Macromolecular Therapeutics Research Articles

Cationic nioplexes-in-polysaccharide-based hydrogels as versatile biodegradable hybrid materials to deliver nucleic acids.

Two polysaccharide-based hydrogels made of only κ-carrageenan (4%; w/v) or of a mixture of methylcellulose:κ-carrageenan (2%; w/v) were used to encapsulate cationic nioplexes. These vesicular particles were made of a synthetic aminolipid and polysorbate-80 (Tween-80), as a non-ionic surfactant agent. According to oscillatory rheological measurements, the presence of nioplexes did not compromise the mechanical integrity of the gels. In vitro niosomal release experiments demonstrated the liberation of nioplexes up to 24 h, and the curves were fitted according to Higuchi, Korsmeyer-Peppas and Weibull equation models, which indicated Fickian-diffusion controlled mechanisms. Besides nioplexes, cervical cancer cells were also entrapped within the biohydrogels. Cell release confirmed that these materials did not affect the cell viability, allowing cells to spread and proliferate after 24 h. The applicability of these biocompatible hydrogels was also extended to gene delivery. In this regard, the best silencing activities were found when cationic niosomes were complexed with antisense oligonucleotides in KC hydrogels. Nioplexes were able to release through the hydrogel and promoted silencing of luciferase expression in the presence of serum without using commercially available cationic lipids. Overall, the formation of such hybrid materials by integrating cationic nioplexes within biodegradable hydrogels provides a new perspective for the delivery of macromolecular therapeutics.

Drug-free macromolecular therapeutics: Impact of structure on induction of apoptosis in Raji B cells

Recently, we developed a new paradigm in macromolecular therapeutics that avoids the use of low molecular weight drugs. The activity of the “drug-free macromolecular therapeutics” is based on the biorecognition of complementary motifs at cell surface resulting in receptor crosslinking and apoptosis induction. The system is composed of two nanoconjugates: (1) a single-stranded morpholino oligonucleotide (MORF1) attached to an anti-CD20 Fab′ fragment (Fab′-MORF1); (2) multiple copies of complementary oligonucleotide MORF2 grafted to a linear polymer of N-(2-hydroxypropyl)methacrylamide (HPMA) – P-(MORF2)x. The two conjugates crosslink CD20 antigens via MORF1-MORF2 hybridization at the surface of CD20+ malignant B-cells and induce apoptosis. Preclinical studies in a murine model of human non-Hodgkin's lymphoma showed cancer cells eradication and long-term survivors. The aim of this study was to determine the relationship between the detailed structure of the nanoconjugates and apoptosis induction in Raji cells to allow system optimization. The factors studied include the length of the MORF sequence, the valence of P-(MORF2)x (varying x), molecular weight of P-(MORF2)x, incorporation of a miniPEG spacer between Fab′ and MORF1 and between polymer backbone and pendant MORF2, and comparison of two Fab′ fragments, one from 1F5 antibody (Fab′1F5), the other from Rituximab (Fab′RTX). The results of apoptosis induction in human Burkitt's B-cell non-Hodgkin's lymphoma (NHL) Raji cells as determined using three apoptotic assays (Annexin V, Caspase 3, and TUNEL) indicated that: a) An improvement of apoptotic activity was observed for a 28 base pair MORF sequence when compared to MORFs composed of 20 and 25 base pairs. The differences depended on type of assay, concentration and exposure schedule (consecutive vs. premixed). b) The higher the valence of P-(MORF2)x the higher the levels of apoptosis. c) Higher molecular weight of P-(MORF2)x induced higher levels of apoptosis. d) A miniPEG8 spacer was effective in enhancing apoptotic levels in contrast to a miniPEG2 spacer. e) There was not a statistically significant difference when comparing Fab′1F5-MORF1 with Fab′RTX-MORF1.

A Redox-Activatable Fluorescent Sensor for the High-Throughput Quantification of Cytosolic Delivery of Macromolecules.

Efficient delivery of biomacromolecules (e.g., proteins, nucleic acids) into cell cytosol remains a critical challenge for the development of macromolecular therapeutics or diagnostics. To date, most common approaches to assess cytosolic delivery rely on fluorescent labeling of macromolecules with an "always on" reporter and subcellular imaging of endolysosomal escape by confocal microscopy. This strategy is limited by poor signal-to-noise ratio and only offers low throughput, qualitative information. Herein we describe a quantitative redox-activatable sensor (qRAS) for the real-time monitoring of cytosolic delivery of macromolecules. qRAS-labeled macromolecules are silent (off) inside the intact endocytic organelles, but can be turned on by redox activation after endolysosomal disruption and delivery into the cytosol, thereby greatly improving the detection accuracy. In addition to confocal microscopy, this quantitative sensing technology allowed for a high-throughput screening of a panel of polymer carriers toward efficient cytosolic delivery of model proteins on a plate reader. The simple and versatile qRAS design offers a useful tool for the investigation of new strategies for endolysosomal escape of biomacromolecules to facilitate the development of macromolecular therapeutics for a variety of disease indications.

A Redox‐Activatable Fluorescent Sensor for the High‐Throughput Quantification of Cytosolic Delivery of Macromolecules

AbstractEfficient delivery of biomacromolecules (e.g., proteins, nucleic acids) into cell cytosol remains a critical challenge for the development of macromolecular therapeutics or diagnostics. To date, most common approaches to assess cytosolic delivery rely on fluorescent labeling of macromolecules with an “always on” reporter and subcellular imaging of endolysosomal escape by confocal microscopy. This strategy is limited by poor signal‐to‐noise ratio and only offers low throughput, qualitative information. Herein we describe a quantitative redox‐activatable sensor (qRAS) for the real‐time monitoring of cytosolic delivery of macromolecules. qRAS‐labeled macromolecules are silent (off) inside the intact endocytic organelles, but can be turned on by redox activation after endolysosomal disruption and delivery into the cytosol, thereby greatly improving the detection accuracy. In addition to confocal microscopy, this quantitative sensing technology allowed for a high‐throughput screening of a panel of polymer carriers toward efficient cytosolic delivery of model proteins on a plate reader. The simple and versatile qRAS design offers a useful tool for the investigation of new strategies for endolysosomal escape of biomacromolecules to facilitate the development of macromolecular therapeutics for a variety of disease indications.

Structural analysis of nanoparticulate carriers for encapsulation of macromolecular drugs

Recently developed macromolecular drugs display strong potential to cure diseases involving genetic components, e.g. Rheumatoid arthritis, diabetes, Alzheimer's disease, Huntington's disease, Duchenne muscular dystrophy, Retinitis pigmentosa, and several types of cancers. Bioavailability and enhanced drug specificity necessitate the preparation of efficient carrier systems for the macromolecular therapeutics. We employ synchrotron small-angle X-ray scattering (SAXS) and cryogenic transmission electron microscopy (Cryo-TEM) to study the nanostructure formation, macromolecular drug upload, and the topological transformations occurring upon complexation of supercoiled plasmid DNA (encoding for the therapeutic protein brain-derived neurotrophic factor, BDNF) with cationic lipid nanocarrier assemblies. Understanding of the liquid crystalline nanostructure formation (hexosomes, cubosomes, inverted hexagonal and intermediate mesophases, or onion-type complexes) enabling efficient delivery of new generation sequencing systems is expected to contribute to the progress in precision nanomedicine and the treatment of various severe diseases.

Peptide-Based Supramolecular Hydrogels for Delivery of Biologics.

The demand for therapeutic biologics has rapidly grown over recent decades, creating a dramatic shift in the pharmaceutical industry from small molecule drugs to biological macromolecular therapeutics. As a result of their large size and innate instability, the systemic, topical, and local delivery of biologic drugs remains a highly challenging task. Although there exist many types of delivery vehicles, peptides and peptide conjugates have received continuously increasing interest as molecular blocks to create a great diversity of supramolecular nanostructures and hydrogels for the effective delivery of biologics, due to their inherent biocompatibility, tunable biodegradability, and responsiveness to various biological stimuli. In this context, we discuss the design principles of supramolecular hydrogels using small molecule peptides and peptide conjugates as molecular building units, and review the recent effort in using these materials for protein delivery and gene delivery.

Nose-to-brain delivery of macromolecules mediated by cell-penetrating peptides

Brain delivery of macromolecular therapeutics (e.g., proteins) remains an unsolved problem because of the formidable blood–brain barrier (BBB). Although a direct pathway of nose-to-brain transfer provides an answer to circumventing the BBB and has already been intensively investigated for brain delivery of small drugs, new challenges arise for intranasal delivery of proteins because of their larger size and hydrophilicity. In order to overcome the barriers and take advantage of available pathways (e.g., epithelial tight junctions, uptake by olfactory neurons, transport into brain tissues, and intra-brain diffusion), a low molecular weight protamine (LMWP) cell-penetrating peptide was utilized to facilitate nose-to-brain transport. Cell-penetrating peptides (CPP) have been widely used to mediate macromolecular delivery through many kinds of biobarriers. Our results show that conjugates of LMWP–proteins are able to effectively penetrate into the brain after intranasal administration. The CPP-based intranasal method highlights a promising solution for protein therapy of brain diseases.

Enhancing insulin oral absorption by using mucoadhesive nanoparticles loaded with LMWP-linked insulin conjugates

Although significant progress has been achieved, effective oral delivery of protein drugs such as insulin by nanoparticle-based carrier systems still faces certain formidable challenges. Considerable amount of protein drug is released from the nanoparticles (NPs) in the gastrointestinal (GI) tract. Because of their low permeability through the intestinal mucosa, the released protein would be soon degraded by the large amount of proteases in the GI tract. Herein, we report an oral insulin delivery system that can overcome the above-mentioned problems by mucoadhesive NPs (MNPs) loaded with cell penetrating peptide-linked insulin conjugates. On one hand, after conjugation with low molecular weight protamine (LMWP), a cell penetrating peptide (CPP), insulin showed greatly improved permeability through intestinal mucus layer and epithelia. On the other hand, the mucoadhesive N-trimethyl chitosan chloride-coated PLGA nanoparticles (MNPs) that were loaded with conjugates enhanced the retention in the intestinal mucus layer. By adopting this delivery strategy, the LMWP-insulin conjugates released from the MNPs could be deprived from enzymatic degradation, due to the short distance in reaching the epithelia and the high permeation of the conjugates through epithelia. The oral delivery system of insulin designed by us showed a long-lasting hypoglycemia effect with a faster onset in diabetic rats. The pharmacological availability of orally delivered conjugates-loaded MNPs was 17.98±5.61% relative to subcutaneously injected insulin solution, with a 2-fold higher improvement over that by MNPs loaded with native insulin. Our results suggested that conjugation with CPP followed by encapsulation in MNPs provides an effective strategy for oral delivery of macromolecular therapeutics.

Stabilising cubosomes with Tween 80 as a step towards targeting lipid nanocarriers to the blood–brain barrier

Coating nanoparticles with the surfactant Tween 80 have been previously shown to enhance drug delivery across the blood–brain barrier (BBB). The aim of this study was to investigate whether Tween 80 could be used to stabilise phytantriol-based cubosomes thereby enabling potential application in delivering macromolecular therapeutics to the brain. Cubosome particles with their large internal and external surface area by virtue of their nanostructure are ideal for delivery of macromolecules. Phase behaviour studies were conducted using a combination of optical microscopy and small-angle X-ray scattering (SAXS) and the addition of Tween 80 to mixtures of phytantriol and water resulted in a rich array of lyotropic mesophases. In particular, a large cubic phase region and a two-phase region of readily dispersed cubosomes is observed. Cubosomes with different concentrations of Tween 80 and phytantriol as the liquid crystal forming lipid were prepared using the solvent precursor method and their physical properties were investigated. A combination of dynamic light scattering, cryogenic electron tomography and SAXS shows formation of well-defined cubosomes with a narrow size distribution and the Im3m cubic structure. Collectively, the results confirm that Tween 80 can effectively stabilize phytantriol cubosomes, opening the possibility for future application in drug delivery across the BBB. Moreover, well-defined, homogenous cubosome formulations prepared using the mild solvent precursor dilution method has significant implications for large-scale production of cubosomes, which currently is a major barrier to the application of cubosomes in the clinic.

Direct quantitative 13 C-filtered 1 H magnetic resonance imaging of PEGylated biomacromolecules in vivo.

1 H MRI is an established diagnostic method that generally relies on detection of water. Imaging specific macromolecules is normally accomplished only indirectly through the use of paramagnetic tags, which alter the water signal in their vicinity. We demonstrate a new approach in which macromolecular constituents, such as proteins and drug delivery systems, are observed directly and quantitatively in vivo using 1 H MRI of 13 C-labeled poly(ethylene glycol) (13 C-PEG) tags. Molecular imaging of 13 C-PEG-labeled species was accomplished by incorporating a modified heteronuclear multiple quantum coherence filter into a gradient echo imaging sequence. We demonstrate the approach by monitoring the real-time distribution of 13 C-PEG and 13 C-PEGylated albumin injected into the hind leg of a mouse. Filtering the 1 H PEG signal through the directly coupled 13 C nuclei largely eliminates background water and fat signals, thus enabling the imaging of molecules using 1 H MRI. PEGylation is widely employed to enhance the performance of a multitude of macromolecular therapeutics and drug delivery systems, and 13 C-filtered 1 H MRI of 13 C-PEG thus offers the possibility of imaging and quantitating their distribution in living systems in real time. Magn Reson Med 77:1553-1561, 2017. © 2016 International Society for Magnetic Resonance in Medicine.

Local Delivery of Neurotrophin-3 and Anti-NogoA Promotes Repair After Spinal Cord Injury.

Tissue and functional repair after spinal cord injury (SCI) continue to elude researchers. Neurotrophin-3 (NT-3) and anti-NogoA have been shown to promote axonal regeneration in animal models of SCI; however, localized and sustained delivery to the central nervous system (CNS) remains a critical challenge for these and other macromolecular therapeutics. An injectable drug delivery system (DDS) has previously been developed, which can provide safe local delivery to the spinal cord. This DDS, composed of poly(lactic-co-glycolic acid) (PLGA) nanoparticles (nps) dispersed in a hyaluronan methylcellulose hydrogel, was adapted for the tunable bioactive delivery of NT-3 and anti-NogoA. Furthermore, the combined delivery of NT-3 and anti-NogoA from the DDS in an impact/compression model of SCI increases axon density and improves locomotor function. The benefits of this np/hydrogel DDS observed for NT-3 and anti-NogoA demonstrate the utility of the DDS as a local delivery strategy for protein therapeutics to the CNS.

Multifunctional Nanotherapeutics with All-in-One Nanoentrapment of Drug/Gene/Inorganic Nanoparticle.

It is challenging but imperative to merge together specific inorganic nanomaterials with macromolecular and small-molecule therapeutics into one nanoentity for all-in-one theranostic/remedy. We establish a versatile nanotechnology to nanoentrap magnetic nanoparticles, doxorubicin, and DNA, thus allowing the combination of magnetic targeting, magnetic resonance (MR) imaging, gene transport, and bioresponsive chemotherapy. We hope this nanotechnology can prompt the development of complex inorganic/organic nanosystems for various applications.

Journey to the Center of the Cell: Current Nanocarrier Design Strategies Targeting Biopharmaceuticals to the Cytoplasm and Nucleus.

New biopharmaceutical molecules, potentially able to provide more personalized and effective treatments, are being identified through the advent of advanced synthetic biology strategies, sophisticated chemical synthesis approaches, and new analytical methods to assess biological potency. However, translation of many of these structures has been significantly limited due to the need for more efficient strategies to deliver macromolecular therapeutics to desirable intracellular sites of action. Engineered nanocarriers that encapsulate peptides, proteins, or nucleic acids are generally internalized into target cells via one of several endocytic pathways. These nanostructures, entrapped within endosomes, must navigate the intracellular milieu to orchestrate delivery to the intended destination, typically the cytoplasm or nucleus. For therapeutics active in the cytoplasm, endosomal escape continues to represent a limiting step to effective treatment, since a majority of nanocarriers trapped within endosomes are ultimately marked for enzymatic degradation in lysosomes. Therapeutics active in the nucleus have the added challenges of reaching and penetrating the nuclear envelope, and nuclear delivery remains a preeminent challenge preventing clinical translation of gene therapy applications. Herein, we review cutting-edge peptide- and polymer-based design strategies with the potential to enable significant improvements in biopharmaceutical efficacy through improved intracellular targeting. These strategies often mimic the activities of pathogens, which have developed innate and highly effective mechanisms to penetrate plasma membranes and enter the nucleus of host cells. Understanding these mechanisms has enabled advances in synthetic peptide and polymer design that may ultimately improve intracellular trafficking and bioavailability, leading to increased access to new classes of biotherapeutics.

Tracking and quantifying polymer therapeutic distribution on a cellular level using 3D dSTORM

We used a single-molecule localization technique called direct stochastic optical reconstruction microscopy (dSTORM) to quantify both colocalization and spatial distribution on a cellular level for two conceptually different N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer conjugates. Microscopy images were acquired of entire cells with resolutions as high as 25nm revealing the nanoscale distribution of the fluorescently labeled therapeutic components. Drug-free macromolecular therapeutics consisting of two self-assembling nanoconjugates showed slight increase in nanoclusters on the cell surface with time. Additionally, dSTORM provided high resolution images of the nanoscale organization of the self-assembling conjugates at the interface between two cells. A conjugate designed for treating ovarian cancer showed that the model drug (Cy3) and polymer bound to Cy5 were colocalized at an early time point before the model drug was enzymatically cleaved from the polymer. Using spatial descriptive statistics it was found that the drug was randomly distributed after 24h while the polymer bound dye remained in clusters. Four different fluorescent dyes were used and two different therapeutic systems were tested to demonstrate the versatility and possible general applicability of dSTORM for use in studying drug delivery systems.

PH responsive supramolecular core-shell protein hybrids

PEGylation of proteins remains an integral part of macromolecular therapeutics due to its well-known benign effects and pharmacokinetic enhancement properties. We report herein that PEGylation can be taken to the next level of complexity and dynamic behaviour by introducing highly stable but responsive supramolecular handles. By attaching small boronic acid groups onto proteins and salicylhydroxamate moiety to end-functionalise PEG chains, we demonstrate a comprehensive study on the facile assembly/disassembly of a core-shell protein–polymer architecture using fluorescence and microscale thermophoresis on a macromolecular level. In addition, we demonstrate that both the activity and cellular transfer of functional proteins remained conserved throughout the assembly process thus establishing a rapid and orthogonal strategy towards protein PEGylation.

Organization of Endothelial Cells, Pericytes, and Astrocytes into a 3D Microfluidic in Vitro Model of the Blood-Brain Barrier.

The endothelial cells lining the capillaries supplying the brain with oxygen and nutrients form a formidable barrier known as the blood-brain barrier (BBB), which exhibits selective permeability to small drug molecules and virtually impermeable to macromolecular therapeutics. Current in vitro BBB models fail to replicate this restrictive behavior due to poor integration of the endothelial cells with supporting cells (pericytes and astrocytes) following the correct anatomical organization observed in vivo. We report the coculture of mouse brain microvascular endothelial cells (b.End3), pericytes, with/without C8-D1A astrocytes in layered microfluidic channels forming three-dimensional (3D) bi- and triculture models of the BBB. The live/dead assay indicated high viability of all cultured cells up to 21 days. Trans-endothelial electrical resistance (TEER) values confirmed the formation of intact monolayers after 3 days in culture and showed statistically higher values for the triculture model compared to the single and biculture models. Screening the permeability of [(14)C]-mannitol and [(14)C]-urea showed the ability of bi- and triculture models to discriminate between different markers based on their size. Further, permeability of [(14)C]-mannitol across the triculture model after 18 days in culture matched its reported permeability across the BBB in vivo. Mathematical calculations also showed that the radius of the tight junctions pores (R) in the triculture model is similar to the reported diameter of the BBB in vivo. Finally, both the bi- and triculture models exhibited functional expression of the P-glycoprotein efflux pump, which increased with the increase in the number of days in culture. These results collectively indicate that the triculture model is a robust in vitro model of the BBB.

Design of smart HPMA copolymer-based nanomedicines

The state-of-the art in water-soluble macromolecular therapeutics has been reviewed. First the design principles for polymer-drug conjugates are discussed followed by two recent developments in the field: a) The design, synthesis and properties of backbone degradable N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-drug conjugates. The enhanced intravascular half-life of such conjugates creates a concentration gradient (blood vs. tumor) for an extended time interval resulting in increased solid tumor accumulation by enhanced permeability and retention (EPR) effect with concomitant increase in efficacy. b) Drug-free macromolecular therapeutics is a new paradigm in macromolecular therapeutics. Apoptosis in malignant cell is induced by crosslinking of cell surface non-internalizing receptors. Crosslinking of receptors is mediated by the biorecognition of two nanoconjugates containing high-fidelity complementary motifs (peptides or oligonucleotides). Results for the treatment of B cell lymphomas in animal models and patient cells demonstrate the high translational potential of this approach.

Preclinical imaging and translational animal models of cancer for accelerated clinical implementation of nanotechnologies and macromolecular agents

The majority of animal models of cancer have performed poorly in terms of predicting clinical performance of new therapeutics, which are most often first evaluated in patients with advanced, metastatic disease. The development and use of metastatic models of cancer may enhance clinical translatability of preclinical studies focused on the development of nanotechnology-based drug delivery systems and macromolecular therapeutics, potentially accelerating their clinical implementation. It is recognized that the development and use of such models are not without challenge. Preclinical imaging tools offer a solution by allowing temporal and spatial characterization of metastatic lesions. This paper provides a review of imaging methods applicable for evaluation of novel therapeutics in clinically relevant models of advanced cancer. An overview of currently utilized models of oncology in small animals is followed by image-based development and characterization of visceral metastatic cancer models. Examples of imaging tools employed for metastatic lesion detection, evaluation of anti-tumor and anti-metastatic potential and biodistribution of novel therapies, as well as the co-development and/or use of imageable surrogates of response, are also discussed. While the focus is on development of macromolecular and nanotechnology-based therapeutics, examples with small molecules are included in some cases to illustrate concepts and approaches that can be applied in the assessment of nanotechnologies or macromolecules.

High-Pressure Transvenous Perfusion of the Upper Extremity in Human Muscular Dystrophy: A Safety Study with 0.9% Saline.

We evaluated safety and feasibility of high-pressure transvenous limb perfusion in an upper extremity of adult patients with muscular dystrophy, after completing a similar study in a lower extremity. A dose escalation study of single-limb perfusion with 0.9% saline was carried out in nine adults with muscular dystrophies under intravenous analgesia. Our study demonstrates that it is feasible and definitely safe to perform high-pressure transvenous perfusion with 0.9% saline up to 35% of limb volume in the upper extremities of young adults with muscular dystrophy. Perfusion at 40% limb volume is associated with short-lived physiological changes in peripheral nerves without clinical correlates in one subject. This study provides the basis for a phase 1/2 clinical trial using pressurized transvenous delivery into upper limbs of nonambulatory patients with Duchenne muscular dystrophy. Furthermore, our results are applicable to other conditions such as limb girdle muscular dystrophy as a method for delivering regional macromolecular therapeutics in high dose to skeletal muscles of the upper extremity.

N-trimethyl chitosan chloride-coated PLGA nanoparticles overcoming multiple barriers to oral insulin absorption.

Although several strategies have been applied for oral insulin delivery to improve insulin bioavailability, little success has been achieved. To overcome multiple barriers to oral insulin absorption simultaneously, insulin-loaded N-trimethyl chitosan chloride (TMC)-coated polylactide-co-glycoside (PLGA) nanoparticles (Ins TMC-PLGA NPs) were formulated in our study. The Ins TMC-PLGA NPs were prepared using the double-emulsion solvent evaporation method and were characterized to determine their size (247.6 ± 7.2 nm), ζ-potential (45.2 ± 4.6 mV), insulin-loading capacity (7.8 ± 0.5%) and encapsulation efficiency (47.0 ± 2.9%). The stability and insulin release of the nanoparticles in enzyme-containing simulated gastrointestinal fluids suggested that the TMC-PLGA NPs could partially protect insulin from enzymatic degradation. Compared with unmodified PLGA NPs, the positively charged TMC-PLGA NPs could improve the mucus penetration of insulin in mucus-secreting HT29-MTX cells, the cellular uptake of insulin via clathrin- or adsorption-mediated endocytosis in Caco-2 cells and the permeation of insulin across a Caco-2 cell monolayer through tight junction opening. After oral administration in mice, the TMC-PLGA NPs moved more slowly through the gastrointestinal tract compared with unmodified PLGA NPs, indicating the mucoadhesive property of the nanoparticles after TMC coating. Additionally, in pharmacological studies in diabetic rats, orally administered Ins TMC-PLGA NPs produced a stronger hypoglycemic effect, with 2-fold higher relative pharmacological availability compared with unmodified NPs. In conclusion, oral insulin absorption is improved by TMC-PLGA NPs with the multiple absorption barriers overcome simultaneously. TMC-PLGA NPs may be a promising drug delivery system for oral administration of macromolecular therapeutics.