasal application of~(125)I-VIP solution. The same improvements in spatial memory and hippocampusconcentration of acetylcholinesterase in ethylcholine aziridium-treated rats were observedfollowing intranasal administration of 25μg/kg and 12.5μg/kg of VIP loaded by NP andWGA-NP, respectively, indicating that WGA-NP might serve as a promising carrierespecially for biotech drugs such as peptides and proteins.
In the fourth part, the transport pathways of WGA-NP from nose to brain wereinvestigated. It was observed that the penetration of WGA-NP into submucosa was fasterthan NP; More fluorescent signals representing WGA-NP were observed in the sieveplate, which was anatomically near the olfactory bulb, suggesting that the absorption ofWGA-NP into the olfactory bulb was faster than that of NP. Distribution profiles ofWGA-NP and UEA-NP in the nasal cavity indicated their higher affinity to the olfactory mucosa than to the respiratory one, which also indicated that WGA-NP in the submucosamight be transported into the CNS through the nerves or the connective tissues around thenerves in the lamina propria. Besides, a novel fluorescent probe with better opticalstability, CdSe quantum dots (QDs), was applied as a fluorescent probe and the transportmechanism of WGA-QDs-NP through the nasal mucosa was investigated using an in situnasal perfusion technique. Inhibition experiment of specific sugar suggested that theinteractions between the nasal mucosa and WGA-NP were due to the immobilization ofcarbohydrate-binding pockets at the surface of the nanoparticles. The addition of ametabolic inhibitor, NAN3, significantly reduced the amount of WGA-NP associated withthe nasal mucosa, indicating that WGA-NP was absorbed into the nasal mucosa throughan active transport pathway. Both PhAsO and chlorpromazine inhibited the association ofWGA-NP with the nasal mucosa while filipin failed to affect the association, suggestingthat clathrin pathway might play an important role in the endocytosis of WGA-NP whilethe caveolae one might not be the main pathway. The addition of both BFA andmonensin also decreased the association of WGA-NP with the nasal mucosa, indicatingthat both Golgi apparatus and lysosome participated in the uptake of WGA-NP. Suchinvestigations on mechanism might provide useful information for the design of noveldrug delivery systems.
In order to evaluate the safety of WGA-NP following intranasal administration, theirinfluences on the nasal cilia, morphology and cells differentiation of the nasal mucosawere investigated. Nasal ciliotoxicity studies were conducted on both toad palate and ratnasal mucosa. The duration of ciliary movement on toad palate was 12 hours and 11.75hours for the WGA-NP-treated and NP-treated mucosa, respectively, which wascomparable with that of the negative control (12.25 hours). The data were consistent withthe results shown by SEM in the rat nasal mucosa, which showed that after intranasaladministration of WGA-NP and NP, no visible change in the morphology and integrity ofthe cilia was observed, suggesting that the nasal ciliotoxicity of WGA-NP and NP werenegligible. Furthermore, it was showed that the morphology and cells differentiation ofthe nasal mucosa were not affected with intact olfactory mucosa of about 40μm, intactand dense cilia on the mucosa and glands, blood vessels and nerve bundles in the laminapropria comparable with those of negative control observed following nasaladministration of WGA-NP for 7 days. These data indicated that WGA-NP was safe following intranasal administration.
鼻脑通路的存在为多肽蛋白药物入脑提供了可能，但多肽蛋白药物的透膜吸收能力差，给药易受鼻腔中酶的降解和鼻纤毛的清除，因此经鼻入脑的药量仍然很低，无法达到有效的临床治疗效果。纳米粒递药系统能够有效减少多肽蛋白类药物的降解，而且有望通过载体的跨膜转运增加药物的吸收。然而，普通纳米粒鼻腔给药仍存在三大问题：①纳米粒的粒径远大于细胞间隙，不易通过细胞间紧密连接被吸收，又无法有效诱导细胞内吞，因而透过鼻粘膜吸收的能力不很理想；②由于受到鼻纤毛的清除，纳米粒在鼻腔中通常仅能滞留15～20 min，因而往往尚未被完全摄取即被清除；③普通纳米粒在鼻粘膜上的吸收缺乏部位选择性：其可能通过呼吸部粘膜吸收增加药物进入血液循环，也可能通过嗅粘膜吸收增加药物入脑，因而脑内递药效率较差。
为了解决上述问题，本课题构建了一种新型的经鼻给药脑内递药系统—凝集素修饰纳米粒载药系统。以纳米粒作为多肽蛋白药物的载体，可保护其体内稳定性；而纳米粒表面修饰的麦胚凝集素(WGA)或荆豆凝集素(UEAI)可分别与鼻腔嗅粘膜上选择性高表达的糖基受体N-乙酰氨基葡萄糖或L-岩藻糖特异结合，延长载药系统在鼻粘膜，尤其是嗅粘膜上的滞留时间，介导鼻粘膜特别是嗅粘膜对载药系统的吸收，选择性地递送更多的药物入脑，提高对中枢疾病的预防和治疗效果，降低全身性的毒副作用。该递药系统的构建模式国内外未见报道，具有较高的创新性。
本文第一部分为凝集素修饰纳米粒的构建和表征。将一定比例的甲氧基聚乙二醇—聚乳酸(MPEG-PLA)和马来酰亚胺聚乙二醇—聚乳酸(Maleimide-PEG-PLA)经复乳／溶媒蒸发法制备得到纳米粒(NP)，通过表面的马来酰亚胺基与巯基化凝集素共价连接得到凝集素修饰纳米粒。所得纳米粒平均粒径100 nm以下，Zeta电位-20 mV。免疫电镜观察证实纳米粒表面连接有凝集素；红细胞凝集实验证实连接于纳米粒表面的凝集素仍具特异性糖基结合活性；以纳米粒的粒径、表面凝集素密度和蛋白连接效率为指标，筛选优化制备条件：MPEG-PLA：Maleimide-PEG-PLA质量比为9：1；巯基化试剂2-IT：蛋白摩尔比为60：1；Maleimide-PEG-PLA：蛋白摩尔比为3：1；反应时间为8-10h。
为了评价凝集素修饰纳米粒经鼻给药后的脑内递药特性，第二部分采用香豆素-6为荧光探针，观察并测定载香豆素-6的凝集素修饰纳米粒经鼻给药后脑组织中的荧光探针强度，并对WGA和UEAI修饰纳米粒鼻腔给药后的脑内靶向递送效果进行评价。首先制备载香豆素-6的凝集素修饰纳米粒，通过体外泄露实验和红细胞凝集试验证实香豆素-6是一种较理想的荧光探针，可用于示踪纳米粒在大鼠体内的转运情况。定性观察发现，鼻腔给予NP和WGA修饰纳米粒(WGA-NP)15 min即可在嗅球、大脑组织切片中观察到纳米粒绿色荧光，说明纳米粒鼻腔给药后可迅速转运入脑，纳米粒经WGA修饰，其入脑量有所提高。定量测定结果显示，WGA和UEAI修饰显著增加包载于纳米粒中的香豆素-6经鼻给药后的入脑量：WGA-NP和UEAI修饰纳米粒(UEA-NP)组脑组织中香豆素-6平均峰浓度分别为NP组的2.26(P＜0.05)和1.33倍，药时曲线下面积分别为NP组的1.97(P＜0.05)和1.70倍(P＜0.05)。3种纳米粒的脑靶向效率依次为：UEA-NP＞WGA-NP＞NP。
第三部分将脑内递药能力较强的WGA-NP用于包载多肽药物—血管活性肠肽(vasoactive intestinal peptide，VIP)，研究其用于多肽蛋白药物脑内递送的可行性。采用复乳／溶媒蒸发法制备载VIP的PEG-PLA纳米粒(VIP-NP)，该纳米粒与巯基化WGA共价连接制得WGA修饰纳米粒(WGA-VIP-NP)，粒径100 nm左右，Zeta电位-20 mV。VIP-NP的包封率70％以上，载药量可达1.4％。在血浆中12 h累积释放约35％，在鼻洗液中8 h释放17％，突释效应不大，显示一定的缓释特性。VIP-NP在血浆、鼻洗液中的稳定性均显著优于游离VIP溶液，为其体内应用提供基础。采用~(125)I标记VIP，HPLC偶联γ计数器检测脑组织中的完整~(125)I-VIP，结果发现VIP溶液剂鼻腔给药、皮下注射后15 min可在脑中检测到完整VIP，随着时间的延长，完整VIP所占比例减小，鼻腔给药后1 h，皮下注射后2 h，即在脑组织检测不到完整VIP。而鼻腔给予~(125)I-VIP-NP和WGA-~(125)I-VIP-NP，直到给药后12 h，仍可在脑中检测到完整VIP，说明采用纳米粒包封VIP，提高其稳定性，大大延长其体内作用时间。VIP-NP鼻腔给药后脑内完整VIP的AUC为VIP溶液鼻腔给药的3.5-4.7倍(P＜0.05)；VIP-NP经WGA修饰后鼻腔给药，入脑量显著提高，脑内完整VIP的AUC为VIP溶液鼻腔给药的5.6-7.7倍(P＜0.05)。药效学研究采用AF64A侧脑室注射构建大鼠中枢胆碱能神经元损毁Alzheimer症动物模型，通过Morris水迷宫实验评价VIP制剂对大鼠空间记忆障碍的改善作用，测定大鼠海马乙酰胆碱酯酶活力评价VIP制剂对中枢胆碱能神经元损毁的保护效果，结果发现VIP-NP鼻腔给药后，能够改善AF64A侧脑室注射所致大鼠空间记忆障碍，提高大鼠海马乙酰胆碱酯酶含量，对中枢胆碱能系统具有一定的保护作用。VIP-NP经WGA修饰后，在剂量减半的情况下仍可产生明显的记忆改善作用，显著提高模型大鼠海马乙酰胆碱酯酶含量，说明WGA-NP是更有效的递药系统，为具有脑内治疗活性药物，特别是稳定性差、难以通过血脑屏障的多肽蛋白药物脑内递送提供了有效的载体，具有很高的应用价值。
第四部分对凝集素修饰纳米粒经鼻入脑转运机理进行研究。首先，以香豆素-6为荧光探针，探讨凝集素修饰纳米粒经鼻入脑转运通路。通过比较WGA-NP和NP不同时间在鼻粘膜不同部位分布情况发现：WGA-NP较NP具有更强的透粘膜吸收能力；在靠近嗅球的筛板处观察到WGA-NP的分布量高于NP，说明WGA-NP更易被摄取进入嗅球。形态学观察结合免疫荧光标记区分嗅粘膜和呼吸部粘膜发现，相对于NP，WGA-NP和UEA-NP均呈现出在嗅粘膜上选择性分布的特点。NP、WGA-NP和UEA-NP主要通过细胞内途径吸收。免疫荧光标记嗅粘膜及粘膜下神经束，初步确定WGA-NP经嗅粘膜上皮细胞或腺体吸收进入粘膜下层，经神经束或神经束周围结缔组织转运入脑。其次，以光稳定性好的CdSe量子点(QDs)作为荧光探针，采用大鼠在体鼻腔灌流技术对WGA-NP鼻粘膜吸收机理进行探讨。结果显示，WGA的特异糖基N-乙酰氨基葡萄糖可显著抑制鼻腔对WGA-QDs-NP的摄取，说明WGA通过与鼻粘膜上的特异糖基结合，增加递药系统在鼻腔的滞留进而介导其吸收。能量代谢抑制剂叠氮钠显著抑制鼻粘膜对WGA-QDs-NP的摄取，提示WGA-QDs-NP以能量依赖方式吸收。包被凹陷内吞抑制剂PhAsO和chlorpromazine显著抑制鼻腔对WGA-QDs-NP的摄取，表明WGA-QDs-NP内吞主要通过细胞膜上的clathrin蛋白构成的包被凹陷介导；而细胞膜穴样内馅caveolae的内吞抑制剂filipin对摄取无显著影响，说明caveolae途径可能并非主要的内吞途径。高尔基体破坏剂BFA抑制鼻腔对WGA-QDs-NP的摄取，说明高尔基体参与WGA-QDs-NP在细胞内转运；溶酶体抑制剂Monensin抑制鼻腔对WGA-QDs-NP的摄取，提示至少部分WGA-QDs-NP在胞内转运过程中经过溶酶体。该机理研究有助于阐明凝集素修饰纳米粒的细胞吸收途径，为后续的递药系统设计提供思路。
为了评价WGA-NP鼻腔应用的生理适应性，第五部分考察了该递药系统对鼻纤毛和鼻粘膜形态及细胞分化情况的影响。采用两种模型评价WGA-NP对鼻粘膜纤毛的影响情况，结果显示：WGA-NP对蟾蜍上颚粘膜纤毛运动持续时间影响较小，与生理盐水组对照，纤毛持续运动时间百分率达90％以上；对大鼠鼻粘膜纤毛形态影响小，持续给药6天后，大鼠鼻粘膜纤毛形态与生理盐水阴性对照组相似，纤毛浓密，未出现明显脱落，说明WGA-NP对鼻纤毛毒性小。鼻粘膜细胞形态观察、嗅粘膜厚度测量和神经特异性烯醇化酶免疫组化观察发现，大鼠鼻腔持续给予WGA-NP，对鼻粘膜形态和细胞分化情况无显著影响，给药7天后，WGA-NP组大鼠鼻粘膜完整，结构清楚，嗅粘膜厚度不变，粘膜上纤毛整齐浓密，粘膜下腺体、血管、神经束清晰可见，与阴性对照组相似，说明WGA-NP鼻腔给药不会引起鼻粘膜形态和细胞分化情况的急性改变，证实该系统具有较高的应用安全性。
Drug delivery into the brain is made difficult by the presence of the blood-brain barrier(BBB), which is formed by tight junctions within the capillary endothelium of thevertebrate brain. The development of neuroscience has facilitated the discoveries ofpeptides and proteins with considerable potential in the treatment of central nervoussystem (CNS) diseases such as Alzheimer's disease and Parkinson's disease. However, asignificant challenge to their clinical administration is their ideal delivery to the CNScrossing the BBB.
Intranasal drug delivery system offers a non-invasive alternative for the delivery oftherapeutics, effectively bypassing the blood-brain barrier. Indeed, the past few yearshave witnessed a sharp increase in the amount of research on the nasal pathway for theCNS drug delivery. However, the total amount of drugs accessing the brain has beenreported to be low, especially in the form of peptides and proteins, which were highlysusceptible to the unfavorable environment of the nasal cavity. The encapsulation ofthese drugs into nanoparticles might be a promising approach, since the colloidalformulations have been shown to protect the drugs from the degrading milieu in the nasalcavity and facilitate their transport across the mucosal barriers. Nevertheless, the amountof nanoparticles accessing the brain is still limited because of the following reasons: firstof all, the penetration of the nanometer-size particles through tight junctions betweencells was negligible and the amount of unmodified nanoparticles endocytosed by thenasal epithelium was limited; secondly, the resident time of nanoparticles in nasal cavityis short because of mucociliary clearance (particles cleared within the nose every 15 to 20min), which is not available for the complete absorption of the formulation; finally,unmodified nanoparticles distributed in the nasal cavity without selectivity, resulting inpoor brain targeting efficiency of the formulation.
To address these problems, novel lectin-modified nanoparticles were constructed.Wheat germ agglutinin (WGA), specifically binding to N-acetyl-D-glucosamine and sialic acid, both of which were abundantly observed in the nasal cavity especially in theolfactory mucosa, and ulex europeus agglutinin I (UEA I), specifically binding toL-fucose, which was largely located in the olfactory epithelium, were selected aspromising targeting ligands. The incorporation of peptides in the nanoparticles mightimprove their stability in vivo while the conjugation of lectin at the nanoparticles surfacemight induce strong mucoadhesion for a longer duration, or a close contact of thenanoparticles with the mucosal cells especially in the olfactory mucosa so as to produce astronger penetration. Such lectin-modified nanoparticles might serve as potential carriersfor brain delivery of peptides and proteins with enhanced brain-targeting efficiency andminimized adverse effects in the peripheral tissues.
In the first part, lectin-conjugated nanoparticles were prepared by incorporatingmaleimide into one end of the PLA-PEG copolymer and taking advantage of its thiolgroup-binding reactivity to conjugate with the lectins thiolated with 2-iminothialane. Themean size of the resulted nanoparticles was about 100 nm and the zeta potential was-20mV. The coupling of WGA with PEG-PLA nanoparticles (NP) was confirmed by theexistence of gold-labeled WGA-NP under TEM. The retention of biorecognitive activityof WGA after the covalent coupling procedure was confirmed by haemagglutination tests.The preparation protocol was optimized based on the following endpoints: lectin densityat the particle surface, particle size and conjugation efficiency, through which the optimalratio of maleimide-PEG-PLA to MePEG-PLA around 1: 9, lectin: 2-iminothiolane 1: 60,thiolated lectin: maleimide 1: 3 and the conjugation time of 8-10 h were obtained.
In order to evaluate the capacity of the lectin-conjugated nanoparticles for drugdelivery into the CNS, in the second part, a lipophilic fluorescent probe with highsensitivity, coumarin-6, was incorporated into the nanoparticles, and the concentrationsof the fluorescent marker in blood and brain tissues following intranasal administration ofWGA-NP were determined and compared with those after nasal delivery of NP. It wassuggested by the in vitro release study and the haemagglutination test that coumarin-6was an appropriate fluorescent probe and the fluorescence signal detected or observedwas mainly attributed to the coumarin-6 encapsulated into the nanoparticles. Strongergreen fluorescent signals were observed on both olfactory bulb and cerebrum slicesfifteen minutes after intranasal administration of WGA-NP than that of NP. In vivopharmacokinetie results in rats suggested that the WGA and UEA I modification at the nanoparticles surface facilitated the absorption of the associated coumarin-6 into thebrain following intranasal administration with significant increase in the peakconcentration (about 2.26 and 1.33 times, respectively) and the area under theconcentration-time curve (about 2 and 1.7 times, respectively) in different brain tissuescompared with that of coumarin-6 incorporated in NP. The brain drug-targetingefficiency of the nanoparticles was in the following order: UEA-NP＞WGA-NP＞NP.
In the third part, WGA-NP was used to deliver vasoactive intestinal peptide (VIP) intothe CNS via nasal administration. VIP was efficiently incorporated into PEG-PLAnanoparticles followed by surface modification with WGA, producing nanoparticles withparticle size of 100 nm, zeta potential of-20 mV, encapsulation efficiency of more than70％and drug loading capacity of 1.4％. In vitro release of VIP from VIP-NP showed aslight burst at the beginning and a long sustained release of about 35％aider incubationin plasma for 12 h and 17％in nasal wash for 8 h. It was also showed that the stability ofentrapped VIP in the plasma and nasal wash was significantly improved compared withthat of VIP. In the pharmacokinetic study, VIP was radio-labeled with Na ~(125)I and intact~(125)I-VIP in the CNS was determined with HPLC coupled with aγ-counter. The amount ofintact VIP detected in the brain tissues suggested that the incorporation of VIP into thenanoparticles significantly increased the stability of VIP in vivo and the area under theconcentration-time curve of intact ~(125)I-VIP in mice brain was significantly enlarged by3.5～4.7 folds and 5.6～7.7 folds following intranasal administration of ~(125)I-VIP carried byNP and WGA-NP, respectively, compared with that after intranasal application of~(125)I-VIP solution. The same improvements in spatial memory and hippocampusconcentration of acetylcholinesterase in ethylcholine aziridium-treated rats were observedfollowing intranasal administration of 25μg/kg and 12.5μg/kg of VIP loaded by NP andWGA-NP, respectively, indicating that WGA-NP might serve as a promising carrierespecially for biotech drugs such as peptides and proteins.
In the fourth part, the transport pathways of WGA-NP from nose to brain wereinvestigated. It was observed that the penetration of WGA-NP into submucosa was fasterthan NP; More fluorescent signals representing WGA-NP were observed in the sieveplate, which was anatomically near the olfactory bulb, suggesting that the absorption ofWGA-NP into the olfactory bulb was faster than that of NP. Distribution profiles ofWGA-NP and UEA-NP in the nasal cavity indicated their higher affinity to the olfactory mucosa than to the respiratory one, which also indicated that WGA-NP in the submucosamight be transported into the CNS through the nerves or the connective tissues around thenerves in the lamina propria. Besides, a novel fluorescent probe with better opticalstability, CdSe quantum dots (QDs), was applied as a fluorescent probe and the transportmechanism of WGA-QDs-NP through the nasal mucosa was investigated using an in situnasal perfusion technique. Inhibition experiment of specific sugar suggested that theinteractions between the nasal mucosa and WGA-NP were due to the immobilization ofcarbohydrate-binding pockets at the surface of the nanoparticles. The addition of ametabolic inhibitor, NAN3, significantly reduced the amount of WGA-NP associated withthe nasal mucosa, indicating that WGA-NP was absorbed into the nasal mucosa throughan active transport pathway. Both PhAsO and chlorpromazine inhibited the association ofWGA-NP with the nasal mucosa while filipin failed to affect the association, suggestingthat clathrin pathway might play an important role in the endocytosis of WGA-NP whilethe caveolae one might not be the main pathway. The addition of both BFA andmonensin also decreased the association of WGA-NP with the nasal mucosa, indicatingthat both Golgi apparatus and lysosome participated in the uptake of WGA-NP. Suchinvestigations on mechanism might provide useful information for the design of noveldrug delivery systems.
In order to evaluate the safety of WGA-NP following intranasal administration, theirinfluences on the nasal cilia, morphology and cells differentiation of the nasal mucosawere investigated. Nasal ciliotoxicity studies were conducted on both toad palate and ratnasal mucosa. The duration of ciliary movement on toad palate was 12 hours and 11.75hours for the WGA-NP-treated and NP-treated mucosa, respectively, which wascomparable with that of the negative control (12.25 hours). The data were consistent withthe results shown by SEM in the rat nasal mucosa, which showed that after intranasaladministration of WGA-NP and NP, no visible change in the morphology and integrity ofthe cilia was observed, suggesting that the nasal ciliotoxicity of WGA-NP and NP werenegligible. Furthermore, it was showed that the morphology and cells differentiation ofthe nasal mucosa were not affected with intact olfactory mucosa of about 40μm, intactand dense cilia on the mucosa and glands, blood vessels and nerve bundles in the laminapropria comparable with those of negative control observed following nasaladministration of WGA-NP for 7 days. These data indicated that WGA-NP was safe following intranasal administration.
引文
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