Size-dependent absorption mechanism of polymeric nanoparticles for oral delivery of protein drugs

Polymeric nanoparticles have been widely applied to oral delivery of protein drugs, however, few studies focused on the systematical elucidation of the size-dependent oral absorption mechanism with well-defined polymeric nanoparticles. Rhodamine B labeled carboxylated chitosan grafted nanoparticles (RhB-CCNP) with different particle sizes (300, 600, and 1000?nm) and similar Zeta potentials (-35?mV) were developed. FITC labeled bovine serum albumin (FITC-BSA) was encapsulated into RhB-CCNP to form drug loaded polymeric nanoparticles (RhB-CCNP-BSA). RhB-CCNP-BSA with uniform particle size and similar surface charge possessed desired structural stability in simulated physiological environment to substantially guarantee the validation of elucidation on size-dependent absorption mechanisms of polymeric nanoparticles using in?vitro, in situ, and ex?vivo models. RhB-CCNP-BSA with smaller sizes (300?nm) demonstrated elevated intestinal absorption, as mechanistically evidenced by higher mucoadhesion in rat ileum, release amount of the payload into the mucus layer, Caco-2 cell internalization, transport across Caco-2 cell monolayers and rat ileum, and systemic biodistribution after oral gavage. Peyer's patches could play a role in the mucoadhesion of nanoparticles, resulting in their close association with the intestinal absorption of nanoparticles. These results provided guidelines for the rational design of oral nanocarriers for protein drugs in terms of particle size.     

The use of deoxycholic acid to enhance the oral bioavailability of biodegradable nanoparticles

Oral delivery of nanoparticles encapsulating drugs and proteins remains a challenging route for administration due to the many barriers in the gastrointestinal tract that limit bioavailability. We hypothesized that bile salts could be used to improve the bioavailability of poly(lactide-co-glycolide) (PLGA) nanoparticles by protecting them during their transport through the gastrointestinal tract and enhancing their absorption by the intestinal epithelia. A deoxycholic acid emulsion is shown to protect PLGA nanoparticles from degradation in acidic conditions and enhance their permeability across a Caco-2 cell monolayer, an in vitro model of human epithelium. Oral administration of loaded PLGA nanoparticles to mice, using a deoxycholic acid emulsion, produced sustained levels of the encapsulant in the blood over 24-48 h with a relative bioavailability of 1.81. Encapsulant concentration was highest in the liver, demonstrating a novel means for targeted delivery to the liver by the oral route.     

Goblet cell-targeting nanoparticles for oral insulin delivery and the influence of mucus on insulin transport

The present study was to demonstrate the effects of goblet cell-targeting nanoparticles on the oral absorption of insulin in vitro, ex vivo and in vivo, and identify the targeting mechanism as well as the influence of mucus. The insulin loaded nanoparticles were prepared using trimethyl chitosan chloride (TMC) modified with a CSKSSDYQC (CSK) targeting peptide. Compared with unmodified nanoparticles, the CSK peptide modification could facilitate the uptake of nanoparticles in villi, enhance the permeation of drugs across the epithelium, meanwhile, induce a significantly higher internalization of drugs via clathrin and caveolae mediated endocytosis on goblet cell-like HT29-MTX cells. In transport studies across Caco-2/HT29-MTX co-cultured cell monolayer (simulating intestinal epithelium), the CSK peptide modification also showed enhanced transport ability, even if the targeting recognition was partially affected by mucus. Moreover, it was found the existence of mucus was propitious to the transport of insulin from both modified and unmodified nanoparticles. In the pharmacological and pharmacokinetic studies in diabetic rats, the orally administrated CSK peptide modified nanoparticles produced a better hypoglycemic effect with a 1.5-fold higher relative bioavailability compared with unmodified ones. In conclusion, CSK peptide modified TMC nanoparticles showed sufficient effectiveness as goblet cell-targeting nanocarriers for oral delivery of insulin.     

Strategies Toward the Improved Oral Delivery of Insulin Nanoparticles via Gastrointestinal Uptake and Translocation

The design of strategies that improve the absorption of insulin through the gastrointestinal tract is a considerable challenge in the pharmaceutical sciences and would significantly enhance the treatment of diabetes mellitus. Several strategies have been devised to overcome physiologic and morphologic barriers to insulin absorption, including the inhibition of acidic and enzymatic degradation, enhancement of membrane permeability or widening of tight junctions, chemical modification of insulin, and the formulation of carrier systems. In particular, the concept of nanoparticulate carriers for oral insulin delivery has evolved through remarkable advances in nanotechnology. Investigations focused on uptake and translocation via Peyer's patches have demonstrated high levels of nanoparticle absorption based on significant alterations in the glycemic response to various glucogenic sources. This paper reviews the mechanisms for insulin and particle uptake and translocation through the gastrointestinal tract, and the potential barriers to this, outlines the design of nanoparticulate carriers for the oral delivery of insulin, and presents prospects for its clinical application.     

Chitosan DNA nanoparticles for oral gene delivery

Gene therapy is a promising technology with potential applications in the treatment of medical conditions, both congenital and acquired. Despite its label as breakthrough technology for the 21^st century, the simple concept of gene therapy - the introduction of a functional copy of desired genes in affected individuals - is proving to be more challenging than expected. Oral gene delivery has shown intriguing results and warrants further exploration. In particular, oral administration of chitosan DNA nanoparticles, one the most commonly used formulations of therapeutic DNA, has repeatedly demonstrated successful in vitro and in vivo gene transfection. While oral gene therapy has shown immense promise as treatment options in a variety of diseases, there are still significant barriers to overcome before it can be considered for clinical applications. In this review we provide an overview of the physiologic challenges facing the use of chitosan DNA nanoparticles for oral gene delivery at both the extracellular and intracellular level. From administration at the oral cavity, chitosan nanoparticles must traverse the gastrointestinal tract and protect its DNA contents from significant jumps in pH levels, various intestinal digestive enzymes, thick mucus layers with high turnover, and a proteinaceous glycocalyx meshwork. Once these extracellular barriers are overcome, chitosan DNA nanoparticles must enter intestinal cells, escape endolysosomes, and disassociate from genetic material at the appropriate time allowing transport of genetic material into the nucleus to deliver a therapeutic effect. The properties of chitosan nanoparticles and modified nanoparticles are discussed in this review. An understanding of the barriers to oral gene delivery and how to overcome them would be invaluable for future gene therapy development.     

口服胰岛素载体的高分子生物材料

背景：利用具有生物相容性和生物可降解性的高分子材料作为载体,通过化学结合或物理包裹胰岛素,可提高胰岛素在体内的稳定性和生物利用度。 目的：从类型、制备方法、特征、药理作用等方面综述国内外口服胰岛素载体的高分子材料的研究进展。 方法：由作者应用计算机检索中国知网数据库、PubMed数据库和Elsevier 数据库2002年1月至2013年2月,与高分子生物材料和口服胰岛素载体相关的文章,中文关键词为“高分子生物材料、口服胰岛素、载体”,英文关键词为“polymeric biomaterials,oral insulin,carrier”。 结果与结论：目前,主要用于口服胰岛素系统控缓释的高分子生物材料可分为天然高分子生物材料和合成高分子生物材料两大类。用于口服胰岛素载体研究的天然高分子材料,以壳聚糖、藻酸盐多见。合成高分子生物材料中聚酯类、聚丙烯酸酯类及其共聚物,因具有良好的生物相容性、生物降解性和生理性能,被用作口服胰岛素制剂的载体材料的研究报道较多。国内外有关口服胰岛素制剂的研究报道虽多,也有一些商品型口服胰岛素进入临床试验阶段,但至今尚未见到实际应用的临床报告。其主要原因是作为载体的高分子材料、胰岛素的生物利用度低、制剂的质量标准及稳定性问题尚未解决。因此,未来的研究将主要集中在：载体材料的选择或者对现有高分子聚合物进行物理和化学的修饰,研发出新型的聚合物基材料作为载体,以避免胃肠道对胰岛素的破坏和改善胰岛素在体内的吸收,获得理想的释药速度和良好缓控释效果。     

Intestinal mucosa permeability following oral insulin delivery using core shell corona nanolipoparticles

Chitosan nanoparticles (NC) have excellent capacity for protein entrapment, favorable epithelial permeability, and are regarded as promising nanocarriers for oral protein delivery. Herein, we designed and evaluated a class of core shell corona nanolipoparticles (CSC) to further improve the absorption through enhanced intestinal mucus penetration. CSC contains chitosan nanoparticles as a core component and pluronic F127-lipid vesicles as a shell with hydrophilic chain and polyethylene oxide PEO as a corona. These particles were developed by hydration of a dry pluronic F127-lipid film with NC suspensions followed by extrusion. Insulin nested inside CSC was well protected from enzymatic degradation. Compared with NC, CSC exhibited significantly higher efficiency of mucosal penetration and, consequently, higher cellular internalization of insulin in mucus secreting E12 cells. The cellular level of insulin after CSC treatment was 36-fold higher compared to treatment with free insulin, and 10-fold higher compared to NC. CSC significantly facilitated the permeation of insulin across the ileum epithelia, as demonstrated in an ex vivo study and an in vivo absorption study. CSC pharmacological studies in diabetic rats showed that the hypoglycemic effects of orally administrated CSC were 2.5-fold higher compared to NC. In conclusion, CSC is a promising oral protein delivery system to enhance the stability, intestinal mucosal permeability, and oral absorption of insulin.     

Intestinal patches with an immobilized solid-in-oil formulation for oral protein delivery

Oral administration of biomolecular drugs such as peptides, proteins, and DNA is an attractive delivery method because of the safety and convenience of delivery in contrast to injection administration. However, oral delivery of biomolecules has several potential barriers such as enzymatic degradation in the gastrointestinal tract and low permeability across an intestinal membrane. In this study, we proposed an intestinal patch system that included surfactant-coated insulin for oral delivery. The intestinal patches, which have mucoadhesive and drug-impermeable layers, induced sustained unidirectional insulin release toward intestinal mucosa and inhibition of insulin leakage from the patches. Moreover, the surfactant-coated insulin, which has high compatibility with cell membranes, enhanced insulin transport across the intestinal membrane. This study demonstrates that the intestinal patches might improve protein permeability in the intestinal mucosa, thereby offering an innovative therapeutic strategy.     

Drug permeability and mucoadhesion properties of thiolated trimethyl chitosan nanoparticles in oral insulin delivery

Trimethyl chitosan-cysteine conjugate (TMC-Cys) was synthesized in an attempt to combine the mucoadhesion and the permeation enhancing effects of TMC and thiolated polymers related to different mechanisms for oral absorption. TMC-Cys with various molecular weights (30, 200, and 500 kDa) and quaternization degrees (15 and 30%) was allowed to form polyelectrolyte nanoparticles with insulin through self-assembly, which demonstrated particle size of 100–200 nm, zeta potential of +12 to +18 mV, and high encapsulation efficiency. TMC-Cys/insulin nanoparticles (TMC-Cys NP) showed a 2.1–4.7-fold increase in mucoadhesion compared to TMC/insulin nanoparticles (TMC NP), which might be partly attributed to disulfide formation between TMC-Cys and mucin as evidenced by DSC measurement. Compared to insulin solution and TMC NP, TMC-Cys NP induced increased insulin transport through rat intestine by 3.3–11.7 and 1.7–2.6 folds, promoted Caco-2 cell internalization by 7.5–12.7 and 1.7–3.0 folds, and augmented uptake in Peyer's patches by 14.7–20.9 and 1.7–5.0 folds, respectively. Such results were further confirmed by in vivo experiment with the optimal TMC-Cys NP. Biocompatibility assessment revealed lack of toxicity of TMC-Cys NP. Therefore, self-assembled nanoparticles between TMC-Cys and protein drugs could be an effective and safe oral delivery system.     

Implication of nanoparticles/microparticles in mucosal vaccine delivery

Although polymeric nanoparticles/microparticles are well established for the mucosal administration of conventional drugs, they have not yet been developed commercially for vaccine delivery. The limitation of the mucosal (particularly oral) route of delivery, including low pH, gastric enzymes, rapid transit and poor absorption of large molecules, has made mucosal vaccine delivery challenging. Nevertheless, several polymeric delivery systems for mucosal vaccine delivery are currently being evaluated. The polymer-based approaches are designed to protect the antigen in the gut, to target the antigen to the gut-associated lymphoid tissue or to increase the residence time of the antigen in the gut through bioadhesion. M-cell targeting is a potential approach for mucosal vaccine delivery, which can be achieved using M-cell-specific lectins, microbial adhesins or immunoglobulins. While many hurdles must be overcome before targeted mucosal vaccine delivery becomes a practical reality, this is a potential area of research that has important implications for future vaccine development. This review comprises various aspects that could be decisive in the development of polymer based mucosal vaccine delivery systems.     

Synthesis and characterization of PEGylated calcium phosphate nanoparticles for oral insulin delivery

The inconvenience of subcutaneous insulin delivery leads to low patient compliance with the dosage regimens. The most desirable form of administration seems to be through the oral route. This work investigates the utility of PEGylated calcium phosphate nanoparticles as oral carriers for insulin. Calcium phosphate nanoparticles (CaP) with an average particle size of 47.9 nm (D50) were synthesized and surface modified by conjugating it with poly(ethylene glycol) (PEG). These modified nanoparticles were having a near zero zeta potential. Protection of insulin from the gastric environment has been achieved by coating the nanoparticles with a pH sensitive polymer that will dissolve in the mildly alkaline pH environment of the intestine. The release profiles of coated nanoparticles exhibited negligible release in acidic (gastric) pH, i.e., only 2% for CaP and 6.5% for PEGylated CaP. However, a sustained release of insulin was observed at neutral (intestinal) pH for over 8 h. The conformation of the released insulin, studied using circular dichroism, was unaltered when compared with native insulin. The released insulin was also stable as it was studied using dynamic light scattering. Radioimmunoassay was performed and the immunoreactivity of the released insulin was found to be intact. These results suggest PEGylated calcium phosphate nanoparticles as an excellent carrier system for insulin toward the development of an oral insulin delivery system.     

Oral Delivery of Peptide Drugs

A wide variety of peptide drugs are now produced on a commercial scale as a result of advances in the biotechnology field. Most of these therapeutic peptides are still administered by the parenteral route because of insufficient absorption from the gastrointestinal tract. Peptide drugs are usually indicated for chronic conditions, and the use of injections on a daily basis during long-term treatment has obvious drawbacks. In contrast to this inconvenient and potentially problematic method of drug administration, the oral route offers the advantages of self-administration with a high degree of patient acceptability and compliance. The main reasons for the low oral bioavailability of peptide drugs are pre-systemic enzymatic degradation and poor penetration of the intestinal mucosa. A considerable amount of research has focused on overcoming the challenges presented by these intestinal absorption barriers to provide effective oral delivery of peptide and protein drugs. Attempts to improve the oral bioavailability of peptide drugs have ranged from changing the physicochemical properties of peptide molecules to the inclusion of functional excipients in specially adapted drug delivery systems. However, the progress in developing an effective peptide delivery system has been hampered by factors such as the inherent toxicities of absorption-enhancing excipients, variation in absorption between individuals, and potentially high manufacturing costs. This review focuses on the intestinal barriers that compromise the systemic absorption of intact peptide and protein molecules and on the advanced technologies that have been developed to overcome the barriers to peptide drug absorption.     

The mucoadhesive and gastroretentive properties of hydrophobin-coated porous silicon nanoparticle oral drug delivery systems

Impediments to intestinal absorption, such as poor solubility and instability in the variable conditions of the gastrointestinal (GI) tract plague many of the current drugs restricting their oral bioavailability. Particulate drug delivery systems hold great promise in solving these problems, but their effectiveness might be limited by their often rapid transit through the GI tract. Here we describe a bioadhesive oral drug delivery system based on thermally-hydrocarbonized porous silicon (THCPSi) functionalized with a self-assembled amphiphilic protein coating consisting of a class II hydrophobin (HFBII) from Trichoderma reesei. The HFBII-THCPSi nanoparticles were found to be non-cytotoxic and mucoadhesive in AGS cells, prompting their use in a biodistribution study in rats after oral administration. The passage of HFBII-THCPSi nanoparticles in the rat GI tract was significantly slower than that of uncoated THCPSi, and the nanoparticles were retained in stomach by gastric mucoadhesion up to 3 h after administration. Upon entry to the small intestine, the mucoadhesive properties were lost, resulting in the rapid transit of the nanoparticles through the remainder of the GI tract. The gastroretentive drug delivery system with a dual function presented here is a viable alternative for improving drug bioavailability in the oral route.     

Polymeric particulate technologies for oral drug delivery and targeting: A pathophysiological perspective

The oral route for delivery of pharmaceuticals is the most widely used and accepted. Nanoparticles and microparticles are increasingly being applied within this arena to optimize drug targeting and bioavailability. Frequently the carrier systems used are either constructed from or contain polymeric materials. Examples of these nanocarriers include polymeric nanoparticles, solid lipid nanocarriers, self-nanoemulsifying drug delivery systems and nanocrystals. It is the purpose of this review to describe these cutting edge technologies and specifically focus on the interaction and fate of these polymers within the gastrointestinal system.     

In vivo evaluation of safety and efficacy of self-assembled nanoparticles for oral insulin delivery

A variety of approaches have been studied in the past to overcome the problems encountered with the oral delivery of insulin, but with little success. In this study, self-assembled nanoparticles (NPs) with a pH-sensitive characteristic were prepared by mixing the anionic poly-γ-glutamic acid solution with the cationic chitosan solution in the presence of MgSO₄ and sodium tripolyphosphate. The in vitro results found that the transport of insulin across Caco-2 cell monolayers by NPs appeared to be pH-dependent; with increasing pH, the amount of insulin transported decreased significantly. An in vivo toxicity study was performed to establish the safety of the prepared NPs after oral administration. Additionally, the impact of orally administered NPs on the pharmacodynamics (PD) and pharmacokinetics (PK) of insulin was evaluated in a diabetic rat model. The in vivo results indicated that the prepared NPs could effectively adhere on the mucosal surface and their constituted components were able to infiltrate into the mucosal cell membrane. The toxicity study indicated that the NPs were well tolerated even at a dose 18 times higher than that used in the PD/PK study. Oral administration of insulin-loaded NPs demonstrated a significant hypoglycemic action for at least 10 h in diabetic rats and the corresponding relative bioavailability of insulin was found to be 15.1 ± 0.9%. These findings suggest that the NPs prepared in the study are a promising vehicle for oral delivery of insulin.     

Self-assembling bubble carriers for oral protein delivery

Successful oral delivery of therapeutic proteins such as insulin can greatly improve the quality of life of patients. This study develops a bubble carrier system by loading diethylene triamine pentaacetic acid (DTPA) dianhydride, a foaming agent (sodium bicarbonate; SBC), a surfactant (sodium dodecyl sulfate; SDS), and a protein drug (insulin) in an enteric-coated gelatin capsule. Following oral administration to diabetic rats, the intestinal fluid that has passed through the gelatin capsule saturates the mixture; concomitantly, DTPA dianhydride produces an acidic environment, while SBC decomposes to form CO₂ bubbles at acidic pH. The gas bubbles grow among the surfactant molecules (SDS) owing to the expansion of the generated CO₂. The walls of the CO₂ bubbles consist of a self-assembled film of water that is in nanoscale and may serve as a colloidal carrier to transport insulin and DTPA. The grown gas bubbles continue to expand until they bump into the wall and burst, releasing their transported insulin, DTPA, and SDS into the mucosal layer. The released DTPA and SDS function as protease inhibitors to protect the insulin molecules as well as absorption enhancers to augment their epithelial permeability and eventual absorption into systemic circulation, exerting their hypoglycemic effects.     

Multistage pH-responsive mucoadhesive nanocarriers prepared by aerosol flow reactor technology: A controlled dual protein-drug delivery system

Nanotechnology based drug delivery systems are anticipated to overcome the persistent challenges in oral protein and peptide administration, and lead to the development of long awaited non-invasive therapies. Herein, an advanced single-step aerosol flow reactor based technology was used to develop a multifunctional site specific dual protein-drug delivery nanosystem. For this purpose, mucoadhesive porous silicon (PSi) nanoparticles encapsulated into a pH-responsive polymeric nanomatrix was developed for advanced oral type 2 diabetes mellitus therapy with an antidiabetic peptide, glucagon like peptide-1 (GLP-1), and the enzyme inhibitor, dipeptidyl peptidase-4 (DPP4). Chitosan surface modification inherited the mucoadhesiveness to the nanosystem which led to enhanced cellular interactions and increased cellular compatibility. An advanced aerosol flow reactor technology was used to encapsulate the chitosan modified nanoparticles into an enteric polymeric nanomatrix. The pH-sensitive polymeric matrix simultaneously prevented the gastric degradation of the encapsulated peptide and also preserved the mucoadhesive functionality of the chitosan-modified PSi nanoparticles in the harsh stomach environment. The multidrug loaded nanosystem showed augmented intestinal permeability of GLP-1, evaluated in an in vitro cell-based intestinal epithelium model, attributed to the permeation enhancer effect of chitosan and inhibition of GLP-1 degradation by the DPP4 inhibitor. The applied technology resulted in the development of a dual-drug delivery nanosystem that synergizes the antidiabetic effect of the loaded peptide and the enzyme inhibitor, thereby indicating high clinical potential of the system and preparation technique.     

聚乙二醇修饰的共聚物纳米粒研究进展

可生物降解的聚合物纳米粒作为药物输送载体有很多优势，如可控释，靶向等。但是，由于聚合物纳米粒经静脉经给药后，数秒或数分钟内会被皮网状系统清除而无法普遍应用，为了克服这一缺点，越来越多的研究者引入亲水性组分聚乙二醇（PEG）对聚合物进行修饰，以避免其被内皮肉状系统摄取。聚乙二醇的引入不仅会影响聚合物纳米粒的生物降解行为，而且会影响药物的释放，体内分布等行为，本文综述了聚乙二醇修饰的共聚物纳米粒的制备，稳定性，载体，体外释药，体内分布，毒性等方面的研究进展，并对其前景进行预测。     

Designing Nanoparticle-based Drug Delivery Systems for Precision Medicine

Traditional drugs are facing bottlenecks of lower solubility, absorption, and especially the inefficient organs or cells targeting during the precision medicine era. It is urgently needed to discover and establish new methods or strategies to modify old drugs or create new ones against the above defects. With the support of nanotechnology, the solubility, absorption and targeting of traditional drugs were greatly improved by modifying and fabricating with various types of nanoparticles to some extent, though many shortages remain. In this mini-review we will focus on advances in several most commonly used nanoparticles, from their nature and design, to drug delivery system and clinical application, that they overcome heterogeneous barriers in precision medicine, thereby ultimately improve patient outcome overall.     

Development of Oral Vaccines to Stimulate Mucosal and Systemic Immunity: Barriers and Novel Strategies

Many questions regarding the induction of mucosal and humoral immunity through oral vaccination exist. Efficacy is dependent on the physicochemical properties of the antigen, the gastrointestinal environment, the presence of adjuvants, and the mode of delivery. Understanding how these factors interrelate will be critical to the development of new oral vaccines. A number of approaches are currently being studied to enhance the immune response. These include chemical conjugation, immunization with recombinant bacteria and viruses, and mucosal adjuvants. Vaccine delivery systems prepared from natural or synthetic polymers is a particularly promising area because many of the current methods to induce mucosal stimulation can be incorporated within these systems. Thus, the polymeric delivery system functions as a platform to facilitate uptake by M-cells and prolong antigen presentation and stimulation of the Peyer's patches. This Review examines some of the physiological and immunological barriers associated with oral vaccination and discusses novel strategies to overcome such barriers.