含羧基小分子药物介导亲水聚合物自组装构建纳米给药系统研究
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摘要
现代新药研发中，大量候选药物由于其低水溶性、低生物利用度或毒性较大等问题而难以走向临床应用。另一方面，目前临床治疗使用的大量小分子药物，由于疏水性导致的溶解度问题和较低的口服生物利用度也大大限制了其更有效而广泛的临床应用。新型药物递送系统为解决此类提供了有效手段，其中纳米释药系统因其微小的粒径和特殊结构而具备一系列独特优势，是目前先进药物递送系统研究的前沿和热点。广泛的研究表明，纳米释药系统可以显著增加难溶性药物的溶解度，同时大大提高许多药物的口服生物利用度。此外，通过被动及主动靶向方式，纳米递送系统可显著提高药物疗效，降低药物毒副作用和不良反应。聚合物自组装体是纳米递送系统中最为广泛研究的微粒系统之一，该类纳米微粒系统被广泛用于包括小分子药物、多肽、蛋白和核酸等在内的各种药物的递送研究中。聚合物自组装体一般是指亲水或两亲性聚合物通过非共价键作用（包括疏水、静电、氢键等）介导的自组装形成的聚合物微粒系统。疏水性药物一般通过疏水作用物理包裹于聚合物纳米组装体的疏水微区中。然而，单一的疏水作用往往导致最终纳米组装体中药物负载量很低，而提高投药量又会导致药物形成结晶。尽管可以通过提高剂量的方式达到治疗目的，但这同时大大增加了聚合物载体材料的用量，使得一方面治疗成本增加，另一方面过多聚合物材料的使用会直接导致毒副作用。此外，制备聚合物自组装体使用的共聚物，其化学结构往往较为复杂，合成过程繁琐，不宜进行准确的结构表征，且大规模合成批次重复性差，因此也是限制聚合物自组装体纳米释药系统临床应用的关键因素之一。
综上所述，构建安全有效的、药物负载性能好、制备简便、且易于规模化制备的聚合物自组装体具有十分重要的意义。本课题创新性地提出通过聚合物和药物间的多重非共价键作用构建聚合物纳米组装体的设想。为了验证该假设，本研究首先选择商品化的聚乙烯亚胺（PEI）均聚物为模型载体材料，选用非甾体类抗炎药吲哚美辛（IND）为模型药物，开展了多重非共价键作用构建聚合物自组装体纳米释药系统的研究。由于PEI同时含有伯胺、仲胺和叔胺基团，IND含有羧基和疏水单元，PEI与IND之间同时存在静电、氢键及疏水相互作用。在深入研究PEI与IND之间不同作用方式，并详细表征所得纳米组装体理化特性的基础上，选择结构不同的模型药物分子证明了该策略的普适性。通过口服给药开展了大鼠体内药动学研究，并通过急性和慢性炎症模型进行了体内药效学评价工作。为探究基于PEI/药物组装体作为口服释药系统的潜在应用，对分子量为800和25000的PEI进行了口服急性毒性评价。在此基础上，为了进一步提高载体材料PEI的生物相容性，在本课题的第二部分研究中，我们设计、合成了β-环糊精键合聚乙烯亚胺(PEI-CD)。β-环糊精的引入一方面可起到降低载体材料毒性作用的目的；另一方面，也赋予了材料与药物之间额外的非共价键作用，即环糊精空腔与疏水基团之间的主-客体相互作用，更有利于纳米粒自组装体的形成。为了实现口服后病灶部位的靶向，在成功构建PEI/IND聚合物纳米组装体的基础上，我们采用酵母细胞壁作为微囊包被PEI/IND纳米微粒。通过酵母细胞壁中β-1，3葡聚糖与巨噬细胞表面β-葡聚糖受体之间的识别作用来靶向巨噬细胞，进而实现巨噬细胞介导的炎症部位靶向。同样，对于酵母细胞壁包裹PEI/IND纳米组装体系统，我们开展了体内药动学和药效学研究。
方法
1.含羧基小分子药物的PEI或PEI-CD纳米组装体微粒的制备
通过透析法来制备PEI和不同小分子羧基药物的纳米组装体，具体方法如下：首先将一定比例的药物和PEI溶于一定体积的亲水性有机溶剂中，得到的溶液于室温下透析去除有机溶剂记得制备得到纳米组装体水溶液。基于PEI-CD的纳米组装体采用改良的透析法来制备，制备时，将溶于水溶性有机溶剂中的药物溶液在超声作用下加入一定浓度的PEI-CD水溶液中，得到的混合体系在水溶液中透析即可得到载药组装体溶液。其中涉及的含羧基的小分子药物包括吲哚美辛、布洛芬、萘普生、二氟尼柳和氟比洛芬等。
2.酵母细胞壁包裹纳米组装体系统的制备
首先通过酸碱处理出去酵母内容物得到酵母细胞壁（YS）。包裹PEI/IND纳米组装体时，称取一定量的酵母细胞，加入PEI水溶液超声摇匀后，于37℃孵育使YS完全溶胀，再加入含有IND的DMSO溶液，摇匀后孵育一定时间，离心洗涤除去DMSO和未包裹PEI/IND，冻干即可得到酵母细胞载药微囊（DL-YS）。
3.释药系统的理化性能表征
其中载药量与包封率采用紫外法（UV）测定。通过扫描电子显微镜(SEM)、透射电子显微镜(TEM)及原子力显微镜(AFM)来观察组装体形貌。动态光散射法(DLS)测定载药组装体微粒形态、大小及分布；表面电位仪测定表面电位。
4.载药组装体中药物存在形式的表征
通过差示扫描量热仪(DSC)和X-射线衍射仪(XRD)以及激光共聚焦显微镜(CLSM)表征载药组装体中药物的存在形式和分布行为。
5.药物-聚合物间多重相互作用的表征
聚合物的结构通过核磁共振光谱(1HNMR)来确定。药物、聚合物、药物聚合物物理混合物及载药组装体分别进行红外光谱(FT-IR)和核磁共振光谱(NMR)测定，以分析其多重相互作用力的存在。
6.β-环糊精键合聚乙烯亚胺（PEI-CD）的合成
通过对甲苯磺酰氯单取代β-环糊精与聚乙烯亚胺之间的亲核取代反应制备β-环糊精键合聚乙烯亚胺（PEI-CD）。
7.体外释放及模拟胃肠道研究
对含有IND的纳米组装体进行了体内释放动力学研究，具体方法如下。取含有一定量药物的载药组装体水溶液进行体外释放实验，同时选择原料药、片剂为对照样品。分别在pH7.4的PBS缓冲液和模拟胃肠道环境的不同释放液中进行释放实验。药物释放量通过UV法测定，计算累计释放百分率，并绘制药物累积释放曲线。
8.大鼠体内药代动力学研究
SD大鼠口服灌胃给药后，预定时间点采集血样，离心后取一定量血浆，通过乙腈沉淀蛋白，内标法测定IND浓度，绘制药时曲线，DAS软件计算主要药动学参数。
9.载药组装体在肠道内保留时间及其对胃肠道刺激研究
使用荧光标记聚合物制备IND载药组装体，SD大鼠灌胃给药后，预定时间点将其处死立即取胃肠道组织，进行病理切片。HE染色后显微镜下观察胃肠道刺激情况，荧光显微镜下观察载药组装体在肠道内的滞留情况。
10.量子点/酵母细胞壁（QD-YS）的制备
通过表面带有正电荷的量子点（QD）的自发沉积来制备载有量子点的酵母细胞微囊。即，取一定量酵母空微囊，加入一定体积量子点在37℃下孵育一段时间，通过离心洗涤除去未包裹量子点。
11.活体成像观察
使用裸鼠来建立关节炎模型，口服一定量QD-YS，24h后取主要脏器和右后爪进行活体成像观察，并对各脏器和足趾部位荧光强度进行定量，以此研究酵母微囊系统的关节炎靶向作用。
12.药效学评价方法
通过角叉菜胶诱导的急性足趾肿胀模型和弗氏佐剂诱导的慢性关节炎模型来评价本研究所构建的不同释药系统的疗效，以预防和治疗两种方式给药进行研究；实验结束后取胃、肠道不同部位和肿胀足趾组织，进行病理切片，HE染色。
13.口服急性毒性评价
选择昆明小鼠，通过灌胃给予不同剂量的PEI，对照组给予生理盐水，连续观察14天。观察的指标包括一般指标（如动物外观、行为、对刺激的反应、分泌物、排泄物等），动物死亡情况（死亡时间、濒死前反应等），动物体重变化（每两天称重一次）等；记录所有的死亡情况、出现的症状，以及症状起始的时间、严重程度、持续时间等。对所有动物进行大体解剖，观察器官变化情况，对任何器官出现体积、颜色、质地的改变，均记录并进行组织病理学检查。
结果
1.通过简便的透析法可以成功制备IND与PEI的纳米组装体，其形态为球形，粒径大小在几十纳米至几百纳米之间，表面带正电荷；其中药物负载量高达80%。采用不同含羧基药物进行的研究表明该自组装方法具有普适性。
2.基于FT-IR和NMR的研究表明，PEI与羧基小分子药物间存在多重非共价键相互作用，包括氢键、静电作用和疏水作用；通过DSC和XRD测定和CLSM观察表明，药物与聚合物组装体中药物以无定形态存在，药物均匀分散于组装体中。
3.体外药物释放研究表明，PEI/IND纳米组装体明显加快药物的溶出和释放，且其释放具有pH响应特性，在胃部几乎不释放，而主要在肠道释放；体内药动学实验结果表明，通过制备成PEI/IND纳米组装体能够有效提高药物的口服生物利用度；基于急性足趾肿胀模型和慢性炎症模型进行的体内药效学研究表明，纳米组装体释药系统大大提高了药物的治疗效果，同时并不会导致明显的毒副作用或不良反应。
4.口服急性毒性评价表明，PEI口服半致死剂量在2.5g/kg以上；脏器指数计算、血常规与肝肾功测定表明，在2.5g/kg以下口服PEI不会产生明显的毒副作用。
5.在PEI中引入β-环糊精后得到的PEI-CD，其与羧基药物组装形成纳米微粒的性能不会改变，PEI-CD/羧基药物组装体同样具有高药物负载量，并能加快药物溶出，提高药物口服生物利用度和体内治疗效果；急性毒性评价表明β-环糊精的引入能明显降低PEI的口服毒性。
6.通过静电作用，酵母空微囊可以成功包裹PEI-IND纳米组装体，由此得到酵母载药微囊，载药量最高达到30%，而包封率可达70%；通过Zeta电位、TEM和CLSM观察证明PEI/IND被成功包被于酵母细胞壁内。
7.大鼠灌胃给药后进行的药动学研究表明，载药酵母微囊IND YS能大大提高药物的口服生物利用度，且IND YS较IND/PEI略有提高；同样，基于角叉菜胶诱导的急性炎症模型及弗氏佐剂诱导的慢性关节炎模型研究亦表明IND YS具有显著提高的疗效。
8.胃肠道刺激评价的结果也表明，不同纳米组装体及纳米组装体酵母微囊在提高IND疗效的同时，可显著的降低由IND引起的胃肠道刺激。
9.通过包裹量子点的酵母微囊（QD YS）进行的活体成像研究表明，口服QD YS4h后，在右后爪炎症部位存在较强的QD荧光，与相同剂量注射QD相比，口服减少了QD的肝脏分布，表明酵母微囊具有非常明显的口服炎症靶向作用。
结论
1.本研究创新性地提出通过多重非共价键作用介导的自组装可形成高载药能力的聚合物自组装体的假设。为验证其可行性，我们通过采用含羧基小分子药物介导聚乙烯亚胺自组装来构建新型纳米释药系统，并对其理化性能、药物负载能力进行表征，同时研究了不同条件下聚合物自组装行为和该简便自组装策略的通用性。研究表明该药物递送系统具有pH响应性，在胃部几乎无药物释放，而在肠道内快速释放药物。通过该组装体纳米释药系统大大提高了药物的口服生物利用度、提高了药物的疗效，同时亦降低胃肠道刺激及毒副作用。口服急性毒性评价表明当PEI的剂量低于2.5g/kg时，不会产生毒副作用或不良反应。
2.为进一步改善PEI分子的生物相容性，以便构建一种更加安全及有效的聚合物自组装体，我们将β-环糊精单元引入PEI得到β-环糊精键合聚乙烯亚胺（PEI-CD）。在提高了PEI生物相容性的同时，β-环糊精的引入同时赋予了PEI-CD与疏水药物分子通过主-客体作用的性能。与PEI相同，PEI-CD能够与不同含羧基小分子药物自组装形成高药物负载性能的纳米微粒，该纳米释药系统能够显著提高药物的口服生物利用度，提高治疗效果的同时降低了原料药对胃肠道的刺激作用。
3.在成功建立了具有pH响应性的聚合自组装体药物传递系统的基础上，我们进一步研究利用酵母微囊来实现口服给药后局部炎症的靶向。结果表明酵母细胞可以有效负载PEI/IND纳米组装体；载药酵母微囊可以通过靶向巨噬细胞实现炎症部位的靶向。本部分研究对于通过生物技术和仿生手段来设计并构建靶向药物递送系统提供了新的思路和有效技术方法。
4.总的来说，本课题通过载体材料与药物分子间多重非共价键作用构建了一种新型纳米释药系统，该一步组装法简单、便利、高效而又易于放大。本研究一方面为新型药物递送系统的设计提供了新思路，同时也为聚合自组装体纳米药物的研究提供了新方向。另外，酵母微囊这一仿生技术的应用，一方面提高了药物微囊的生物相容性，另一方面创新性地实现了口服后药物对于炎症部位的靶向。
In modern drug devolvement, the low water solubility, lowbioavailability,andtoxicityissueshave prohibited the clinical application of a large number of drugcandidates. In addition, the poorsolubility ofmany clinically used smallmoleculedrugsrestricts their broad applications. To solve these problems, aseries of newdrug deliverysystems such as nanoparticles-based drug delivery platforms have been developed, whichhave many unique advantagesbecauseoftheirsmallparticle size andspecial structure.Numerous studies have indicated that nanocarriers can significantly increasethesolubilityofpoorly soluble drugs, and therefore greatly improve their oral bioavailability. Inaddition,nanovehicles can significantly improve the drug’s efficacyand reduce its sideeffectsor adverse reactions by their passive or active targeting. Self-assembled polymernanocarriers, one ofthe most widelystudiednanosystems, havebeen widelyusedfor deliveryof avarietyoftherapeutics including small molecule drugs, peptides, proteins,and nucleicacids. Polymeric assemblies are generally assembly by hydrophilicoramphiphilicpolymersvia non-covalentinteractions like hydrophobic, electrostatic, andhydrogen bonding forces. Hydrophobic drugsaregenerallywrappedinthehydrophobicdomainofpolymerassemblies based on hydrophobic interaction. However, hydrophobicinteraction aloneoften leads tolowdrugloading, while enhancing drug feeding will result inthe formation of drug crystals.Although the expected efficacy may be achieved by increaseddrug dose, the concomitant increase in the dose of polymer vehicles would raisethecost oftreatmentand produce side effects. Besides, amphiphilic copolymers employed forself-assembly of drug delivery nanoplatforms often possess complex chemical structureandneed to be synthesized by multiple processes, which strongly hinder thebench-to-bedside translation of nanomedicines based on polymer assemblies.
Accordingly, it is highly necessary to develop an effective, high drug loading, and facilestrategy to fabricate nanoplatforms by polymer self-assembly. To circumvent this issue, we hypothesize that multiple non-convalent interactions between polymer and drug mayenhance the encapsulation of drug molecules into nanoassemblies.In this study, we firstlyselected indomethacin (IND) as a model drug and homopolymer polyethyleneimine (PEI)as a modelcarrier material to prove this concept. Since PEI contains primary, secondary, andtertiary amines as well as long carbon chain, while IND contains carboxyl group andhydrophobicunit, there should be electrostatic, hydrogen-bonding, and hydrophobicinteractions between PEI and IND. In addition, the hydrophobic interactions of drugmolecules may also facilitate the construction of highly efficient nanomedicines. Carefulcharacterization was performed to elucidate the physicochemical properties and IND/PEInanoassemblies and interactions between IND and PEI.By using a series ofcarboxyl-containing drugs with different structures, we proved the universality of this facileassembly approach.In addition to pharmacokinetic studies,in vivo pharmacodynamicevaluations were conducted based on acute and chronic inflammation models.Furthermore,acute toxicity of1800PEI and25000PEI was evaluated after oral administration to addressthe potential clinical applications of these assembled PEI nanomedicines.
Based on above results, β-cyclodextrin (β-CD) conjugated PEI (PEI-CD) was synthesizedto improve the biocompatibility of PEI. On the one hand,the introduction of β-CDmayreduce the toxic effects of the PEI carrier material. On the other hand, it can provideadditional non-covalent force between the carrier material and drug, i.e. the host-guestinteraction between cyclodextrin cavity and hydrophobic group, which is beneficial to theformation of nanoassemblies.
In order to achieve oral targeting, we used the yeast wall to entrap IND/PEInanoparticles.Targeting of inflammatory sites may be achieved by recognition of β-1,3glucan on the yeast wall with its receptor on macrophages. Similarly, the pharmacokineticand pharmacodynamic studies were carried out for IND/PEI nanoassemblies loaded yeastcapsules.
Methods
1. Fabrication of nanoassemblies based on PEI or PEI-CD
PEI/drug nanoassemblies were prepared through a dialysis method. First, a certainproportion of drug and PEI was dissolved in a hydrophilic organic solvent, and the resultingsolution was dialyzed against deionized water at room temperature to remove the organic solvent. To fabricate drug/PEI-CD nanomedicines, a modified dialysis process was used.Briefly, an appropriate amount of drug dissolved in DMSO was gradually added into anaqueous solution of PEI-CD (10.0mg/mL) under bath sonication. The obtained mixture wasthen dialyzed against deionized water to form drug-containing nanoassemblies. Theinvolved drugs include IND, naproxen, ibuprofen, flurbiprofen, and diflunisal.
2. Preparation of YS and drug loaded YS
Yeast shells were prepared from yeast cells by treatment using acid, alkali, and organicsolvent to remove yeast cell contents. After YS were dried, they were mixed with PEIaqueous solution to minimally hydrate the particles and incubated for2h to allow YScompletely swelled and adsorb PEI molecules. Then IND in DMSO solution was added andthe particles were resuspended by homogenization or sonication. The suspension wascentrifuged to remove DMSO and uncoated IND and PEI, the precipitate was freeze-driedto give drug-loaded YS.
3. Physicochemical characterization of drug delivery systems
The drug loading and encapsulation efficiency were detected by UV method. Themorphology of nanoparticles was observed by scanning electron microscopy (SEM),transmission electron microscopy (TEM), and atomic force microscopy (AFM). Dynamiclight scattering (DLS) was used for characterize the size distribution and surface potentialof nanoparticles.
4. Characterization of the drug form in assemblies
Raw IND, IND/PEI or IND/PEI-CD physical mixture, and IND/PEI or IND/PEI-CDassemblies containing various contents of IND were examined by DSC and XRDmeasurement.
5. Characterization of multiple interactions between polymer and drug
Raw IND, IND/PEI or IND/PEI-CD physical mixture, and IND/PEI or IND/PEI-CDassemblies containing various contents of IND were examined by FT-IR,1H and1H-1HRoesy NMR measurements.
6. Synthesis and characterization of β-cyclodextrin-conjugated polyethyleneimine(PEI-CD)
PEI-CD was synthesized by a nucleophilic substitution reaction between branched PEIand6-monotosyl β-CD. The structure of PEI-CD was characterized by1H-NMR and FT-IR. The molar ratio of ethyleneimine units in PEI to β-CD groups was calculated via1H-NMRspectrum
7. In vitro release tests
For in vitro release study,0.5mL of IND formations was placed into dialysis tubing,which was immerged into40mL of PBS (pH7.4). At predetermined time intervals,4.0mLof release medium was withdrawn, and fresh PBS was added.
To simulate release profiles under GI tract conditions, aqueous solution of HCl (pH1.2)was employed within the first two hours, and then the release medium was switched into0.01M PBS (pH7.4). IND concentration in release buffer was quantified by UV at310nm.
8. Pharmacokinetic study
IND formations were orally administered via gastric gavage. Blood samples werecollected at specific time points post-dose. Plasma was obtained after10min ofcentrifugation at3000rpm. Then,100mL of acetonitrile and400mL of acetonitrile wasadded into50μL plasma, followed by votexing for1min, and the supernatant waswithdrawn after centrifugation at8000rpm for10min. After being dried under N2atmosphere,100μL of mobile phase was added for sampling. IND concentration in wasquantified by high performance liquid chromatography. The chromatographic conditionsare as follows: mobile phase, acetonitrile-6μM H3PO4(55:45, v:v); eluent rate,1.0mL/min;column temperature,40°C; detection wavelength,245nm; sample volume,20μL; column,C18reverse column (5μm×250mm).
9. Study on the retention and gastrointestinal irritation
IND/PEI-CD assemblies were orally administered to SD male rats at10mg/kg. Atpredetermined time points, they were euthanized and isolated segments of stomach andsmall intestine tissues were fixed. Histological sections were made and stained withhematoxylin-eosin (HE). Using the fluorescence-labeled PEI-CD, the distribution ofIND-containing assemblies in intestinal tract was detected at various time points.
10. Preparation of quantum dots-yeast shell (QDs-YS)
The QD-YS were prepared by the spontaneous deposition of the quantum dots (QDs)with positive surface charge. To this end, YS were mixed with a certain volume of quantumdots, and incubated at37°C for a period of time. After centrifugation, they were washedwith deionized water to remove free quantum dots.
11. Ex vivo imaging
The nude mice with acute paw edema were orally administrated with a certain amountof QDs-YS. The major organs and right hind paws were collected after24h for ex vivoimaging. After fluorescence imaging, the fluorescence intensity of various parts werequantified to evaluate the arthritis targeting capability of the yeast microcapsules.
12. In vivo pharmacodynamic study
In order to substantiate the therapeutic advantages of newly fabricated IND/PEInanomedicines, preliminary pharmacodynamic evaluation was carried out based on acarrageenan-induced acute inflammation and Freund's adjuvant induced arthritis model inrats. The anti-inflammatory efficacy was evaluated by the swelling degree defined as thedifference between the paw volume before and after inflammation.
Swelling degree=Vt-V0(mL)
Where V0and Vtrepresent the paw volume before and after inflammation,respectively.
13. Acute toxicity evaluation.
Male Kunming mice (22-27g) were randomly assigned into4groups (n=6),including three experimental groups and one control group. In the experimental groups,mice were administered via oral gavage of PEI aqueous solution (1.0mL) at doses rangingfrom0.625,1.25, to2.5g/kg. Mice in the control group were orally administered with1.0mL of saline solution. Each day post-injection, mice were weighed and their behaviors wereobserved for any signs of illness. After14days, animals were sacrificed by cervicaldislocation after anaesthesia. Blood samples were collected for the quantification ofhematological parameters and biochemical markers relevant to liver/kidney functions.Organs including heart, liver, kidney, lung, and spleen were harvested and weighed tocalculate the organ index. Histopathological sections of organs and gastrointestinal tissueswere made and stained with HE.
14. Statistical Analysis.
Statistical analysis was performed by SPSS12.0using one-way ANOVA test forexperiments consisting of more than two groups, and with a two-tailed, unpaired t-test inexperiments with two groups. Statistical significance was assessed at p <0.05.
Results
1. Nanoassemblies based on carboxyl-containing drugs and PEI or PEI-CD werefacilely prepared by dialysis, taking advantage of multiple interactions between carriermaterials and drugs. Morphology observation combined with size determination revealedthus obtained nanoassemblies displayed spherical shape with size controllable viaprocessing parameters. In addition, these drug-containing assemblies are positively chargedaccording to the zeta-potential measurement. For IND assemblies, they exhibited drugloading content higher than71%and encapsulation efficiency>90%. The similarassembling profiles were found in other carboxyl-containing drugs like naproxen, diflunisal,ibuprofen, and flurbiprofen.
2. Measurements based on FT-IR,1H and1H-1H Roesy NMR confirmed the presenceof non-covalent multiple interactions between IND and PEI or PEI-CD, includinghydrophobic, host-guest recognition, hydrogen-bonding, and electrostatic forces. DSCand XRD measurements as well as CLSM observation indicated the IND moleculesencapsulated in nanoparticles were essentially amorphous other than drug crystal.
3. For IND/PEI nanomedicines, fast release of IND was achieved at pH7.4. In vitrorelease in media simulating GI conditions suggested that the drug release was largelysuppressed in acidic conditions, which can be dramatically accelerated when the releasemedium was switched into release buffer of pH7.4. In addition, the release of IND wasfaster than that of raw IND and commercial tablet. Consistent with in vitro release profiles,in vivo pharmacokinetic study in rats after oral administration indicated that the area underthe plasma concentration-time curve (AUC) of nanomedicines was120%larger than that ofraw IND. This implied that the oral bioavailability of IND was significantly enhanced byformulating into nanomedicines via molecular self-assembly. In vivo therapeutic effect washighly improved compared to the raw IND control, while no significant toxicities werefound.
4. No animal death occurred at PEI doses lower than2.5g/kg, and the median lethaldose (LD50) was higher than2.5g/kg. Calculation of the organ index, measurements oftypical hematological parameters, and biomarkers relevant to liver/kidney functionsshowed no significant toxicities at PEI doses lower than2.5g/kg.
5. Similarly, high drug-loading property,rapid drug release, high bioavailability, andimproved therapeutic effect were found for IND/PEI-CD nanoparticles. Moreover, PEI-CD was proved to be more biocompatible than PEI, because of the introuction of β-CD units.
6. IND YS and QDs-YS were successfully prepared as demonstrated by measurementsof zeta-potential, TEM, and fluorescence microscopy. IND loading capacity was up to30%,with an entrapment efficiency of70%. Pharmacokinetic study showed that drug in IND YSwas rapidly released at pH7.4, while almost no release occurred at pH1.2.
7. The bioavailability of IND in the case of the yeast system was highly improvedwhen compared with raw IND. In vivo evaluation based on models of carrageenan inducedacute paw edema and Freund's adjuvant induced arthritis suggested that theanti-inflammatory efficacy was also highly improved after microencapsulation of IND inYS.
8. Pathological study showed nanoassemblies can significantly suppress the GIirritation of IND, while improving its anti-inflammatory efficacy
9. En vivo imaging indicated that oral administration of QDs-YS presented strongfluorescence intensity, which is comparable to that by i.v. injection of QDs, while thedistribution in liver was significantly deceased.
Conclusions
1. In this study, we proposed innovatively that polymer assemblies with high drugloading can be achieved by multiple non-covalent forces. In order to verify this hypothesis,we constructed IND/PEI nanomedicines by one-pot self-assembly mediated via multipleinteractions between drug and PEI. The physicochemical properties and drug loadingcapacity of obtained nanoassemblies were carefully characterized. Also, the self-assemblyprofiles and the versatility of this facile assembly strategy under different conditions wereinvestigated. In vitro release study showed that the new nanosystem displayed a significantpH-sensitivity. Virtually no drug release occurred in the stomach, while rapid release couldbe achieved in the intestine. The oral bioavailability and therapeutic efficacy of the loadeddrug were greatly improved through the assembly of nanosystems, while thegastrointestinal irritation and toxicity were reduced. Acute oral toxicity evaluation of PEIshowed that no side effects or adverse reactions were produced at PEI dose less than2.5g/kg.
2. To further improve the biocompatibility of PEI for more safety and effectivepolymer nanoassemblies, we introduced β-cyclodextrin unit into PEI, resulting in a new polymer of β-cyclodextrin conjugated PEI (PEI-CD). The introduction of β-CD cansignificantly improve the biocompatibility of PEI, and provide additional host-guestinteractions for assembly of PEI-CD and various drugs. Similarly, efficient drug loading,rapid cargo release, high bioavailability, and improved therapeutic effect were achieved byPEI-CD/IND nanoparticles.
3. After the assembled pH-responsive polymeric nanomedicines were establishedsuccessfully, we further used the yeast wall as microcapsules to entrap IND/PEInanoparticles to achieve inflammation targeting by macrophage surface receptor wich canrecognize β-1,3glucan on the yeast wall. This part of study provided a new way ofdesigning orally targeted drug delivery system through biotechnology and bioengineeringapproaches.
4. In summary, we discovered a facile, convenient, cost-effective, and easily scalableone-pot assembly strategy to formulate various lipophilic therapeutics bearing carboxylgroup into nanomedicines. The present study provides new insights into the design of novelnanomedicines, and opens a new direction for the molecular self-assembly of polymerdriven by small molecules. Additionally, the application of biomimetic technology in yeastmicrocapsules can highly improve the biocompatibility of drug payload, and innovativelyachieve the oral targeting of drugs to inflammatory sites.
综上所述，构建安全有效的、药物负载性能好、制备简便、且易于规模化制备的聚合物自组装体具有十分重要的意义。本课题创新性地提出通过聚合物和药物间的多重非共价键作用构建聚合物纳米组装体的设想。为了验证该假设，本研究首先选择商品化的聚乙烯亚胺（PEI）均聚物为模型载体材料，选用非甾体类抗炎药吲哚美辛（IND）为模型药物，开展了多重非共价键作用构建聚合物自组装体纳米释药系统的研究。由于PEI同时含有伯胺、仲胺和叔胺基团，IND含有羧基和疏水单元，PEI与IND之间同时存在静电、氢键及疏水相互作用。在深入研究PEI与IND之间不同作用方式，并详细表征所得纳米组装体理化特性的基础上，选择结构不同的模型药物分子证明了该策略的普适性。通过口服给药开展了大鼠体内药动学研究，并通过急性和慢性炎症模型进行了体内药效学评价工作。为探究基于PEI/药物组装体作为口服释药系统的潜在应用，对分子量为800和25000的PEI进行了口服急性毒性评价。在此基础上，为了进一步提高载体材料PEI的生物相容性，在本课题的第二部分研究中，我们设计、合成了β-环糊精键合聚乙烯亚胺(PEI-CD)。β-环糊精的引入一方面可起到降低载体材料毒性作用的目的；另一方面，也赋予了材料与药物之间额外的非共价键作用，即环糊精空腔与疏水基团之间的主-客体相互作用，更有利于纳米粒自组装体的形成。为了实现口服后病灶部位的靶向，在成功构建PEI/IND聚合物纳米组装体的基础上，我们采用酵母细胞壁作为微囊包被PEI/IND纳米微粒。通过酵母细胞壁中β-1，3葡聚糖与巨噬细胞表面β-葡聚糖受体之间的识别作用来靶向巨噬细胞，进而实现巨噬细胞介导的炎症部位靶向。同样，对于酵母细胞壁包裹PEI/IND纳米组装体系统，我们开展了体内药动学和药效学研究。
方法
1.含羧基小分子药物的PEI或PEI-CD纳米组装体微粒的制备
通过透析法来制备PEI和不同小分子羧基药物的纳米组装体，具体方法如下：首先将一定比例的药物和PEI溶于一定体积的亲水性有机溶剂中，得到的溶液于室温下透析去除有机溶剂记得制备得到纳米组装体水溶液。基于PEI-CD的纳米组装体采用改良的透析法来制备，制备时，将溶于水溶性有机溶剂中的药物溶液在超声作用下加入一定浓度的PEI-CD水溶液中，得到的混合体系在水溶液中透析即可得到载药组装体溶液。其中涉及的含羧基的小分子药物包括吲哚美辛、布洛芬、萘普生、二氟尼柳和氟比洛芬等。
2.酵母细胞壁包裹纳米组装体系统的制备
首先通过酸碱处理出去酵母内容物得到酵母细胞壁（YS）。包裹PEI/IND纳米组装体时，称取一定量的酵母细胞，加入PEI水溶液超声摇匀后，于37℃孵育使YS完全溶胀，再加入含有IND的DMSO溶液，摇匀后孵育一定时间，离心洗涤除去DMSO和未包裹PEI/IND，冻干即可得到酵母细胞载药微囊（DL-YS）。
3.释药系统的理化性能表征
其中载药量与包封率采用紫外法（UV）测定。通过扫描电子显微镜(SEM)、透射电子显微镜(TEM)及原子力显微镜(AFM)来观察组装体形貌。动态光散射法(DLS)测定载药组装体微粒形态、大小及分布；表面电位仪测定表面电位。
4.载药组装体中药物存在形式的表征
通过差示扫描量热仪(DSC)和X-射线衍射仪(XRD)以及激光共聚焦显微镜(CLSM)表征载药组装体中药物的存在形式和分布行为。
5.药物-聚合物间多重相互作用的表征
聚合物的结构通过核磁共振光谱(1HNMR)来确定。药物、聚合物、药物聚合物物理混合物及载药组装体分别进行红外光谱(FT-IR)和核磁共振光谱(NMR)测定，以分析其多重相互作用力的存在。
6.β-环糊精键合聚乙烯亚胺（PEI-CD）的合成
通过对甲苯磺酰氯单取代β-环糊精与聚乙烯亚胺之间的亲核取代反应制备β-环糊精键合聚乙烯亚胺（PEI-CD）。
7.体外释放及模拟胃肠道研究
对含有IND的纳米组装体进行了体内释放动力学研究，具体方法如下。取含有一定量药物的载药组装体水溶液进行体外释放实验，同时选择原料药、片剂为对照样品。分别在pH7.4的PBS缓冲液和模拟胃肠道环境的不同释放液中进行释放实验。药物释放量通过UV法测定，计算累计释放百分率，并绘制药物累积释放曲线。
8.大鼠体内药代动力学研究
SD大鼠口服灌胃给药后，预定时间点采集血样，离心后取一定量血浆，通过乙腈沉淀蛋白，内标法测定IND浓度，绘制药时曲线，DAS软件计算主要药动学参数。
9.载药组装体在肠道内保留时间及其对胃肠道刺激研究
使用荧光标记聚合物制备IND载药组装体，SD大鼠灌胃给药后，预定时间点将其处死立即取胃肠道组织，进行病理切片。HE染色后显微镜下观察胃肠道刺激情况，荧光显微镜下观察载药组装体在肠道内的滞留情况。
10.量子点/酵母细胞壁（QD-YS）的制备
通过表面带有正电荷的量子点（QD）的自发沉积来制备载有量子点的酵母细胞微囊。即，取一定量酵母空微囊，加入一定体积量子点在37℃下孵育一段时间，通过离心洗涤除去未包裹量子点。
11.活体成像观察
使用裸鼠来建立关节炎模型，口服一定量QD-YS，24h后取主要脏器和右后爪进行活体成像观察，并对各脏器和足趾部位荧光强度进行定量，以此研究酵母微囊系统的关节炎靶向作用。
12.药效学评价方法
通过角叉菜胶诱导的急性足趾肿胀模型和弗氏佐剂诱导的慢性关节炎模型来评价本研究所构建的不同释药系统的疗效，以预防和治疗两种方式给药进行研究；实验结束后取胃、肠道不同部位和肿胀足趾组织，进行病理切片，HE染色。
13.口服急性毒性评价
选择昆明小鼠，通过灌胃给予不同剂量的PEI，对照组给予生理盐水，连续观察14天。观察的指标包括一般指标（如动物外观、行为、对刺激的反应、分泌物、排泄物等），动物死亡情况（死亡时间、濒死前反应等），动物体重变化（每两天称重一次）等；记录所有的死亡情况、出现的症状，以及症状起始的时间、严重程度、持续时间等。对所有动物进行大体解剖，观察器官变化情况，对任何器官出现体积、颜色、质地的改变，均记录并进行组织病理学检查。
结果
1.通过简便的透析法可以成功制备IND与PEI的纳米组装体，其形态为球形，粒径大小在几十纳米至几百纳米之间，表面带正电荷；其中药物负载量高达80%。采用不同含羧基药物进行的研究表明该自组装方法具有普适性。
2.基于FT-IR和NMR的研究表明，PEI与羧基小分子药物间存在多重非共价键相互作用，包括氢键、静电作用和疏水作用；通过DSC和XRD测定和CLSM观察表明，药物与聚合物组装体中药物以无定形态存在，药物均匀分散于组装体中。
3.体外药物释放研究表明，PEI/IND纳米组装体明显加快药物的溶出和释放，且其释放具有pH响应特性，在胃部几乎不释放，而主要在肠道释放；体内药动学实验结果表明，通过制备成PEI/IND纳米组装体能够有效提高药物的口服生物利用度；基于急性足趾肿胀模型和慢性炎症模型进行的体内药效学研究表明，纳米组装体释药系统大大提高了药物的治疗效果，同时并不会导致明显的毒副作用或不良反应。
4.口服急性毒性评价表明，PEI口服半致死剂量在2.5g/kg以上；脏器指数计算、血常规与肝肾功测定表明，在2.5g/kg以下口服PEI不会产生明显的毒副作用。
5.在PEI中引入β-环糊精后得到的PEI-CD，其与羧基药物组装形成纳米微粒的性能不会改变，PEI-CD/羧基药物组装体同样具有高药物负载量，并能加快药物溶出，提高药物口服生物利用度和体内治疗效果；急性毒性评价表明β-环糊精的引入能明显降低PEI的口服毒性。
6.通过静电作用，酵母空微囊可以成功包裹PEI-IND纳米组装体，由此得到酵母载药微囊，载药量最高达到30%，而包封率可达70%；通过Zeta电位、TEM和CLSM观察证明PEI/IND被成功包被于酵母细胞壁内。
7.大鼠灌胃给药后进行的药动学研究表明，载药酵母微囊IND YS能大大提高药物的口服生物利用度，且IND YS较IND/PEI略有提高；同样，基于角叉菜胶诱导的急性炎症模型及弗氏佐剂诱导的慢性关节炎模型研究亦表明IND YS具有显著提高的疗效。
8.胃肠道刺激评价的结果也表明，不同纳米组装体及纳米组装体酵母微囊在提高IND疗效的同时，可显著的降低由IND引起的胃肠道刺激。
9.通过包裹量子点的酵母微囊（QD YS）进行的活体成像研究表明，口服QD YS4h后，在右后爪炎症部位存在较强的QD荧光，与相同剂量注射QD相比，口服减少了QD的肝脏分布，表明酵母微囊具有非常明显的口服炎症靶向作用。
结论
1.本研究创新性地提出通过多重非共价键作用介导的自组装可形成高载药能力的聚合物自组装体的假设。为验证其可行性，我们通过采用含羧基小分子药物介导聚乙烯亚胺自组装来构建新型纳米释药系统，并对其理化性能、药物负载能力进行表征，同时研究了不同条件下聚合物自组装行为和该简便自组装策略的通用性。研究表明该药物递送系统具有pH响应性，在胃部几乎无药物释放，而在肠道内快速释放药物。通过该组装体纳米释药系统大大提高了药物的口服生物利用度、提高了药物的疗效，同时亦降低胃肠道刺激及毒副作用。口服急性毒性评价表明当PEI的剂量低于2.5g/kg时，不会产生毒副作用或不良反应。
2.为进一步改善PEI分子的生物相容性，以便构建一种更加安全及有效的聚合物自组装体，我们将β-环糊精单元引入PEI得到β-环糊精键合聚乙烯亚胺（PEI-CD）。在提高了PEI生物相容性的同时，β-环糊精的引入同时赋予了PEI-CD与疏水药物分子通过主-客体作用的性能。与PEI相同，PEI-CD能够与不同含羧基小分子药物自组装形成高药物负载性能的纳米微粒，该纳米释药系统能够显著提高药物的口服生物利用度，提高治疗效果的同时降低了原料药对胃肠道的刺激作用。
3.在成功建立了具有pH响应性的聚合自组装体药物传递系统的基础上，我们进一步研究利用酵母微囊来实现口服给药后局部炎症的靶向。结果表明酵母细胞可以有效负载PEI/IND纳米组装体；载药酵母微囊可以通过靶向巨噬细胞实现炎症部位的靶向。本部分研究对于通过生物技术和仿生手段来设计并构建靶向药物递送系统提供了新的思路和有效技术方法。
4.总的来说，本课题通过载体材料与药物分子间多重非共价键作用构建了一种新型纳米释药系统，该一步组装法简单、便利、高效而又易于放大。本研究一方面为新型药物递送系统的设计提供了新思路，同时也为聚合自组装体纳米药物的研究提供了新方向。另外，酵母微囊这一仿生技术的应用，一方面提高了药物微囊的生物相容性，另一方面创新性地实现了口服后药物对于炎症部位的靶向。
In modern drug devolvement, the low water solubility, lowbioavailability,andtoxicityissueshave prohibited the clinical application of a large number of drugcandidates. In addition, the poorsolubility ofmany clinically used smallmoleculedrugsrestricts their broad applications. To solve these problems, aseries of newdrug deliverysystems such as nanoparticles-based drug delivery platforms have been developed, whichhave many unique advantagesbecauseoftheirsmallparticle size andspecial structure.Numerous studies have indicated that nanocarriers can significantly increasethesolubilityofpoorly soluble drugs, and therefore greatly improve their oral bioavailability. Inaddition,nanovehicles can significantly improve the drug’s efficacyand reduce its sideeffectsor adverse reactions by their passive or active targeting. Self-assembled polymernanocarriers, one ofthe most widelystudiednanosystems, havebeen widelyusedfor deliveryof avarietyoftherapeutics including small molecule drugs, peptides, proteins,and nucleicacids. Polymeric assemblies are generally assembly by hydrophilicoramphiphilicpolymersvia non-covalentinteractions like hydrophobic, electrostatic, andhydrogen bonding forces. Hydrophobic drugsaregenerallywrappedinthehydrophobicdomainofpolymerassemblies based on hydrophobic interaction. However, hydrophobicinteraction aloneoften leads tolowdrugloading, while enhancing drug feeding will result inthe formation of drug crystals.Although the expected efficacy may be achieved by increaseddrug dose, the concomitant increase in the dose of polymer vehicles would raisethecost oftreatmentand produce side effects. Besides, amphiphilic copolymers employed forself-assembly of drug delivery nanoplatforms often possess complex chemical structureandneed to be synthesized by multiple processes, which strongly hinder thebench-to-bedside translation of nanomedicines based on polymer assemblies.
Accordingly, it is highly necessary to develop an effective, high drug loading, and facilestrategy to fabricate nanoplatforms by polymer self-assembly. To circumvent this issue, we hypothesize that multiple non-convalent interactions between polymer and drug mayenhance the encapsulation of drug molecules into nanoassemblies.In this study, we firstlyselected indomethacin (IND) as a model drug and homopolymer polyethyleneimine (PEI)as a modelcarrier material to prove this concept. Since PEI contains primary, secondary, andtertiary amines as well as long carbon chain, while IND contains carboxyl group andhydrophobicunit, there should be electrostatic, hydrogen-bonding, and hydrophobicinteractions between PEI and IND. In addition, the hydrophobic interactions of drugmolecules may also facilitate the construction of highly efficient nanomedicines. Carefulcharacterization was performed to elucidate the physicochemical properties and IND/PEInanoassemblies and interactions between IND and PEI.By using a series ofcarboxyl-containing drugs with different structures, we proved the universality of this facileassembly approach.In addition to pharmacokinetic studies,in vivo pharmacodynamicevaluations were conducted based on acute and chronic inflammation models.Furthermore,acute toxicity of1800PEI and25000PEI was evaluated after oral administration to addressthe potential clinical applications of these assembled PEI nanomedicines.
Based on above results, β-cyclodextrin (β-CD) conjugated PEI (PEI-CD) was synthesizedto improve the biocompatibility of PEI. On the one hand,the introduction of β-CDmayreduce the toxic effects of the PEI carrier material. On the other hand, it can provideadditional non-covalent force between the carrier material and drug, i.e. the host-guestinteraction between cyclodextrin cavity and hydrophobic group, which is beneficial to theformation of nanoassemblies.
In order to achieve oral targeting, we used the yeast wall to entrap IND/PEInanoparticles.Targeting of inflammatory sites may be achieved by recognition of β-1,3glucan on the yeast wall with its receptor on macrophages. Similarly, the pharmacokineticand pharmacodynamic studies were carried out for IND/PEI nanoassemblies loaded yeastcapsules.
Methods
1. Fabrication of nanoassemblies based on PEI or PEI-CD
PEI/drug nanoassemblies were prepared through a dialysis method. First, a certainproportion of drug and PEI was dissolved in a hydrophilic organic solvent, and the resultingsolution was dialyzed against deionized water at room temperature to remove the organic solvent. To fabricate drug/PEI-CD nanomedicines, a modified dialysis process was used.Briefly, an appropriate amount of drug dissolved in DMSO was gradually added into anaqueous solution of PEI-CD (10.0mg/mL) under bath sonication. The obtained mixture wasthen dialyzed against deionized water to form drug-containing nanoassemblies. Theinvolved drugs include IND, naproxen, ibuprofen, flurbiprofen, and diflunisal.
2. Preparation of YS and drug loaded YS
Yeast shells were prepared from yeast cells by treatment using acid, alkali, and organicsolvent to remove yeast cell contents. After YS were dried, they were mixed with PEIaqueous solution to minimally hydrate the particles and incubated for2h to allow YScompletely swelled and adsorb PEI molecules. Then IND in DMSO solution was added andthe particles were resuspended by homogenization or sonication. The suspension wascentrifuged to remove DMSO and uncoated IND and PEI, the precipitate was freeze-driedto give drug-loaded YS.
3. Physicochemical characterization of drug delivery systems
The drug loading and encapsulation efficiency were detected by UV method. Themorphology of nanoparticles was observed by scanning electron microscopy (SEM),transmission electron microscopy (TEM), and atomic force microscopy (AFM). Dynamiclight scattering (DLS) was used for characterize the size distribution and surface potentialof nanoparticles.
4. Characterization of the drug form in assemblies
Raw IND, IND/PEI or IND/PEI-CD physical mixture, and IND/PEI or IND/PEI-CDassemblies containing various contents of IND were examined by DSC and XRDmeasurement.
5. Characterization of multiple interactions between polymer and drug
Raw IND, IND/PEI or IND/PEI-CD physical mixture, and IND/PEI or IND/PEI-CDassemblies containing various contents of IND were examined by FT-IR,1H and1H-1HRoesy NMR measurements.
6. Synthesis and characterization of β-cyclodextrin-conjugated polyethyleneimine(PEI-CD)
PEI-CD was synthesized by a nucleophilic substitution reaction between branched PEIand6-monotosyl β-CD. The structure of PEI-CD was characterized by1H-NMR and FT-IR. The molar ratio of ethyleneimine units in PEI to β-CD groups was calculated via1H-NMRspectrum
7. In vitro release tests
For in vitro release study,0.5mL of IND formations was placed into dialysis tubing,which was immerged into40mL of PBS (pH7.4). At predetermined time intervals,4.0mLof release medium was withdrawn, and fresh PBS was added.
To simulate release profiles under GI tract conditions, aqueous solution of HCl (pH1.2)was employed within the first two hours, and then the release medium was switched into0.01M PBS (pH7.4). IND concentration in release buffer was quantified by UV at310nm.
8. Pharmacokinetic study
IND formations were orally administered via gastric gavage. Blood samples werecollected at specific time points post-dose. Plasma was obtained after10min ofcentrifugation at3000rpm. Then,100mL of acetonitrile and400mL of acetonitrile wasadded into50μL plasma, followed by votexing for1min, and the supernatant waswithdrawn after centrifugation at8000rpm for10min. After being dried under N2atmosphere,100μL of mobile phase was added for sampling. IND concentration in wasquantified by high performance liquid chromatography. The chromatographic conditionsare as follows: mobile phase, acetonitrile-6μM H3PO4(55:45, v:v); eluent rate,1.0mL/min;column temperature,40°C; detection wavelength,245nm; sample volume,20μL; column,C18reverse column (5μm×250mm).
9. Study on the retention and gastrointestinal irritation
IND/PEI-CD assemblies were orally administered to SD male rats at10mg/kg. Atpredetermined time points, they were euthanized and isolated segments of stomach andsmall intestine tissues were fixed. Histological sections were made and stained withhematoxylin-eosin (HE). Using the fluorescence-labeled PEI-CD, the distribution ofIND-containing assemblies in intestinal tract was detected at various time points.
10. Preparation of quantum dots-yeast shell (QDs-YS)
The QD-YS were prepared by the spontaneous deposition of the quantum dots (QDs)with positive surface charge. To this end, YS were mixed with a certain volume of quantumdots, and incubated at37°C for a period of time. After centrifugation, they were washedwith deionized water to remove free quantum dots.
11. Ex vivo imaging
The nude mice with acute paw edema were orally administrated with a certain amountof QDs-YS. The major organs and right hind paws were collected after24h for ex vivoimaging. After fluorescence imaging, the fluorescence intensity of various parts werequantified to evaluate the arthritis targeting capability of the yeast microcapsules.
12. In vivo pharmacodynamic study
In order to substantiate the therapeutic advantages of newly fabricated IND/PEInanomedicines, preliminary pharmacodynamic evaluation was carried out based on acarrageenan-induced acute inflammation and Freund's adjuvant induced arthritis model inrats. The anti-inflammatory efficacy was evaluated by the swelling degree defined as thedifference between the paw volume before and after inflammation.
Swelling degree=Vt-V0(mL)
Where V0and Vtrepresent the paw volume before and after inflammation,respectively.
13. Acute toxicity evaluation.
Male Kunming mice (22-27g) were randomly assigned into4groups (n=6),including three experimental groups and one control group. In the experimental groups,mice were administered via oral gavage of PEI aqueous solution (1.0mL) at doses rangingfrom0.625,1.25, to2.5g/kg. Mice in the control group were orally administered with1.0mL of saline solution. Each day post-injection, mice were weighed and their behaviors wereobserved for any signs of illness. After14days, animals were sacrificed by cervicaldislocation after anaesthesia. Blood samples were collected for the quantification ofhematological parameters and biochemical markers relevant to liver/kidney functions.Organs including heart, liver, kidney, lung, and spleen were harvested and weighed tocalculate the organ index. Histopathological sections of organs and gastrointestinal tissueswere made and stained with HE.
14. Statistical Analysis.
Statistical analysis was performed by SPSS12.0using one-way ANOVA test forexperiments consisting of more than two groups, and with a two-tailed, unpaired t-test inexperiments with two groups. Statistical significance was assessed at p <0.05.
Results
1. Nanoassemblies based on carboxyl-containing drugs and PEI or PEI-CD werefacilely prepared by dialysis, taking advantage of multiple interactions between carriermaterials and drugs. Morphology observation combined with size determination revealedthus obtained nanoassemblies displayed spherical shape with size controllable viaprocessing parameters. In addition, these drug-containing assemblies are positively chargedaccording to the zeta-potential measurement. For IND assemblies, they exhibited drugloading content higher than71%and encapsulation efficiency>90%. The similarassembling profiles were found in other carboxyl-containing drugs like naproxen, diflunisal,ibuprofen, and flurbiprofen.
2. Measurements based on FT-IR,1H and1H-1H Roesy NMR confirmed the presenceof non-covalent multiple interactions between IND and PEI or PEI-CD, includinghydrophobic, host-guest recognition, hydrogen-bonding, and electrostatic forces. DSCand XRD measurements as well as CLSM observation indicated the IND moleculesencapsulated in nanoparticles were essentially amorphous other than drug crystal.
3. For IND/PEI nanomedicines, fast release of IND was achieved at pH7.4. In vitrorelease in media simulating GI conditions suggested that the drug release was largelysuppressed in acidic conditions, which can be dramatically accelerated when the releasemedium was switched into release buffer of pH7.4. In addition, the release of IND wasfaster than that of raw IND and commercial tablet. Consistent with in vitro release profiles,in vivo pharmacokinetic study in rats after oral administration indicated that the area underthe plasma concentration-time curve (AUC) of nanomedicines was120%larger than that ofraw IND. This implied that the oral bioavailability of IND was significantly enhanced byformulating into nanomedicines via molecular self-assembly. In vivo therapeutic effect washighly improved compared to the raw IND control, while no significant toxicities werefound.
4. No animal death occurred at PEI doses lower than2.5g/kg, and the median lethaldose (LD50) was higher than2.5g/kg. Calculation of the organ index, measurements oftypical hematological parameters, and biomarkers relevant to liver/kidney functionsshowed no significant toxicities at PEI doses lower than2.5g/kg.
5. Similarly, high drug-loading property,rapid drug release, high bioavailability, andimproved therapeutic effect were found for IND/PEI-CD nanoparticles. Moreover, PEI-CD was proved to be more biocompatible than PEI, because of the introuction of β-CD units.
6. IND YS and QDs-YS were successfully prepared as demonstrated by measurementsof zeta-potential, TEM, and fluorescence microscopy. IND loading capacity was up to30%,with an entrapment efficiency of70%. Pharmacokinetic study showed that drug in IND YSwas rapidly released at pH7.4, while almost no release occurred at pH1.2.
7. The bioavailability of IND in the case of the yeast system was highly improvedwhen compared with raw IND. In vivo evaluation based on models of carrageenan inducedacute paw edema and Freund's adjuvant induced arthritis suggested that theanti-inflammatory efficacy was also highly improved after microencapsulation of IND inYS.
8. Pathological study showed nanoassemblies can significantly suppress the GIirritation of IND, while improving its anti-inflammatory efficacy
9. En vivo imaging indicated that oral administration of QDs-YS presented strongfluorescence intensity, which is comparable to that by i.v. injection of QDs, while thedistribution in liver was significantly deceased.
Conclusions
1. In this study, we proposed innovatively that polymer assemblies with high drugloading can be achieved by multiple non-covalent forces. In order to verify this hypothesis,we constructed IND/PEI nanomedicines by one-pot self-assembly mediated via multipleinteractions between drug and PEI. The physicochemical properties and drug loadingcapacity of obtained nanoassemblies were carefully characterized. Also, the self-assemblyprofiles and the versatility of this facile assembly strategy under different conditions wereinvestigated. In vitro release study showed that the new nanosystem displayed a significantpH-sensitivity. Virtually no drug release occurred in the stomach, while rapid release couldbe achieved in the intestine. The oral bioavailability and therapeutic efficacy of the loadeddrug were greatly improved through the assembly of nanosystems, while thegastrointestinal irritation and toxicity were reduced. Acute oral toxicity evaluation of PEIshowed that no side effects or adverse reactions were produced at PEI dose less than2.5g/kg.
2. To further improve the biocompatibility of PEI for more safety and effectivepolymer nanoassemblies, we introduced β-cyclodextrin unit into PEI, resulting in a new polymer of β-cyclodextrin conjugated PEI (PEI-CD). The introduction of β-CD cansignificantly improve the biocompatibility of PEI, and provide additional host-guestinteractions for assembly of PEI-CD and various drugs. Similarly, efficient drug loading,rapid cargo release, high bioavailability, and improved therapeutic effect were achieved byPEI-CD/IND nanoparticles.
3. After the assembled pH-responsive polymeric nanomedicines were establishedsuccessfully, we further used the yeast wall as microcapsules to entrap IND/PEInanoparticles to achieve inflammation targeting by macrophage surface receptor wich canrecognize β-1,3glucan on the yeast wall. This part of study provided a new way ofdesigning orally targeted drug delivery system through biotechnology and bioengineeringapproaches.
4. In summary, we discovered a facile, convenient, cost-effective, and easily scalableone-pot assembly strategy to formulate various lipophilic therapeutics bearing carboxylgroup into nanomedicines. The present study provides new insights into the design of novelnanomedicines, and opens a new direction for the molecular self-assembly of polymerdriven by small molecules. Additionally, the application of biomimetic technology in yeastmicrocapsules can highly improve the biocompatibility of drug payload, and innovativelyachieve the oral targeting of drugs to inflammatory sites.
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