用于改善生物大分子药物功效的超多孔水凝胶、纳米粒新型给药载体
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摘要
随着科学技术的迅猛发展,以往单一学科及其技术难以解决的关键科学问题经多学科交叉及技术的使用获得突破。本文运用生物科学、材料科学、纳米科学及药剂学等学科的理论和方法,研究可显著改善蛋白质多肽类药物及DNA、siRNA功效的新型给药载体及其作用机理。
蛋白质多肽、核酸等生物大分子药物药理活性强、特异性高,在肿瘤、糖尿病、感染性疾病等重大疾病的治疗中显示出巨大潜力,但其临床应用主要为注射剂型,多数药物半衰期短,长期用药患者顺应性差。而非注射给药特别是口服给药时,此类亲水性生物大分子药物不易被亲脂性的生物膜摄取,易被体内各种酶降解导致活性降低或失活,生物利用度低。利用给药系统(Drug Delivery System, DDS)可提高生物大分子药物的体内外稳定性、促进药物吸收、改善药物体内作用功效。其中水凝胶给药载体还可控制药物释放,具有生物黏附、生物相容和生物可降解等特性；纳米给药载体还可增溶难溶性药物,缓控释药物和靶向给药。
同时具有抑制蛋白酶活性、促进药物渗透及黏膜黏附等性质的多功能聚合物给药载体可显著提高蛋白质多肽类药物的口服吸收,能有效突破胞外屏障(吞噬系统、核酸酶)和胞内屏障(细胞膜、内涵体、溶酶体、核膜)的多功能非病毒基因载体有望持续、高效地将基因导入靶细胞和靶组织。据此,设计并研究新型互穿网络聚合物超多孔水凝胶(SPH-IPN),以胰岛素为模型药物,研究SPH-IPN促进蛋白质多肽类药物的口服吸收及作用机理;依据壳聚糖季胺盐(TMC)与巯基化聚合物黏膜黏附及促渗特点,设计一种新型壳聚糖多功能衍生物—巯基化壳聚糖季胺盐(TMC-Cys),以胰岛素和pEGFP分别作为模型蛋白质药物和模型基因,研究其自组装纳米载体促进蛋白质药物口服吸收、基因转染及其作用机理：对TMC-Cys纳米载体进行甘露糖配体修饰,以TNF-αsiRNA为靶基因,研究甘露糖修饰的TMC-Cys (MTC)纳米载体对小肠M细胞、巨噬细胞的主动靶向作用及增加siRNA口服给药功效与作用机理。
以丙烯酸(AA)和丙烯酰胺(AM)为单体,过硫酸铵(APS)/N,N,N',N'-四甲基乙二胺(TEMED)为引发体系,NaHCO3为起泡剂,N,N'-亚甲基-双丙烯酰胺(Bis)为交联剂,溶液聚合制得聚(丙烯酸-丙烯酰胺)(P(AA-co-AM))超多孔水凝胶；采用分步互穿网络聚合物技术,聚合时加入O-羧甲基壳聚糖(O-CMC),胶凝后以戊二醛(GA)交联O-CMC,制得SPH-IPN。傅立叶变换红外光谱(FTIR)、核磁共振(13C NMR)、差示扫描量热分析(DSC)等研究表明SPH-IPN中含有P(AA-co-AM)和交联的O-CMC;扫描电镜(SEM)、光镜、共聚焦激光扫描显微镜(CLSM)观察表明SPH-IPN含有大量相互连接、直径100-300μm的孔隙,O-CMC围绕孔隙边缘分布。SPH-IPN孔隙率大于80%,水中可快速溶胀,平衡溶胀比30-80,SPH-IPN的溶胀比随O-CMC含量增加、交联度增加、交联时间延长而降低。引入互穿网络聚合物(IPN)结构可显著提高SPH-IPN的压缩模量和拉伸模量；压缩模量和拉伸模量均随O-CMC含量增加而显著提高;压缩模量随O-CMC交联度增加、交联时间延长而显著提高。
考察pH、离子强度和温度对SPH-IPN溶胀行为的影响；以胰岛素为模型药物,研究SPH-IPN的载药量、载胰岛素SPH-IPN在不同介质中的体外释放行为、载药前后胰岛素的稳定性及聚合物-药物相互作用；研究SPH-IPN中水的状态及保水性。
研究表明,SPH-IPN的溶胀具有离子强度、pH和温度敏感性。离子强度≥0.01mol·L-1时,SPH-IPN的溶胀比随离子强度增大而减小;离子强度＜0.001mol·L-1时,溶胀不受影响。pH≤3.0时SPH-IPN几乎不溶胀；3.0≤pH≤6.2时随pH升高溶胀速率加快,溶胀比增大;pH≥6.2时溶胀充分,溶胀行为无显著改变;SPH-IPN对脉冲式pH改变可快速响应,呈现可逆的溶胀-去溶胀行为。温度升高可促进SPH-IPN的溶胀。SPH-IPN对胰岛素吸附载药的载药量为4%-7%,显著高于传统超多孔水凝胶(CSPH);载药量随O-CMC含量增加而略有降低。胰岛素体外释放对离子强度、pH和温度敏感。圆二色谱分析和生物活性检测结果表明载药前后胰岛素的构象和生物活性无显著变化。SPH-IPN对胰岛素的实际载药率显著高于理论载药率,pH 7.4 PBS介质中药物可快速、完全释放,空白SPH-IPN和释药后SPH-IPN的FTIR图谱相似,表明SPH-IPN与胰岛素间存在较强的物理相互作用,不存在化学共价连接。溶胀的SPH-IPN中可冻结水占主要部分；SPH-IPN与水分子形成氢键的作用随O-CMC含量和胰岛素载药量增加而减弱。外加压力与37℃孵育时,SPH-IPN保水性较好,保水性随O-CMC含量增加而提高。
考察载胰岛素SPH-IPN正常大鼠口服给药和回肠给药的生物利用度,以及糖尿病模型大鼠口服给药降血糖效果;从黏膜黏附、蛋白酶抑制、渗透促进、小肠滞留等方面探讨SPH-IPN促胰岛素肠道吸收机理；考察聚合物结构完整性对SPH-IPN抑酶、促渗、小肠滞留及胰岛素口服吸收的影响。
正常大鼠口服载胰岛素完整的SPH-IPN (I-SPH-IPN)后药物吸收和降血糖效果显著,血糖最低降至初始值的65%,相对皮下注射的口服生物利用度为5.0%,药理生物利用度为6.3%；口服载胰岛素粉碎的SPH-IPN (P-SPH-IPN)后药物吸收和降血糖效果均不明显；回肠给予载胰岛素I-SPH-IPN和P-SPH-IPN时,药物吸收和降血糖效果显著,血糖最低降至初始值的30%,二者效果相当,均优于口服给药；糖尿病模型大鼠口服载胰岛素I-SPH-IPN后降血糖效果显著。SPH-IPN可通过非特异性作用和机械作用黏附至小肠黏膜,黏附力随O-CMC含量增加而增大。SPH-IPN可通过捕获蛋白酶溶液和络合Ca2+抑制肠腔蛋白酶(胰蛋白酶、糜蛋白酶)活性,且SPH-IPN络合二价金属离子的能力随O-CMC含量增加而增强,抑酶作用相应增强。I-SPH-IPN和P-SPH-IPN的体外抑酶作用相当,I-SPH-IPN可减少药物在肠腔中的释放,增加黏液层和黏膜中的释放,从而降低肠腔蛋白酶对药物的降解,I-SPH-IPN对胰岛素的保护作用强于P-SPH-IPN。SPH-IPN通过机械作用可逆打开小肠上皮细胞间紧密连接,促进胰岛素的胞间转运；加入I-SPH-IPN和P-SPH-IPN, Caco-2细胞单层的跨膜电阻最低分别降至初始值的25%和50%,FITC-胰岛素在Caco-2细胞单层中的转运量分别提高至未加聚合物的4.9和1.9倍,离体小肠中的Papp分别提高至4.2和1.8倍,表明I-SPH-IPN打开上皮细胞间紧密连接和促胰岛素渗透的能力强于P-SPH-IPN。I-SPH-IPN通过机械作用固定于大鼠小肠壁,小肠滞留时间超过8h；P-SPH-IPN在小肠中易分散,无法通过机械作用固定于肠壁,滞留时间短于4h。
考察SPH-IPN的细胞毒性、基因(遗传)毒性、小肠组织相容性、口服急性、亚急性毒性和血液相容性,从整体动物、组织、细胞和分子水平评价SPH-IPN的安全性;HPLC测定SPH-IPN中单体和交联剂的残留量,为生物相容性评价提供依据。
FDA/PI双染色、LDH、中性红、蛋白质含量测定试验结果表明RBL-2H3和Caco-2细胞与SPH-IPN及其浸提液(10、1、0.1 mg·mL-1)短时程(24h)接触后,胞外LDH释放量、胞内中性红摄入量、蛋白质含量均无显著变化,细胞活力无显著影响;MTT试验结果表明二种细胞与SPH-IPN及其浸提液长时程(7d)接触后,细胞增殖率无显著影响,SPH-IPN细胞毒性低。DNA Ladder、流式细胞仪检测、单细胞凝胶电泳和小鼠骨髓微核试验结果表明SPH-IPN不会引起RBL-2H3和Caco-2细胞凋亡、DNA断裂和小鼠骨髓微核发生,基因毒性低。SPH-IPN不会引起大鼠小肠黏膜组织损伤,组织相容性良好。SPH-IPN浸提液小鼠灌胃给药最大耐受剂量为1000 mg·kg-1;亚急性毒性试验中连续28天每天一次灌胃给予SPH-IPN浸提液(500、200、100mg·kg-1),小鼠体重增长正常,血液学和血清生化指标正常,肝、肾、脾重量正常,组织切片中未见炎症、坏死、水肿等病理现象,肝、肾、脾中ACP、ALKP、GPT、GOT和LPO含量正常,表明SPH-IPN口服安全性好。SPH-IPN的溶血率低于5%,动态凝血率低于硅化玻璃,血小板黏附率低,具有一定的抗凝血效果,血液相容性良好。SPH-IPN中AA、AM和GA的残留量分别为1.4±0.6、2.0±0.2、<0.2 ppm,符合其限量规定。
壳聚糖经季胺化和巯基化修饰制备TMC-Cys,表征理化性质：聚电解质(PEC)法制备TMC-Cys/胰岛素自组装纳米粒(TMC-Cys NP)并表征理化性质;考察TMC-Cys NP正常大鼠口服给药和回肠给药的降血糖效果,研究TMC-Cys NP促胰岛素口服吸收机理。
壳聚糖(Mw 30、200、500 kDa)与碘甲烷反应合成季胺化度分别为15%和30%的TMC,TMC与Cys经EDAC/NHS催化的缩合反应合成TMC-Cys。400-500μmol·g-1 Cys共价连接至TMC,其中约35%为游离巯基,其余为二硫键；游离巯基在中性条件下可氧化形成二硫键。FTIR和13C NMR表明TMC与Cys以酰胺键连接,DSC和TGA结果表明季胺化和巯基化修饰可改变壳聚糖的结晶度,降低其热稳定性。TMC-Cys的抑菌作用与TMC相近,清除自由基作用强于TMC,季胺化度较高时抑菌作用较强,清除自由基能力减弱。荷正电的TMC-Cys与荷负电的胰岛素经静电作用自组装形成TMC-Cys NP,纳米粒呈球形,分散度良好,粒径为100-170 nm,Zeta电势为+12-+18 mV,胰岛素包封率达90%。纳米粒粒径、Zeta电势和包封率受胰岛素溶液pH、TMC-Cys/胰岛素质量比和离子强度影响;胰岛素体外释放受壳聚糖分子量、季胺化度、释放介质离子强度和离子类型影响。TMC-Cys NP的小肠黏膜黏附力和黏蛋白黏附率较TMC NP分别提高2.1-4.7倍和1.5-2.2倍,黏蛋白黏附率随壳聚糖分子量或季胺化度升高而增加。DSC分析表明TMC-Cys通过与黏蛋白间形成二硫键提高黏附力。与TMC NP相比,TMC-Cys NP胰岛素的离体小肠Papp提高1.7-26倍,Caco-2细胞摄取量提高1.7-3.0倍,Peyer's结摄取量提高1.7-5.0倍,其中TMC-Cys(200,30) NP的促渗作用最强。大鼠口服和回肠给药时,TMC-Cys NP的降血糖效果优于TMC NP,血糖最低分别降至初始值的65%和30%。Caco-2细胞MTT试验和大鼠回肠LDH试验结果表明TMC-Cys NP细胞毒性低,安全性良好。
以增强型绿荧光蛋白表达质粒(pEGFP)为模式质粒,PEC法制备TMC-Cys/pEGFP自组装纳米复合物(TMC-Cys NC)并表征理化性质；考察TMC-Cys NC在HEK293细胞和小鼠胫前肌中的体内外转染效率,研究其转染机制。
TMC-Cys NC呈球形,分散度良好,粒径为150-400 nm,Zeta电势为+14-+20mV；凝胶阻滞试验结果表明TMC-Cys可通过静电作用缩合pDNA; TMC-Cys NC可有效保护pDNA免受核酶降解。TMC-Cys NC的细胞黏附率较TMC NC显著提高；TMC-Cys NC的HEK293细胞摄取率分别提高至TMC NC和Lipofectamine2000的1.4-3.0倍和1.6-4.4倍；4℃时TMC-Cys NC的摄取量为37℃时的25%,叠氮钠和氯丙嗪处理分别使摄取量减少40%和70%,表明TMC-Cys NC主要经能量依赖的网格蛋白介导的细胞内吞途径进胞；松胞素D和染料木素对TMC-Cys NC的摄取无显著影响,表明进胞机理与细胞骨架的重构和窖蛋白介导的通路无关。TMC-Cys NC的释放行为呈现谷胱甘肽(GSH)浓度依赖性,胞外GSH浓度下缓慢释放pEGFP,胞内GSH浓度下快速释放pEGFP并转运进核,细胞核中pEGFP的含量为TMC NC组的3.7倍。TMC-Cys NC在HEK293细胞中的转染效率提高至TMC NC的1.4-3.2倍,其中TMC-Cys(100,30) NC的转染效率最高(约35%),为Lipofectamine2000的1.5倍。TMC-Cys(100,30) NC的体内转染效率分别提高至TMC NC和Lipofectamine2000的2.3倍和4.1倍。
制备MTC及其纳米粒,研究纳米粒的理化性质;以TNF-a siRNA为靶基因,研究MTC纳米粒对小肠M细胞、巨噬细胞的主动靶向作用和提高siRNA口服给药功效及其作用机理。
TMC (200、500 kDa)经甘露糖修饰和巯基化修饰制得甘露糖修饰度约20%的MTC；捕获法、吸附法和自组装法分别制备载siRNA MTC纳米粒(en-MTC-TPP NP、ad-MTC-TPP NP和MTC-SANP),纳米粒为球形或亚球形,分散良好,粒径为130-230 nm,Zeta电势为正值,siRNA包封率为70-80%,纳米粒可显著提高siRNA的血清稳定性;纳米粒粒径随壳聚糖分子量增大而增大：ad-MTC-TPP NP和MTC-SANP受离子强度影响较大,siRNA包封率随离子强度升高而显著降低,但en-MTC-TPP NP受离子强度影响较小。与载NC siRNA的巯基化壳聚糖季胺盐纳米粒(en-TC-TPP NP)相比,en-MTC-TPP NP的Caco-2细胞和Raw 264.7细胞黏附力显著提高,siRNA在Raw 264.7细胞中的摄取量提高2.0-24倍,Peyer's结摄取量提高19-24倍,体外M细胞模型和Caco-2细胞单层转运量提高1.2-2.1倍,离体小肠转运量提高6.9-11.0倍,且M细胞模型中的转运量显著高于Caco-2细胞单层中的转运量,含Peyer's结离体小肠的转运量高于不含Peyer's结离体小肠的转运量,表明甘露糖配体修饰载体可通过特异性配体-受体结合作用显著提高纳米粒对M细胞和巨噬细胞的主动靶向作用,从而促进siRNA的小肠摄取和转运。以TNF-αsiRNA为靶基因,en-MTC-TPP NP在Raw 264.7细胞中的干扰效率显著强于en-TC-TPP NP和Lipofectamine2000;相同干扰效率(70%)时,en-MTC-TPP NP的siRNA剂量较Lipofectamine2000降低200倍。en-MTC-TPP NP在Raw 264.7细胞和Caco-2细胞中基本无毒性,表明纳米粒在发挥基因沉默作用时不会造成细胞毒性。小鼠口服en-MTC-TPP NP可显著降低血清TNF-α含量。
With the rapid development of science and technology, multi-interdisciplinary cooperation and technological application have demonstrated an effective artifice in overcoming the difficulties that have long been laid unsolved by an individual subject or technique. The current thesis provides a systemic investigation on the development of novel delivery carriers for significantly improving the effectiveness of protein, peptide, DNA, and siRNA, and elucidation of the underlying mechanisms, taking full advantages of the theories and experimental strategies in biological sciences, material sciences, nano sciences, and pharmaceutical sciences.
Protein, peptide and nucleic acid drugs have been exploited which possess potent pharmacological activity as well as high specificity, and have shown great potentials in the treatment of diseases such as cancer, diabetes, and infectious diseases. However, their clinical applications are mostly restricted to injection formulations, which lead to short half-life and undesired patient compliance. When parenterally delivered, especially orally delivered, these hydrophilic biomacromolecules are barely absorbed by the lipophilic biological membranes, and are readily degraded by various enzymes, resulting in poor bioavailability. Therefore, development of drug delivery systems (DDS) is attracting more attention, which aims at improving in vivo drug stability, promoting drug absorption, and enhancing in vivo drug efficacy. Hydrogel delivery carriers are distinguished for its bioadhesion, biocompatibility, biodegradability, and a manner for controlled drug release. Nanoparticulate delivery carriers show distinct beneficial attributes, including solubilization of insoluble drugs, controlled and sustained drug release and targeted drug delivery. The characteristics of delivery systems are largely dependent on the polymeric carriers used.
Consequently, multi-functional carriers that exhibited mucoadhesion, enzymatic inhibition, and absorption enhancement would be an efficient strategy in facilitating oral absorption of protein and peptide drugs. In addition, multi-functional non-viral gene carriers to allow overcoming of the extracellular barrier (phagocyte system and nuclease) and intracellular barrier (cell membrane, endosome, lysosome, nucleus membrane) could deliver the genes into target cells and tissues in an efficient, sustained, and targeted manner. The current investigation aims at constructing novel superporous hydrogels containing interpenetrating polymer networks (SPH-IPN), investigating efficacy of SPH-IPN as oral delivery vehicles for protein drugs with insulin as a model drug, and elucidation the absorption mechanisms. Besides, thiolated trimethyl chitosan (TMC-Cys) as a novel multifunctional chitosan derivative was synthesized to combine the advantages of TMC and thiolated polymers. With insulin and pEGFP as model protein drug and model pDNA, TMC-Cys was subjected to formation of TMC-Cys/insulin nanoparticles (TMC-Cys NP) and TMC-Cys/pEGFP nanocomplexes (TMC-Cys NC) through self-assembly, which were evaluated as oral delivery vehicles for protein drugs and gene delivery carriers, respectively. Besides, the mechanisms underlying the drug absorption enhancement and gene transfection elevation were elucidated. TMC-Cys nanocarriers were further modified with mannose, and the resultant mannose-modified TMC-Cys (MTC) nanocarriers were investigated for its active targeting towards intestinal M cells as well as macrophages with TNF-a siRNA as the target gene. Besides, RNAi efficacy was evaluated via oral delivery along with elucidation of the underlying mechanisms.
Superporous hydrogels of P(AA-co-AM) was synthesized through solution copolymerization of acrylic acid (AA) and acrylamide (AM) with APS/TEMED as initiators, NaHCO3 as foaming agent, and Bis as cross-linker. O-CMC was allowed to be well distributed in SPH along with the polymerization reaction, and was further cross-linked by glutaraldehyde (GA) after SPH was synthesized, thus achieving the SPH-IPN. The structures of the SPH-IPNs were characterized with FTIR,13C NMR, and DSC. SEM, CLSM and light images revealed that the SPH-IPNs possessed both the IPN network and large numbers of pores with diameters of 100-300μm, and the cross-linked O-CMC molecules were located on the peripheries of these pores. SPH-IPN quickly swelled in water with equilibrium swelling ratio of 30-80, and an increase in O-CMC content, GA amount and cross-linking time led to slower swelling behavior. Due to the cross-linked O-CMC network, compression and tensile modulus of SPH-IPN were greatly improved, and an increase in O-CMC content, GA amount and cross-linking time was favorable for enhanced mechanical strength.
Swelling behaviors of SPH-IPN were measured at different pH, ionic strength, and temperature. With insulin as a model drug, loading capacity of the SPH-IPN was determined. Release profiles were also evaluated at different pH, ionic strength, and temperature. Stability of insulin following drug loading and polymer-drug interactions were also investigated. State of water in SPH-IPN and water retention capacities were examined.
Swelling of SPH-IPN was sensitive towards of pH, ionic strength, and temperature. An increase in the ionic strength within the range of 0.001-1M yields a significant decrease in the swelling ratio, while it brought about an insignificant difference in the swelling behavior of the polymer when the ionic strength was no higher than 0.001M. A drastic increase in the swelling was observed within the pH range of 3.0-6.2, whereas at pH> 6.2 or pH< 3.0, the change in the swelling behavior was slight. SPH-IPN could rapidly respond to pulsatile alternation of pH values between 1.2 and 7.4, thereby exhibiting fast swelling and de-swelling. An increase in temperature could further facilitate swelling of SPH-IPN. Insulin loading capacity of SPH-IPN was 4%-7%, which was notably higher than CSPH, and an increase in O-CMC content yielded slightly deceased loading capacity. Insulin release also exhibited sensitivity towards pH, ionic strength, and temperature. After drug loading and release, the circular dichroism (CD) spectra revealed that conformation of insulin had no significant alteration and bioactivity of insulin was well preserved according to hypoglycaemic effect in mice. Great discrepancies were noted between the theoretical and experimental loading levels of SPH-IPN for insulin, and fast and complete drug release was observed in pH 7.4 PBS. Besides, FTIR spectra of blank SPH-IPN and washed SPH-IPN after drug release were similar, which suggested strong physical interactions rather than chemical linkage between the polymer and the drug. Freezing water was the majority of the imbibed water in the swollen SPH-IPN, and the ability of SPH-IPN to form hydrogen bonding with water molecules was weakened as the O-CMC content and insulin loading amount increased. SPH-IPN showed desired water retention capacities against compression and exposure at 37℃, which further improved as the amount of O-CMC network increased.
Pharmacokinetics and pharmacokinetics were investigated following oral and ileal administration of insulin-loaded SPH-IPN in normal rats, and the hypoglycemic effect of insulin-loaded SPH-IPN was also monitored in diabetic rats following oral delivery. Mucoadhesion, enzymatic inhibition, insulin permeation enhancing effect, and intestinal retention of SPH-IPN were evaluated to provide an insight into the intestinal absorption mechanisms of insulin in the presence of SPH-IPN. Besides, the effect of polymer integrity on the enzymatic inhibition, permeation enhancement, intestinal retention, and oral absorption of insulin was investigated.
Oral administration of insulin-loaded integral SPH-IPN (I-SPH-IPN) led to marked insulin absorption and hypoglycemic effect in normal rats with F and PA of 5.0% and 6.3%, respectively. In comparison, minimal insulin absorption and unappreciable hypoglycemic effect were observed following powdered SPH-IPN (P-SPH-IPN) delivery. When ileally administered, both I-SPH-IPN and P-SPH-IPN yielded a faster and more potent insulin absorption and blood glucose depression, with the minimal blood glucose level of 30% of initial values. Notable hypoglycemic effect was also observed following oral administration of insulin-loaded I-SPH-IPN in diabetic rats. SPH-IPN could adhere to the intestinal mucosa through non-specific binding and mechanical fixation, and an increase in O-CMC yielded a higher muadhesion capacity. SPH-IPN exhibited potent enzymatic inhibitory effect towards luminal proteolytic enzymes (trypsin andα-chymotrypsin) via enzyme entrapment and Ca2+ binding. A higher O-CMC content correlated to enhanced Ca2+ binding capacities, which accounted for enhanced enzymatic inhibitory effect. Although I-SPH-IPN and P-SPH-IPN possessed equivalent enzymatic inhibition capacity in vitro, I-SPH-IPN outperformed P-SPH-IPN in protecting insulin from proteolytic hydrolysis under the in vivo conditions, because I-SPH-IPN released more insulin in the mucus layer rather than in the intestinal lumen and thereby prevented degradation by luminally secreted proteases. SPH-IPN facilitated paracellular transport of insulin through reversible opening of epithelial tight junctions as a result of mechanical pressure, and I-SPH-IPN exhibited potent permeation enhancing effect than P-SPH-IPN in that I-SPH-IPN and P-SPH-IPN led to a depression in TEER values of Caco-2 cell monlayers to 25% and 50% of initial values, an increase in FITC-insulin transport in Caco-2 cell monlayers by 4.9 and 1.9 folds, and an enhancement in FITC-insulin transport in excised rat ileum by 4.2 and 1.8 folds, respectively. Through mechanical fixation onto the gut wall, I-SPH-IPN showed a prolonged intestinal retention of more than 8 h, while P-SPH-IPN was cleared from the intestinal within 4 h due to failure in mechanical fixation.
Biocompatibility of SPH-IPN was explored at molecular, cellular, tissue, and animal levels in terms of cytotoxicity, genetoxicity, tissue toxicity, oral acute and sub-acute toxicity, and blood compatibility. Residual monomers and cross-linkers in SPH-IPN were quantified by HPLC to provide evidences for biocompatibility assessment.
Results of FDA/PI double staining assay, LDH assay, neutral red assay, and protein assay in RBL-2H3 and Caco-2 cells revealed lack of cytotoxicity of SPH-IPN and SPH-IPN extract (10,1,0.1 mg·mL-1) following short-time exposure (24 h), and MTT assay further evidenced that SPH-IPN did not interfere with cell proliferation following long-time treatment (7 d). DNA ladder assay, cytometry assay and comet assay in the above two cell lines and in vivo micronucleus (MN) assay in mice showed that SPH-IPN did not induce cell apoptosis, DNA breakage, and MN formation, suggesting lack of genotoxicity. SPH-IPN did not induce impairment to the intestinal mucous, indicating good tissue compatibility. Oral administration of SPH-IPN extract (1000 mg·kg-1) resulted in unappreciable acute toxicity, and in the 28-day sub-acute toxicity study (500,200,100 mg·kg-1), body weight of I-SPH-IPN extract treated mice gradually increased with no appreciable difference to control animals, weights of mouse livers, kidneys, and spleens were found to be normal, and in the histological examination no necrosis, inflammation, edema or other pathological signs were detected. With regard to the hematological parameters and biochemical assays, no statistically significant difference was observed against control. LPO, GOP, GPT, ACP, and AKLP activities in mouse livers, kidneys, and spleens showed no abnormality, either. Hemolysis ratio of SPH-IPN was lower than 5%, the dynamic blood clotting ratio was lower than silicated glass, and platelet adsorption ratio was low, suggesting its desired anticoagulant capacities. Residual amount of AA, AM and GA in SPH-IPN was quantified to be 1.4±0.6,2.0±0.2, and<0.2 ppm, which was within the safety range.
TMC-Cys was synthesized through trimethylation of chitosan and thiolation of TMC, and TMC-Cys NP was prepared using the PEC method. It was thereafter evaluated as an oral delivery vehicle for protein drugs, and the mechanisms of oral absorption of insulin was elucidated.
Chitosan (30、200、500 kDa) was allowed to react with CH3I to achieve TMC with quaterization degree (DQ) of 15% and 30%, and TMC was subsequently modified with Cys via amide bond formation between the residual primary amino groups on TMC and carboxyl groups on Cys as mediated by EDAC/NHS. About 400-500 mmol·g-1 of sulphydryl was immobilized on TMC-Cys, with approximately one-third remaining the free thiol groups while two thirds being oxidized to the disulfide. FTIR and 13C NMR confirmed covalent conjugation between TMC and Cys via amide bonding, while DSC and TGA evidenced reduced crystallinity and decreased thermal stability of the polymer following trimethylation and thiolation. TMC-Cys exhibited comparable antimicrobial effect to TMC while stronger scavenging effect against free radicals. TMC-Cys NP was prepared through electrostatic interactions between oppositely charged TMC-Cys and insulin, which demonstrated particle size of 100-170 nm, Zeta potential of+12-+18 mV, and high encapsulation efficiency of 90%. Particle size, Zeta potential, and insulin encapsulation efficiency were largely dependent on pH of the insulin solution, TMC-Cys/insulin weight ratio, and ionic strength. Besides, chitosan Mw, DQ of TMC, ionic strength and ion types of the dissolution medium also exerted appreciable effect on in vitro release profiles of insulin. TMC-Cys NP showed a 2.1-4.7-fold and a 1.5-2.2-fold increase in intestinal mucoadhesion and mucin adsorption compared to TMC NP, and higher Mw or DQ was favorable for mucin adsorption. DSC measurement evidenced disulfide formation between TMC-Cys and mucin. Compared to TMC NP, TMC-Cys NP induced increased insulin transport through rat intestine by 1.7-2.6 folds, promoted Caco-2 cell internalization by 1.7-3.0 folds, and augmented uptake in Peyer's patches by 1.7-5.0 folds, respectively, among which TMC-Cys(200,30) NP exhibited the optimal permeation enhancing effect. Such results were further confirmed by in vivo experiment. MTT assay in Caco-2 cells and LDH assay in rat intestine revealed lack of toxicity of TMC-Cys NP.
With pEGFP as model pDNA, TMC-Cys NC was prepared using the PEC method and subjected to measurement of size and Zeta potential, and morphology observation using SEM and AFM. In vitro and in vivo transfection efficiency of TMC-Cys NC were monitored in HEK293 cells and mouse posterior tibialis muscles, respectively, and the transfection mechanisms were elucidated. TMC-Cys showed potent condensation capacity towards pDNA as evidenced by EB exclusion and gel retardation assays, and the resultant TMC-Cys NC demonstrated diameters of 150-400 nm and Zeta potentials of+14-+20 mV. TMC-Cys NC could also prevent degradation of pDNA by nucleases. Cell binding and mucin adsorption of TMC-Cys NC were enhanced 2.4-3.0 and 1.2-1.7 folds, respectively, compared to TMC NC, and cellular uptake of TMC-Cys NC was enhanced 1.4-3.0 fold and 1.6-4.4 fold compared to TMC NC and Lipofectamine2000. Lowering the temperature from 37 to 4℃substantially reduced uptake of TMC-Cys(100,30) NC by approximately 75%, and pretreatment of sodium azide or chlorpromazine yielded a depression in the complex internalization by approximately 30% and 70%, respectively, suggesting that an energy-dependent clathrin-mediated endocytic process was involved in the complex uptake. Comparatively, cytochalasin D and genistein exerted unappreciable effect on the cellular uptake of nanocomplexes, which indicated that complex uptake was not associated with cytoskeleton recognization and the caveolin-mediated pathway. pEGFP was slowly released from TMC-Cys NC at extracellular GSH concentrations, while was quickly released at the intracellular concentration and transported to the nuclei, leading to a 3.7-fold enhancement in nuclear accumulation of pEGFP compared to TMC NC. Besides, the relative amount of nuclear pEGFP increased with incubated time, which reached a plateau of 40% at 4 h. Consequently, TMC-Cys NC showed a 1.4 to 3.2-fold increase in the transfection efficiency in HEK293 cells as compared to TMC NC and the optimal TMC-Cys(100,30) NC showed a 1.5-fold enhancement than Lipofectamine2000. Such results were further confirmed by in vivo transfection with a 2.3-fold and 4.1-fold higher transfection efficiency of TMC-Cys(100,30) NC than TMC(100,30) NC and Lipofectamine2000, respectively.
MTC was synthesized and MTC nanoparticles were prepared and characterized. With TNF-a siRNA as a target gene, MTC nanoparticles were evaluated for their capacity of actively targeting towards intestinal M cells and macrophages, improving oral RNAi effect and the underlying mechanisms.
MTC with mannose modification degree of about 20% was synthesized through modification of TMC (Mw 200,500 kDa) with mannopyranosylphenylisothiocyanate and subsequent thiolation with cysteine. siRNA loaded MTC nanoparticles were prepared via the entrapment method, adsorption method, and self-assembly method. The nanoparticles were spherical or sub-spherical in shape, demonstrating diameters of 130-230 nm, positive Zeta potentials, and siRNA encapsulation efficiency of 70-80%. Serum stability of siRNA was significantly enhanced following nanoparticle encapsulation. Higher chitosan MW led to larger particle sizes; dilution led to augmentation of particle size and polydispersity of MTC-SA NP, and an increase in ionic strength resulted in notably decreased encapsulation efficiency of ad-MTC-TPP NP and MTC-SA NP. Comparatively, en-MTC-TPP NP was superior in resistance towards ionic strength. As compared to en-TC-TPP NP, en-MTC-TPP NP exhibited significantly higher cell binding capacity towards Caco-2 cells and Raw 264.7 cells, enhanced uptake level in Raw264.7 cells by 2.0-2.4 folds, increased Peyer's patch uptake by 1.9-2.4 folds, augmented Caco-2 cell monolayer transport of 1.2-2.1 folds, and elevated intestinal transport by 6.9-11.0 folds. Besides, transport in co-cultures of Caco-2 cell monolayers was higher than that in mono-cultures, and transport in excised rat intestine with Peyer's patches was higher than that in rat intestine without Peyer's patches, what suggested that mannose modification remarkably promoted active targeting of nanoparticles towards M cells and macrophages and thereby facilitating siRNA uptake and intestinal transport. With TNF-a siRNA as the target gene, en-MTC-TPP NP demonstrated potent silencing capacity in Raw 264.7 cells than en-TC-TPP NP and Lipofectamine2000, wherein en-MTC-TPP NP showed comparable silencing effect to Lipofectamine2000 at a reduced siRNA dose of 200 folds. en-MTC-TPP NP exhibited minimal cytoxicity in Raw264.7 cells and Caco-2 cells, suggesting lack of toxicity of the nanoparticles when exerting potent gene silencing effect. Oral delivery of en-MTC-TPP NP resulted in notable inhition of LPS-induced serum TNF-αproduction.
蛋白质多肽、核酸等生物大分子药物药理活性强、特异性高,在肿瘤、糖尿病、感染性疾病等重大疾病的治疗中显示出巨大潜力,但其临床应用主要为注射剂型,多数药物半衰期短,长期用药患者顺应性差。而非注射给药特别是口服给药时,此类亲水性生物大分子药物不易被亲脂性的生物膜摄取,易被体内各种酶降解导致活性降低或失活,生物利用度低。利用给药系统(Drug Delivery System, DDS)可提高生物大分子药物的体内外稳定性、促进药物吸收、改善药物体内作用功效。其中水凝胶给药载体还可控制药物释放,具有生物黏附、生物相容和生物可降解等特性；纳米给药载体还可增溶难溶性药物,缓控释药物和靶向给药。
同时具有抑制蛋白酶活性、促进药物渗透及黏膜黏附等性质的多功能聚合物给药载体可显著提高蛋白质多肽类药物的口服吸收,能有效突破胞外屏障(吞噬系统、核酸酶)和胞内屏障(细胞膜、内涵体、溶酶体、核膜)的多功能非病毒基因载体有望持续、高效地将基因导入靶细胞和靶组织。据此,设计并研究新型互穿网络聚合物超多孔水凝胶(SPH-IPN),以胰岛素为模型药物,研究SPH-IPN促进蛋白质多肽类药物的口服吸收及作用机理;依据壳聚糖季胺盐(TMC)与巯基化聚合物黏膜黏附及促渗特点,设计一种新型壳聚糖多功能衍生物—巯基化壳聚糖季胺盐(TMC-Cys),以胰岛素和pEGFP分别作为模型蛋白质药物和模型基因,研究其自组装纳米载体促进蛋白质药物口服吸收、基因转染及其作用机理：对TMC-Cys纳米载体进行甘露糖配体修饰,以TNF-αsiRNA为靶基因,研究甘露糖修饰的TMC-Cys (MTC)纳米载体对小肠M细胞、巨噬细胞的主动靶向作用及增加siRNA口服给药功效与作用机理。
以丙烯酸(AA)和丙烯酰胺(AM)为单体,过硫酸铵(APS)/N,N,N',N'-四甲基乙二胺(TEMED)为引发体系,NaHCO3为起泡剂,N,N'-亚甲基-双丙烯酰胺(Bis)为交联剂,溶液聚合制得聚(丙烯酸-丙烯酰胺)(P(AA-co-AM))超多孔水凝胶；采用分步互穿网络聚合物技术,聚合时加入O-羧甲基壳聚糖(O-CMC),胶凝后以戊二醛(GA)交联O-CMC,制得SPH-IPN。傅立叶变换红外光谱(FTIR)、核磁共振(13C NMR)、差示扫描量热分析(DSC)等研究表明SPH-IPN中含有P(AA-co-AM)和交联的O-CMC;扫描电镜(SEM)、光镜、共聚焦激光扫描显微镜(CLSM)观察表明SPH-IPN含有大量相互连接、直径100-300μm的孔隙,O-CMC围绕孔隙边缘分布。SPH-IPN孔隙率大于80%,水中可快速溶胀,平衡溶胀比30-80,SPH-IPN的溶胀比随O-CMC含量增加、交联度增加、交联时间延长而降低。引入互穿网络聚合物(IPN)结构可显著提高SPH-IPN的压缩模量和拉伸模量；压缩模量和拉伸模量均随O-CMC含量增加而显著提高;压缩模量随O-CMC交联度增加、交联时间延长而显著提高。
考察pH、离子强度和温度对SPH-IPN溶胀行为的影响；以胰岛素为模型药物,研究SPH-IPN的载药量、载胰岛素SPH-IPN在不同介质中的体外释放行为、载药前后胰岛素的稳定性及聚合物-药物相互作用；研究SPH-IPN中水的状态及保水性。
研究表明,SPH-IPN的溶胀具有离子强度、pH和温度敏感性。离子强度≥0.01mol·L-1时,SPH-IPN的溶胀比随离子强度增大而减小;离子强度＜0.001mol·L-1时,溶胀不受影响。pH≤3.0时SPH-IPN几乎不溶胀；3.0≤pH≤6.2时随pH升高溶胀速率加快,溶胀比增大;pH≥6.2时溶胀充分,溶胀行为无显著改变;SPH-IPN对脉冲式pH改变可快速响应,呈现可逆的溶胀-去溶胀行为。温度升高可促进SPH-IPN的溶胀。SPH-IPN对胰岛素吸附载药的载药量为4%-7%,显著高于传统超多孔水凝胶(CSPH);载药量随O-CMC含量增加而略有降低。胰岛素体外释放对离子强度、pH和温度敏感。圆二色谱分析和生物活性检测结果表明载药前后胰岛素的构象和生物活性无显著变化。SPH-IPN对胰岛素的实际载药率显著高于理论载药率,pH 7.4 PBS介质中药物可快速、完全释放,空白SPH-IPN和释药后SPH-IPN的FTIR图谱相似,表明SPH-IPN与胰岛素间存在较强的物理相互作用,不存在化学共价连接。溶胀的SPH-IPN中可冻结水占主要部分；SPH-IPN与水分子形成氢键的作用随O-CMC含量和胰岛素载药量增加而减弱。外加压力与37℃孵育时,SPH-IPN保水性较好,保水性随O-CMC含量增加而提高。
考察载胰岛素SPH-IPN正常大鼠口服给药和回肠给药的生物利用度,以及糖尿病模型大鼠口服给药降血糖效果;从黏膜黏附、蛋白酶抑制、渗透促进、小肠滞留等方面探讨SPH-IPN促胰岛素肠道吸收机理；考察聚合物结构完整性对SPH-IPN抑酶、促渗、小肠滞留及胰岛素口服吸收的影响。
正常大鼠口服载胰岛素完整的SPH-IPN (I-SPH-IPN)后药物吸收和降血糖效果显著,血糖最低降至初始值的65%,相对皮下注射的口服生物利用度为5.0%,药理生物利用度为6.3%；口服载胰岛素粉碎的SPH-IPN (P-SPH-IPN)后药物吸收和降血糖效果均不明显；回肠给予载胰岛素I-SPH-IPN和P-SPH-IPN时,药物吸收和降血糖效果显著,血糖最低降至初始值的30%,二者效果相当,均优于口服给药；糖尿病模型大鼠口服载胰岛素I-SPH-IPN后降血糖效果显著。SPH-IPN可通过非特异性作用和机械作用黏附至小肠黏膜,黏附力随O-CMC含量增加而增大。SPH-IPN可通过捕获蛋白酶溶液和络合Ca2+抑制肠腔蛋白酶(胰蛋白酶、糜蛋白酶)活性,且SPH-IPN络合二价金属离子的能力随O-CMC含量增加而增强,抑酶作用相应增强。I-SPH-IPN和P-SPH-IPN的体外抑酶作用相当,I-SPH-IPN可减少药物在肠腔中的释放,增加黏液层和黏膜中的释放,从而降低肠腔蛋白酶对药物的降解,I-SPH-IPN对胰岛素的保护作用强于P-SPH-IPN。SPH-IPN通过机械作用可逆打开小肠上皮细胞间紧密连接,促进胰岛素的胞间转运；加入I-SPH-IPN和P-SPH-IPN, Caco-2细胞单层的跨膜电阻最低分别降至初始值的25%和50%,FITC-胰岛素在Caco-2细胞单层中的转运量分别提高至未加聚合物的4.9和1.9倍,离体小肠中的Papp分别提高至4.2和1.8倍,表明I-SPH-IPN打开上皮细胞间紧密连接和促胰岛素渗透的能力强于P-SPH-IPN。I-SPH-IPN通过机械作用固定于大鼠小肠壁,小肠滞留时间超过8h；P-SPH-IPN在小肠中易分散,无法通过机械作用固定于肠壁,滞留时间短于4h。
考察SPH-IPN的细胞毒性、基因(遗传)毒性、小肠组织相容性、口服急性、亚急性毒性和血液相容性,从整体动物、组织、细胞和分子水平评价SPH-IPN的安全性;HPLC测定SPH-IPN中单体和交联剂的残留量,为生物相容性评价提供依据。
FDA/PI双染色、LDH、中性红、蛋白质含量测定试验结果表明RBL-2H3和Caco-2细胞与SPH-IPN及其浸提液(10、1、0.1 mg·mL-1)短时程(24h)接触后,胞外LDH释放量、胞内中性红摄入量、蛋白质含量均无显著变化,细胞活力无显著影响;MTT试验结果表明二种细胞与SPH-IPN及其浸提液长时程(7d)接触后,细胞增殖率无显著影响,SPH-IPN细胞毒性低。DNA Ladder、流式细胞仪检测、单细胞凝胶电泳和小鼠骨髓微核试验结果表明SPH-IPN不会引起RBL-2H3和Caco-2细胞凋亡、DNA断裂和小鼠骨髓微核发生,基因毒性低。SPH-IPN不会引起大鼠小肠黏膜组织损伤,组织相容性良好。SPH-IPN浸提液小鼠灌胃给药最大耐受剂量为1000 mg·kg-1;亚急性毒性试验中连续28天每天一次灌胃给予SPH-IPN浸提液(500、200、100mg·kg-1),小鼠体重增长正常,血液学和血清生化指标正常,肝、肾、脾重量正常,组织切片中未见炎症、坏死、水肿等病理现象,肝、肾、脾中ACP、ALKP、GPT、GOT和LPO含量正常,表明SPH-IPN口服安全性好。SPH-IPN的溶血率低于5%,动态凝血率低于硅化玻璃,血小板黏附率低,具有一定的抗凝血效果,血液相容性良好。SPH-IPN中AA、AM和GA的残留量分别为1.4±0.6、2.0±0.2、<0.2 ppm,符合其限量规定。
壳聚糖经季胺化和巯基化修饰制备TMC-Cys,表征理化性质：聚电解质(PEC)法制备TMC-Cys/胰岛素自组装纳米粒(TMC-Cys NP)并表征理化性质;考察TMC-Cys NP正常大鼠口服给药和回肠给药的降血糖效果,研究TMC-Cys NP促胰岛素口服吸收机理。
壳聚糖(Mw 30、200、500 kDa)与碘甲烷反应合成季胺化度分别为15%和30%的TMC,TMC与Cys经EDAC/NHS催化的缩合反应合成TMC-Cys。400-500μmol·g-1 Cys共价连接至TMC,其中约35%为游离巯基,其余为二硫键；游离巯基在中性条件下可氧化形成二硫键。FTIR和13C NMR表明TMC与Cys以酰胺键连接,DSC和TGA结果表明季胺化和巯基化修饰可改变壳聚糖的结晶度,降低其热稳定性。TMC-Cys的抑菌作用与TMC相近,清除自由基作用强于TMC,季胺化度较高时抑菌作用较强,清除自由基能力减弱。荷正电的TMC-Cys与荷负电的胰岛素经静电作用自组装形成TMC-Cys NP,纳米粒呈球形,分散度良好,粒径为100-170 nm,Zeta电势为+12-+18 mV,胰岛素包封率达90%。纳米粒粒径、Zeta电势和包封率受胰岛素溶液pH、TMC-Cys/胰岛素质量比和离子强度影响;胰岛素体外释放受壳聚糖分子量、季胺化度、释放介质离子强度和离子类型影响。TMC-Cys NP的小肠黏膜黏附力和黏蛋白黏附率较TMC NP分别提高2.1-4.7倍和1.5-2.2倍,黏蛋白黏附率随壳聚糖分子量或季胺化度升高而增加。DSC分析表明TMC-Cys通过与黏蛋白间形成二硫键提高黏附力。与TMC NP相比,TMC-Cys NP胰岛素的离体小肠Papp提高1.7-26倍,Caco-2细胞摄取量提高1.7-3.0倍,Peyer's结摄取量提高1.7-5.0倍,其中TMC-Cys(200,30) NP的促渗作用最强。大鼠口服和回肠给药时,TMC-Cys NP的降血糖效果优于TMC NP,血糖最低分别降至初始值的65%和30%。Caco-2细胞MTT试验和大鼠回肠LDH试验结果表明TMC-Cys NP细胞毒性低,安全性良好。
以增强型绿荧光蛋白表达质粒(pEGFP)为模式质粒,PEC法制备TMC-Cys/pEGFP自组装纳米复合物(TMC-Cys NC)并表征理化性质；考察TMC-Cys NC在HEK293细胞和小鼠胫前肌中的体内外转染效率,研究其转染机制。
TMC-Cys NC呈球形,分散度良好,粒径为150-400 nm,Zeta电势为+14-+20mV；凝胶阻滞试验结果表明TMC-Cys可通过静电作用缩合pDNA; TMC-Cys NC可有效保护pDNA免受核酶降解。TMC-Cys NC的细胞黏附率较TMC NC显著提高；TMC-Cys NC的HEK293细胞摄取率分别提高至TMC NC和Lipofectamine2000的1.4-3.0倍和1.6-4.4倍；4℃时TMC-Cys NC的摄取量为37℃时的25%,叠氮钠和氯丙嗪处理分别使摄取量减少40%和70%,表明TMC-Cys NC主要经能量依赖的网格蛋白介导的细胞内吞途径进胞；松胞素D和染料木素对TMC-Cys NC的摄取无显著影响,表明进胞机理与细胞骨架的重构和窖蛋白介导的通路无关。TMC-Cys NC的释放行为呈现谷胱甘肽(GSH)浓度依赖性,胞外GSH浓度下缓慢释放pEGFP,胞内GSH浓度下快速释放pEGFP并转运进核,细胞核中pEGFP的含量为TMC NC组的3.7倍。TMC-Cys NC在HEK293细胞中的转染效率提高至TMC NC的1.4-3.2倍,其中TMC-Cys(100,30) NC的转染效率最高(约35%),为Lipofectamine2000的1.5倍。TMC-Cys(100,30) NC的体内转染效率分别提高至TMC NC和Lipofectamine2000的2.3倍和4.1倍。
制备MTC及其纳米粒,研究纳米粒的理化性质;以TNF-a siRNA为靶基因,研究MTC纳米粒对小肠M细胞、巨噬细胞的主动靶向作用和提高siRNA口服给药功效及其作用机理。
TMC (200、500 kDa)经甘露糖修饰和巯基化修饰制得甘露糖修饰度约20%的MTC；捕获法、吸附法和自组装法分别制备载siRNA MTC纳米粒(en-MTC-TPP NP、ad-MTC-TPP NP和MTC-SANP),纳米粒为球形或亚球形,分散良好,粒径为130-230 nm,Zeta电势为正值,siRNA包封率为70-80%,纳米粒可显著提高siRNA的血清稳定性;纳米粒粒径随壳聚糖分子量增大而增大：ad-MTC-TPP NP和MTC-SANP受离子强度影响较大,siRNA包封率随离子强度升高而显著降低,但en-MTC-TPP NP受离子强度影响较小。与载NC siRNA的巯基化壳聚糖季胺盐纳米粒(en-TC-TPP NP)相比,en-MTC-TPP NP的Caco-2细胞和Raw 264.7细胞黏附力显著提高,siRNA在Raw 264.7细胞中的摄取量提高2.0-24倍,Peyer's结摄取量提高19-24倍,体外M细胞模型和Caco-2细胞单层转运量提高1.2-2.1倍,离体小肠转运量提高6.9-11.0倍,且M细胞模型中的转运量显著高于Caco-2细胞单层中的转运量,含Peyer's结离体小肠的转运量高于不含Peyer's结离体小肠的转运量,表明甘露糖配体修饰载体可通过特异性配体-受体结合作用显著提高纳米粒对M细胞和巨噬细胞的主动靶向作用,从而促进siRNA的小肠摄取和转运。以TNF-αsiRNA为靶基因,en-MTC-TPP NP在Raw 264.7细胞中的干扰效率显著强于en-TC-TPP NP和Lipofectamine2000;相同干扰效率(70%)时,en-MTC-TPP NP的siRNA剂量较Lipofectamine2000降低200倍。en-MTC-TPP NP在Raw 264.7细胞和Caco-2细胞中基本无毒性,表明纳米粒在发挥基因沉默作用时不会造成细胞毒性。小鼠口服en-MTC-TPP NP可显著降低血清TNF-α含量。
With the rapid development of science and technology, multi-interdisciplinary cooperation and technological application have demonstrated an effective artifice in overcoming the difficulties that have long been laid unsolved by an individual subject or technique. The current thesis provides a systemic investigation on the development of novel delivery carriers for significantly improving the effectiveness of protein, peptide, DNA, and siRNA, and elucidation of the underlying mechanisms, taking full advantages of the theories and experimental strategies in biological sciences, material sciences, nano sciences, and pharmaceutical sciences.
Protein, peptide and nucleic acid drugs have been exploited which possess potent pharmacological activity as well as high specificity, and have shown great potentials in the treatment of diseases such as cancer, diabetes, and infectious diseases. However, their clinical applications are mostly restricted to injection formulations, which lead to short half-life and undesired patient compliance. When parenterally delivered, especially orally delivered, these hydrophilic biomacromolecules are barely absorbed by the lipophilic biological membranes, and are readily degraded by various enzymes, resulting in poor bioavailability. Therefore, development of drug delivery systems (DDS) is attracting more attention, which aims at improving in vivo drug stability, promoting drug absorption, and enhancing in vivo drug efficacy. Hydrogel delivery carriers are distinguished for its bioadhesion, biocompatibility, biodegradability, and a manner for controlled drug release. Nanoparticulate delivery carriers show distinct beneficial attributes, including solubilization of insoluble drugs, controlled and sustained drug release and targeted drug delivery. The characteristics of delivery systems are largely dependent on the polymeric carriers used.
Consequently, multi-functional carriers that exhibited mucoadhesion, enzymatic inhibition, and absorption enhancement would be an efficient strategy in facilitating oral absorption of protein and peptide drugs. In addition, multi-functional non-viral gene carriers to allow overcoming of the extracellular barrier (phagocyte system and nuclease) and intracellular barrier (cell membrane, endosome, lysosome, nucleus membrane) could deliver the genes into target cells and tissues in an efficient, sustained, and targeted manner. The current investigation aims at constructing novel superporous hydrogels containing interpenetrating polymer networks (SPH-IPN), investigating efficacy of SPH-IPN as oral delivery vehicles for protein drugs with insulin as a model drug, and elucidation the absorption mechanisms. Besides, thiolated trimethyl chitosan (TMC-Cys) as a novel multifunctional chitosan derivative was synthesized to combine the advantages of TMC and thiolated polymers. With insulin and pEGFP as model protein drug and model pDNA, TMC-Cys was subjected to formation of TMC-Cys/insulin nanoparticles (TMC-Cys NP) and TMC-Cys/pEGFP nanocomplexes (TMC-Cys NC) through self-assembly, which were evaluated as oral delivery vehicles for protein drugs and gene delivery carriers, respectively. Besides, the mechanisms underlying the drug absorption enhancement and gene transfection elevation were elucidated. TMC-Cys nanocarriers were further modified with mannose, and the resultant mannose-modified TMC-Cys (MTC) nanocarriers were investigated for its active targeting towards intestinal M cells as well as macrophages with TNF-a siRNA as the target gene. Besides, RNAi efficacy was evaluated via oral delivery along with elucidation of the underlying mechanisms.
Superporous hydrogels of P(AA-co-AM) was synthesized through solution copolymerization of acrylic acid (AA) and acrylamide (AM) with APS/TEMED as initiators, NaHCO3 as foaming agent, and Bis as cross-linker. O-CMC was allowed to be well distributed in SPH along with the polymerization reaction, and was further cross-linked by glutaraldehyde (GA) after SPH was synthesized, thus achieving the SPH-IPN. The structures of the SPH-IPNs were characterized with FTIR,13C NMR, and DSC. SEM, CLSM and light images revealed that the SPH-IPNs possessed both the IPN network and large numbers of pores with diameters of 100-300μm, and the cross-linked O-CMC molecules were located on the peripheries of these pores. SPH-IPN quickly swelled in water with equilibrium swelling ratio of 30-80, and an increase in O-CMC content, GA amount and cross-linking time led to slower swelling behavior. Due to the cross-linked O-CMC network, compression and tensile modulus of SPH-IPN were greatly improved, and an increase in O-CMC content, GA amount and cross-linking time was favorable for enhanced mechanical strength.
Swelling behaviors of SPH-IPN were measured at different pH, ionic strength, and temperature. With insulin as a model drug, loading capacity of the SPH-IPN was determined. Release profiles were also evaluated at different pH, ionic strength, and temperature. Stability of insulin following drug loading and polymer-drug interactions were also investigated. State of water in SPH-IPN and water retention capacities were examined.
Swelling of SPH-IPN was sensitive towards of pH, ionic strength, and temperature. An increase in the ionic strength within the range of 0.001-1M yields a significant decrease in the swelling ratio, while it brought about an insignificant difference in the swelling behavior of the polymer when the ionic strength was no higher than 0.001M. A drastic increase in the swelling was observed within the pH range of 3.0-6.2, whereas at pH> 6.2 or pH< 3.0, the change in the swelling behavior was slight. SPH-IPN could rapidly respond to pulsatile alternation of pH values between 1.2 and 7.4, thereby exhibiting fast swelling and de-swelling. An increase in temperature could further facilitate swelling of SPH-IPN. Insulin loading capacity of SPH-IPN was 4%-7%, which was notably higher than CSPH, and an increase in O-CMC content yielded slightly deceased loading capacity. Insulin release also exhibited sensitivity towards pH, ionic strength, and temperature. After drug loading and release, the circular dichroism (CD) spectra revealed that conformation of insulin had no significant alteration and bioactivity of insulin was well preserved according to hypoglycaemic effect in mice. Great discrepancies were noted between the theoretical and experimental loading levels of SPH-IPN for insulin, and fast and complete drug release was observed in pH 7.4 PBS. Besides, FTIR spectra of blank SPH-IPN and washed SPH-IPN after drug release were similar, which suggested strong physical interactions rather than chemical linkage between the polymer and the drug. Freezing water was the majority of the imbibed water in the swollen SPH-IPN, and the ability of SPH-IPN to form hydrogen bonding with water molecules was weakened as the O-CMC content and insulin loading amount increased. SPH-IPN showed desired water retention capacities against compression and exposure at 37℃, which further improved as the amount of O-CMC network increased.
Pharmacokinetics and pharmacokinetics were investigated following oral and ileal administration of insulin-loaded SPH-IPN in normal rats, and the hypoglycemic effect of insulin-loaded SPH-IPN was also monitored in diabetic rats following oral delivery. Mucoadhesion, enzymatic inhibition, insulin permeation enhancing effect, and intestinal retention of SPH-IPN were evaluated to provide an insight into the intestinal absorption mechanisms of insulin in the presence of SPH-IPN. Besides, the effect of polymer integrity on the enzymatic inhibition, permeation enhancement, intestinal retention, and oral absorption of insulin was investigated.
Oral administration of insulin-loaded integral SPH-IPN (I-SPH-IPN) led to marked insulin absorption and hypoglycemic effect in normal rats with F and PA of 5.0% and 6.3%, respectively. In comparison, minimal insulin absorption and unappreciable hypoglycemic effect were observed following powdered SPH-IPN (P-SPH-IPN) delivery. When ileally administered, both I-SPH-IPN and P-SPH-IPN yielded a faster and more potent insulin absorption and blood glucose depression, with the minimal blood glucose level of 30% of initial values. Notable hypoglycemic effect was also observed following oral administration of insulin-loaded I-SPH-IPN in diabetic rats. SPH-IPN could adhere to the intestinal mucosa through non-specific binding and mechanical fixation, and an increase in O-CMC yielded a higher muadhesion capacity. SPH-IPN exhibited potent enzymatic inhibitory effect towards luminal proteolytic enzymes (trypsin andα-chymotrypsin) via enzyme entrapment and Ca2+ binding. A higher O-CMC content correlated to enhanced Ca2+ binding capacities, which accounted for enhanced enzymatic inhibitory effect. Although I-SPH-IPN and P-SPH-IPN possessed equivalent enzymatic inhibition capacity in vitro, I-SPH-IPN outperformed P-SPH-IPN in protecting insulin from proteolytic hydrolysis under the in vivo conditions, because I-SPH-IPN released more insulin in the mucus layer rather than in the intestinal lumen and thereby prevented degradation by luminally secreted proteases. SPH-IPN facilitated paracellular transport of insulin through reversible opening of epithelial tight junctions as a result of mechanical pressure, and I-SPH-IPN exhibited potent permeation enhancing effect than P-SPH-IPN in that I-SPH-IPN and P-SPH-IPN led to a depression in TEER values of Caco-2 cell monlayers to 25% and 50% of initial values, an increase in FITC-insulin transport in Caco-2 cell monlayers by 4.9 and 1.9 folds, and an enhancement in FITC-insulin transport in excised rat ileum by 4.2 and 1.8 folds, respectively. Through mechanical fixation onto the gut wall, I-SPH-IPN showed a prolonged intestinal retention of more than 8 h, while P-SPH-IPN was cleared from the intestinal within 4 h due to failure in mechanical fixation.
Biocompatibility of SPH-IPN was explored at molecular, cellular, tissue, and animal levels in terms of cytotoxicity, genetoxicity, tissue toxicity, oral acute and sub-acute toxicity, and blood compatibility. Residual monomers and cross-linkers in SPH-IPN were quantified by HPLC to provide evidences for biocompatibility assessment.
Results of FDA/PI double staining assay, LDH assay, neutral red assay, and protein assay in RBL-2H3 and Caco-2 cells revealed lack of cytotoxicity of SPH-IPN and SPH-IPN extract (10,1,0.1 mg·mL-1) following short-time exposure (24 h), and MTT assay further evidenced that SPH-IPN did not interfere with cell proliferation following long-time treatment (7 d). DNA ladder assay, cytometry assay and comet assay in the above two cell lines and in vivo micronucleus (MN) assay in mice showed that SPH-IPN did not induce cell apoptosis, DNA breakage, and MN formation, suggesting lack of genotoxicity. SPH-IPN did not induce impairment to the intestinal mucous, indicating good tissue compatibility. Oral administration of SPH-IPN extract (1000 mg·kg-1) resulted in unappreciable acute toxicity, and in the 28-day sub-acute toxicity study (500,200,100 mg·kg-1), body weight of I-SPH-IPN extract treated mice gradually increased with no appreciable difference to control animals, weights of mouse livers, kidneys, and spleens were found to be normal, and in the histological examination no necrosis, inflammation, edema or other pathological signs were detected. With regard to the hematological parameters and biochemical assays, no statistically significant difference was observed against control. LPO, GOP, GPT, ACP, and AKLP activities in mouse livers, kidneys, and spleens showed no abnormality, either. Hemolysis ratio of SPH-IPN was lower than 5%, the dynamic blood clotting ratio was lower than silicated glass, and platelet adsorption ratio was low, suggesting its desired anticoagulant capacities. Residual amount of AA, AM and GA in SPH-IPN was quantified to be 1.4±0.6,2.0±0.2, and<0.2 ppm, which was within the safety range.
TMC-Cys was synthesized through trimethylation of chitosan and thiolation of TMC, and TMC-Cys NP was prepared using the PEC method. It was thereafter evaluated as an oral delivery vehicle for protein drugs, and the mechanisms of oral absorption of insulin was elucidated.
Chitosan (30、200、500 kDa) was allowed to react with CH3I to achieve TMC with quaterization degree (DQ) of 15% and 30%, and TMC was subsequently modified with Cys via amide bond formation between the residual primary amino groups on TMC and carboxyl groups on Cys as mediated by EDAC/NHS. About 400-500 mmol·g-1 of sulphydryl was immobilized on TMC-Cys, with approximately one-third remaining the free thiol groups while two thirds being oxidized to the disulfide. FTIR and 13C NMR confirmed covalent conjugation between TMC and Cys via amide bonding, while DSC and TGA evidenced reduced crystallinity and decreased thermal stability of the polymer following trimethylation and thiolation. TMC-Cys exhibited comparable antimicrobial effect to TMC while stronger scavenging effect against free radicals. TMC-Cys NP was prepared through electrostatic interactions between oppositely charged TMC-Cys and insulin, which demonstrated particle size of 100-170 nm, Zeta potential of+12-+18 mV, and high encapsulation efficiency of 90%. Particle size, Zeta potential, and insulin encapsulation efficiency were largely dependent on pH of the insulin solution, TMC-Cys/insulin weight ratio, and ionic strength. Besides, chitosan Mw, DQ of TMC, ionic strength and ion types of the dissolution medium also exerted appreciable effect on in vitro release profiles of insulin. TMC-Cys NP showed a 2.1-4.7-fold and a 1.5-2.2-fold increase in intestinal mucoadhesion and mucin adsorption compared to TMC NP, and higher Mw or DQ was favorable for mucin adsorption. DSC measurement evidenced disulfide formation between TMC-Cys and mucin. Compared to TMC NP, TMC-Cys NP induced increased insulin transport through rat intestine by 1.7-2.6 folds, promoted Caco-2 cell internalization by 1.7-3.0 folds, and augmented uptake in Peyer's patches by 1.7-5.0 folds, respectively, among which TMC-Cys(200,30) NP exhibited the optimal permeation enhancing effect. Such results were further confirmed by in vivo experiment. MTT assay in Caco-2 cells and LDH assay in rat intestine revealed lack of toxicity of TMC-Cys NP.
With pEGFP as model pDNA, TMC-Cys NC was prepared using the PEC method and subjected to measurement of size and Zeta potential, and morphology observation using SEM and AFM. In vitro and in vivo transfection efficiency of TMC-Cys NC were monitored in HEK293 cells and mouse posterior tibialis muscles, respectively, and the transfection mechanisms were elucidated. TMC-Cys showed potent condensation capacity towards pDNA as evidenced by EB exclusion and gel retardation assays, and the resultant TMC-Cys NC demonstrated diameters of 150-400 nm and Zeta potentials of+14-+20 mV. TMC-Cys NC could also prevent degradation of pDNA by nucleases. Cell binding and mucin adsorption of TMC-Cys NC were enhanced 2.4-3.0 and 1.2-1.7 folds, respectively, compared to TMC NC, and cellular uptake of TMC-Cys NC was enhanced 1.4-3.0 fold and 1.6-4.4 fold compared to TMC NC and Lipofectamine2000. Lowering the temperature from 37 to 4℃substantially reduced uptake of TMC-Cys(100,30) NC by approximately 75%, and pretreatment of sodium azide or chlorpromazine yielded a depression in the complex internalization by approximately 30% and 70%, respectively, suggesting that an energy-dependent clathrin-mediated endocytic process was involved in the complex uptake. Comparatively, cytochalasin D and genistein exerted unappreciable effect on the cellular uptake of nanocomplexes, which indicated that complex uptake was not associated with cytoskeleton recognization and the caveolin-mediated pathway. pEGFP was slowly released from TMC-Cys NC at extracellular GSH concentrations, while was quickly released at the intracellular concentration and transported to the nuclei, leading to a 3.7-fold enhancement in nuclear accumulation of pEGFP compared to TMC NC. Besides, the relative amount of nuclear pEGFP increased with incubated time, which reached a plateau of 40% at 4 h. Consequently, TMC-Cys NC showed a 1.4 to 3.2-fold increase in the transfection efficiency in HEK293 cells as compared to TMC NC and the optimal TMC-Cys(100,30) NC showed a 1.5-fold enhancement than Lipofectamine2000. Such results were further confirmed by in vivo transfection with a 2.3-fold and 4.1-fold higher transfection efficiency of TMC-Cys(100,30) NC than TMC(100,30) NC and Lipofectamine2000, respectively.
MTC was synthesized and MTC nanoparticles were prepared and characterized. With TNF-a siRNA as a target gene, MTC nanoparticles were evaluated for their capacity of actively targeting towards intestinal M cells and macrophages, improving oral RNAi effect and the underlying mechanisms.
MTC with mannose modification degree of about 20% was synthesized through modification of TMC (Mw 200,500 kDa) with mannopyranosylphenylisothiocyanate and subsequent thiolation with cysteine. siRNA loaded MTC nanoparticles were prepared via the entrapment method, adsorption method, and self-assembly method. The nanoparticles were spherical or sub-spherical in shape, demonstrating diameters of 130-230 nm, positive Zeta potentials, and siRNA encapsulation efficiency of 70-80%. Serum stability of siRNA was significantly enhanced following nanoparticle encapsulation. Higher chitosan MW led to larger particle sizes; dilution led to augmentation of particle size and polydispersity of MTC-SA NP, and an increase in ionic strength resulted in notably decreased encapsulation efficiency of ad-MTC-TPP NP and MTC-SA NP. Comparatively, en-MTC-TPP NP was superior in resistance towards ionic strength. As compared to en-TC-TPP NP, en-MTC-TPP NP exhibited significantly higher cell binding capacity towards Caco-2 cells and Raw 264.7 cells, enhanced uptake level in Raw264.7 cells by 2.0-2.4 folds, increased Peyer's patch uptake by 1.9-2.4 folds, augmented Caco-2 cell monolayer transport of 1.2-2.1 folds, and elevated intestinal transport by 6.9-11.0 folds. Besides, transport in co-cultures of Caco-2 cell monolayers was higher than that in mono-cultures, and transport in excised rat intestine with Peyer's patches was higher than that in rat intestine without Peyer's patches, what suggested that mannose modification remarkably promoted active targeting of nanoparticles towards M cells and macrophages and thereby facilitating siRNA uptake and intestinal transport. With TNF-a siRNA as the target gene, en-MTC-TPP NP demonstrated potent silencing capacity in Raw 264.7 cells than en-TC-TPP NP and Lipofectamine2000, wherein en-MTC-TPP NP showed comparable silencing effect to Lipofectamine2000 at a reduced siRNA dose of 200 folds. en-MTC-TPP NP exhibited minimal cytoxicity in Raw264.7 cells and Caco-2 cells, suggesting lack of toxicity of the nanoparticles when exerting potent gene silencing effect. Oral delivery of en-MTC-TPP NP resulted in notable inhition of LPS-induced serum TNF-αproduction.
引文
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