Intestinal permeation enhancers enable oral delivery of macromolecules up to 70 kDa in size.

The decades-long effort to deliver peptide drugs orally has resulted in several clinically successful formulations. These formulations are enabled by the inclusion of permeation enhancers that facilitate the intestinal absorption of peptides. Thus far, these oral peptide drugs have been limited to peptides less than 5 kDa, and it is unclear whether there is an upper bound of protein size that can be delivered with permeation enhancers. In this work, we examined two permeation enhancers, 1-phenylpiperazine (PPZ) and sodium deoxycholate (SDC), for their ability to increase intestinal transport of a model macromolecule (FITC-Dextran) as a function of its size. Specifically, the permeability of dextrans with molecular weights of 4, 10, 40, and 70 kDa was assessed in an in vitro and in vivo model of the intestine. In Caco-2 monolayers, both PPZ and SDC significantly increased the permeability of only FD4 and FD10. However, in mice, PPZ and SDC behaved differently. While SDC improved the absorption of all tested sizes of dextrans, PPZ was effective only for FD4 and FD10. This work is the first report of PPZ as a permeation enhancer in vivo, and it highlights the ability of permeation enhancers to improve the absorption of macromolecules across a broad range of sizes relevant for protein drugs.

Controlling the semi-permeability of protein nanocapsules influences the cellular response to macromolecular payloads.

Nanocapsules are an excellent platform for the delivery of macromolecular payloads such as proteins, nucleic acids or polyprodrugs, since they can both protect the sensitive cargo and target its delivery to the desired site of action. However, the release of macromolecules from nanocapsules remains a challenge due to their restricted diffusion through the nanoshell compared to small molecule cargo. Here, we designed degradable protein nanocapsules with varying crosslinking densities of the nanoshell to control the release of model macromolecules. While the crosslinking did not influence the degradability of the capsules by natural proteases, it significantly affected the release profiles. Furthermore, the optimized protein nanocapsules were successfully used to deliver and effectively release a bioactive macromolecular vaccine adjuvant in vitro and, thus, can be used as an efficient platform for the design of potential nanovaccines.

Injectable Magnetic-Responsive Short-Peptide Supramolecular Hydrogels: Ex Vivo and In Vivo Evaluation.

The inclusion of magnetic nanoparticles (MNP) in a hydrogel matrix to produce magnetic hydrogels has broadened the scope of these materials in biomedical research. Embedded MNP offer the possibility to modulate the physical properties of the hydrogel remotely and on demand by applying an external magnetic field. Moreover, they enable permanent changes in the mechanical properties of the hydrogel, as well as alterations in the micro- and macroporosity of its three-dimensional (3D) structure, with the associated potential to induce anisotropy. In this work, the behavior of biocompatible and biodegradable hydrogels made with Fmoc-diphenylalanine (Fmoc-FF) (Fmoc = fluorenylmethoxycarbonyl) and Fmoc-arginine-glycine-aspartic acid (Fmoc-RGD) short peptides to which MNP were incorporated was studied in detail with physicochemical, mechanical, and biological methods. The resulting hybrid hydrogels showed enhance mechanical properties and withstood injection without phase disruption. In mice, the hydrogels showed faster and improved self-healing properties compared to their nonmagnetic counterparts. Thanks to these superior physical properties and stability during culture, they can be used as 3D scaffolds for cell growth. Additionally, magnetic short-peptide hydrogels showed good biocompatibility and the absence of toxicity, which together with their enhanced mechanical stability and excellent injectability make them ideal biomaterials for in vivo biomedical applications with minimally invasive surgery. This study presents a new approach to improving the physical and mechanical properties of supramolecular hydrogels by incorporating MNP, which confer structural reinforcement and stability, remote actuation by magnetic fields, and better injectability. Our approach is a potential catalyst for expanding the biomedical applications of supramolecular short-peptide hydrogels.

Glycomacromolecules: Addressing challenges in drug delivery and therapeutic development.

Carbohydrate-based materials offer exciting opportunities for drug delivery. They present readily available, biocompatible components for the construction of macromolecular systems which can be loaded with cargo, and can enable targeting of a payload to particular cell types through carbohydrate recognition events established in biological systems. These systems can additionally be engineered to respond to environmental stimuli, enabling triggered release of payload, to encompass multiple modes of therapeutic action, or to simultaneously fulfil a secondary function such as enabling imaging of target tissue. Here, we will explore the use of glycomacromolecules to deliver therapeutic benefits to address key health challenges, and suggest future directions for development of next-generation systems.

A Macromolecular Drug for Cancer Therapy via Extracellular Calcification.

Cancer chemotherapy typically relies on drug endocytosis and inhibits tumor cell proliferation via intracellular pathways; however, severe side effects may arise. In this study, we performed a first attempt to develop macromolecular-induced extracellular chemotherapy involving biomineralization by absorbing calcium from the blood through a new type of drug, polysialic acid conjugated with folate (folate-polySia), which selectively induces biogenic mineral formation on tumor cells and results in the pathological calcification of tumors. The macromolecule-initiated extracellular calcification causes cancer cell death mainly by intervening with the glycolysis process in cancer cells. Systemic administration of folate-polySia inhibited cervical and breast tumor growth and dramatically improved survival rates in mice. This study provides an extracellular therapeutic approach for malignant tumor diseases via calcification that is ready for clinical trials and offers new insights into macromolecular anticancer drug discovery.

Optimizing synthetic nucleic acid and protein nanocarriers: The chemical evolution approach.

Optimizing synthetic nanocarriers is like searching for a needle in a haystack. How to find the most suitable carrier for intracellular delivery of a specified macromolecular nanoagent for a given disease target location? Here, we review different synthetic 'chemical evolution' strategies that have been pursued. Libraries of nanocarriers have been generated either by unbiased combinatorial chemistry or by variation and novel combination of known functional delivery elements. As in natural evolution, definition of nanocarriers as sequences, as barcode or design principle, may fuel chemical evolution. Screening in appropriate test system may not only provide delivery candidates, but also a refined understanding of cellular delivery including novel, unpredictable mechanisms. Combined with rational design and computational algorithms, candidates can be further optimized in subsequent evolution cycles into nanocarriers with improved safety and efficacy. Optimization of nanocarriers differs for various cargos, as illustrated for plasmid DNA, siRNA, mRNA, proteins, or genome-editing nucleases.

Influence of Bile Composition on Membrane Incorporation of Transient Permeability Enhancers.

Transient permeability enhancers (PEs), such as caprylate, caprate, and salcaprozate sodium (SNAC), improve the bioavailability of poorly permeable macromolecular drugs. However, the effects are variable across individuals and classes of macromolecular drugs and biologics. Here, we examined the influence of bile compositions on the ability of membrane incorporation of three transient PEs-caprylate, caprate, and SNAC-using coarse-grained molecular dynamics (CG-MD). The availability of free PE monomers, which are important near the absorption site, to become incorporated into the membrane was higher in fasted-state fluids than that in fed-state fluids. The simulations also showed that transmembrane perturbation, i.e., insertion of PEs into the membrane, is a key mechanism by which caprylate and caprate increase permeability. In contrast, SNAC was mainly adsorbed onto the membrane surface, indicating a different mode of action. Membrane incorporation of caprylate and caprate was also influenced by bile composition, with more incorporation into fasted- than fed-state fluids. The simulations of transient PE interaction with membranes were further evaluated using two experimental techniques: the quartz crystal microbalance with dissipation technique and total internal reflection fluorescence microscopy. The experimental results were in good agreement with the computational simulations. Finally, the kinetics of membrane insertion was studied with CG-MD. Variation in micelle composition affected the insertion rates of caprate monomer insertion and expulsion from the micelle surface. In conclusion, this study suggests that the bile composition and the luminal composition of the intestinal fluid are important factors contributing to the interindividual variability in the absorption of macromolecular drugs administered with transient PEs.

Polysaccharide nanoparticles for oral controlled drug delivery: the role of drug-polymer and interpolymer interactions.

Introduction: The oral route still represents the most popular way of administering drugs; nowadays oral administration faces new challenges, in particular with regards to the delivery of APIs that are poorly absorbed and sensitive to degradation such as macromolecules and biotechnological drugs. Nanoparticles are promising tools for the efficient delivery of these drugs to the gastrointestinal tract. Areas covered:Approaches and techniques for the formulation of drugs, with particular focus on the preparation of polysaccharide nanoparticles obtained by non-covalent interactions. Expert opinion:Polysaccharide-based nanoparticulate systems offer the opportunity to address some of the issues posed by biotechnological drugs, as well as by small molecules, with problems of stability/intestinal absorption, by exploiting the capability of the polymer to establish non-covalent bonds with functional groups in the chemical structure of the API. This area of research will continue to grow, provided that these drug delivery technologies will efficaciously be translated into systems that can be manufactured on a large scale under GMP conditions. Industrial scale-up represents the biggest obstacle to overcome in view of the transformation of very promising results obtained on lab scale into medicinal products. To do that, an effort toward the simplification of the process and technologies is necessary.

Role of pharmacokinetic consideration for the development of drug delivery systems: A historical overview.

Drug delivery system is defined as a system or technology to achieve optimum therapeutic effects of drugs through precise control of their movements in the body. In order to optimize function of drug delivery systems aiming at targeting, their whole-body distribution profiles should be systematically evaluated and analyzed, where pharmacokinetic analysis based on the clearance concepts plays important role. Organ perfusion experiments combined with statistical moment analysis further supply detailed information on drug disposition at organ and cellular levels. Based on general relationship between physicochemical properties and distribution profile, macromolecular prodrugs or polymer conjugates of proteins are rationally designed and further introduction of ligand structure brings cell-specific delivery for them. These approaches are also applicable for particulate carriers such as liposomes and offer various opportunities for biological drugs such as nucleic acid drugs for their delivery. Mechanistic approach for dermal absorption analysis based on physiological skin model offers another opportunity in rational design of drug delivery. Potential of drug delivery technology in future medicines such as cell therapy and nanomaterial platform application is further discussed in relation to pharmacokinetic consideration.

[Drug Delivery to Various Body Organs via Non-blood Circulatory Pathway].

Delivery of nucleic acid therapeutics to target body organs requires injection of nanocarriers into the bloodstream. However, as such nanocarriers would also be delivered to non-target organs, low delivery efficiency to target organs and risk of unexpected effects are clear limitations of this technology. We recently applied iontophoresis (IP) for direct delivery of nucleic acid therapeutics to various organs. IP relies on a weak electric current for noninvasive transdermal drug delivery. We found that IP can deliver hydrophilic macromolecules and nanoparticles into the skin. We previously succeeded in transdermal delivery of siRNA, and subsequent knockdown (70%) of target mRNA levels in the skin via IP of siRNA (Int. J. Pharm., 383, 2010, Kigasawa et al.). Moreover, we found that cell signal activation and cleavage of intercellular junctions are induced by IP (J. Biol. Chem., 289, 2014, Hama et al.). We hypothesized that this phenomenon should be observed in not only skin but also other organs, and subsequently carried out IP of nucleic acid therapeutics to various body organs including liver, pancreas and kidney. This technique resulted in delivery of nucleic acid therapeutics into the various target body organs, and subsequent knockdown of target genes. These results suggest that direct delivery to target body organs via non-blood circulatory pathway is possible. This technology may offer a solution to the various limitations associated with current drug delivery systems (DDS).

Influence of molecular weight on transdermal delivery of model macromolecules using hydrogel-forming microneedles: potential to enhance the administration of novel low molecular weight biotherapeutics.

With a view to improve the current monoclonal antibody-based therapies dominating the pharmaceutical market, low molecular weight (MW) protein-based macromolecules, such as recombinant antibody fragments, typically within the range of 10-70 kDa, have been developed. Previously, our group successfully delivered Avastin®, a monoclonal antibody (mAb) across the skin using hydrogel-forming microneedles (MN). However, it is thought that this delivery system can be further enhanced using novel, lower MW biomolecules. To address this perception, in the current study, FITC-dextran of different MWs (10, 70 and 150 kDa) was used to model the transdermal delivery of low MW biotherapeutics and mAbs with MWs of approximately 150 kDa. Conversely, fluorescein sodium was the compound selected to model hydrophilic, low MW drugs. As expected, fluorescein sodium produced the greatest cumulative permeation (637.4 ± 42.69 µg). The amounts of FITC-dextran 10 kDa and 150 kDa which permeated across neonatal porcine skin in vitro were 462.17 ± 65.85 µg and 213.54 ± 15.19 µg after 24 h, respectively. The results collated here suggest that the delivery of emerging novel biotherapeutics, via'super swelling' hydrogel-forming MNs, have the potential to result in greater permeation across human skin, compared to the delivery of mAbs delivered via the same route.

Transscleral Iontophoresis for Noninvasive Ocular Drug Delivery of Macromolecules.

Purpose: The objectives were to investigate the effect of transscleral iontophoresis of macromolecules in vitro and in vivo, to study the importance of electroosmosis on macromolecules of low charge to mass ratio, and to evaluate transscleral iontophoresis efficacy in a choroidal neovascularization (CNV) animal model. Methods: Through in vitro transport experiments, the permeability coefficients of macromolecules [eg, immunoglobulin G (IgG), dextran 70 kDa] were determined under different conditions. The effect of ionic strength formulations and iontophoretic conditions was studied on the distribution of IgG and bevacizumab into the eye in vivo. Magnetic resonance imaging (MRI) was utilized to evaluate in vivo real time distribution of gadolinium-labeled albumin (Galbumin) following iontophoresis. The efficacy between no treatment, intravitreal injection (IVT), and iontophoresis of bevacizumab on a CNV model of subretinal injection of adeno-associated virus encoding human VEGF-165 was investigated. Results: The permeability data suggested a significant effect of ionic strength on the iontophoretic transport of macromolecules. Transscleral iontophoresis of IgG at 4 mA with a low ionic strength formulation was about 600 times greater than passive diffusion and 14-fold over a conventional formulation in vitro. Approximately 0.6 mg of bevacizumab can be delivered into the rabbit eye in vivo with a 20-min treatment of iontophoresis. MRI showed that Galbumin was in the posterior tissues after iontophoresis. In the CNV model, the iontophoresis and IVT methods of bevacizumab delayed retinal neovascularization by 4 and 8 weeks, respectively. Conclusions: Transscleral iontophoresis is capable of delivering macromolecule drugs through the conjunctiva and sclera, eventually exposing the retina/choroid to the drugs.

Policaptil Gel Retard Intake Reduces Postprandial Triglycerides, Ghrelin and Appetite in Obese Children: A Clinical Trial.

The aim of this study is to test the hypothesis that the intake of Policaptil Gel Retard® (PGR) is able to affect appetite, metabolic and hormonal postprandial profile in obese children. 46 obese children were randomly assigned to treatment with PGR or placebo, in a double blind clinical trial. Two PGR tablets or placebo were given in fasting condition, before the ingestion of a mixed meal (15 kcal/kg lean body mass). Blood samples were taken at baseline and for 4 hours, for measuring blood lipids, glucose, insulin, ghrelin, and glucagon like peptide-1 (GLP-1). Appetite was quantified using a visual analog scale. Children assuming PGR had a significantly lower increase of postprandial triglycerides (area under the curve (AUC): 3021 (2879) vs. 5038 (3738) mg × 240 min/Dl) and appetite (-234 (274) vs. 36 (329)) than children assuming placebo. The AUC of ghrelin was significantly lower after PGR ingestion, than after placebo (-8179 (8073) vs. -2800 (7579) pg × 240 min/mL). Blood glucose, insulin, non-esterified fatty acids (NEFA) and GLP-1 profiles were not significantly different in the two groups. In conclusion, a single intake of two tablets of PGR was associated with a significant reduction of appetite, ghrelin, and triglycerides in the postprandial period in obese children. Further investigation will assess if a chronic intake of PGR may affect body weight and glucose metabolism.

Distribution and Diffusion of Macromolecule Delivery to the Brain via Focused Ultrasound using Magnetic Resonance and Multispectral Fluorescence Imaging.

Focused ultrasound (FUS), in combination with microbubble contrast agents, can be used to transiently open the blood-brain barrier (BBB) to allow intravascular agents to cross into the brain. Often, FUS is carried out in conjunction with magnetic resonance imaging (MRI) to evaluate BBB opening to gadolinium-based MRI contrast agents. Although MRI allows direct visualization of the distribution of gadolinium-based contrast agents in the brain parenchyma, it does not allow measurements of the distribution of other molecules crossing the BBB. Therapeutic molecules (e.g., monoclonal antibodies) are much different in size than MRI contrast agents and have been found to have different distributions in the brain after FUS-mediated BBB opening. In the work described here, we combined in vivo MRI and ex vivo multispectral fluorescence imaging to compare the distributions of MRI contrast and dextran molecules of different molecular weights (3, 70 and 500 kDa) after FUS-mediated BBB opening through a range of ultrasound pressures (0.18-0.46 MPa) in laboratory mice. The volume of brain exposed was calculated from the MRI and fluorescence images and was significantly dependent on both molecular weight and ultrasound pressure. Diffusion coefficients of the different-molecular-weight dextran molecules in the brain parenchyma were also calculated from the fluorescence images and were negatively correlated with the molecular weight of the dextran molecules. The results of this work build on a body of knowledge that is critically important for the FUS technique to be used in clinical delivery of therapeutics to the brain.

A Renal-Clearable Macromolecular Reporter for Near-Infrared Fluorescence Imaging of Bladder Cancer.

Bladder cancer (BC) is a prevalent disease with high morbidity and mortality; however, in vivo optical imaging of BC remains challenging because of the lack of cancer-specific optical agents with high renal clearance. Herein, a macromolecular reporter (CyP1) was synthesized for real-time near-infrared fluorescence (NIRF) imaging and urinalysis of BC in living mice. Because of the high renal clearance (ca. 94 % of the injection dosage at 24 h post-injection) and its cancer biomarker (APN=aminopeptidase N) specificity, CyP1 can be efficiently transported to the bladder and specially turn on its NIRF signal to report the detection of BC in living mice. Moreover, CyP1 can be used for optical urinalysis, permitting the ex vivo tracking of tumor progression for therapeutic evaluation and easy translation of CyP2 as an in vitro diagnostic assay. This study not only provides new opportunities for non-invasive diagnosis of BC, but also reveals useful guidelines for the development of molecular reporters for the detection of bladder diseases.

An anionic, endosome-escaping polymer to potentiate intracellular delivery of cationic peptides, biomacromolecules, and nanoparticles.

Peptides and biologics provide unique opportunities to modulate intracellular targets not druggable by conventional small molecules. Most peptides and biologics are fused with cationic uptake moieties or formulated into nanoparticles to facilitate delivery, but these systems typically lack potency due to low uptake and/or entrapment and degradation in endolysosomal compartments. Because most delivery reagents comprise cationic lipids or polymers, there is a lack of reagents specifically optimized to deliver cationic cargo. Herein, we demonstrate the utility of the cytocompatible polymer poly(propylacrylic acid) (PPAA) to potentiate intracellular delivery of cationic biomacromolecules and nano-formulations. This approach demonstrates superior efficacy over all marketed peptide delivery reagents and enhances delivery of nucleic acids and gene editing ribonucleoproteins (RNPs) formulated with both commercially-available and our own custom-synthesized cationic polymer delivery reagents. These results demonstrate the broad potential of PPAA to serve as a platform reagent for the intracellular delivery of cationic cargo.

Enzyme-Assisted Photoinjection of Megadalton Molecules into Intact Plant Cells Using Femtosecond Laser Amplifier.

Femtosecond laser photoporation has become a popular method to deliver various kinds of molecules such as genes, proteins, and fluorescent dyes into single mammalian cells. However, this method is not easily applied to plant cells because their cell wall and turgor pressure prevent the delivery, especially for larger molecules than the mesh size of the cell wall. This work is the first demonstration of the efficient photoinjection of megadalton molecules into a cytoplasm of an intact single plant cell by employing a femtosecond laser amplifier under moderate enzyme treatment conditions. The intense femtosecond laser pulse effectively formed a pore on the cell wall and membrane of Tobacco BY-2, and 2 MDa dextran molecules were introduced through the pore. Along with the pore formation, induced mechanical tensile stresses on BY-2 cells were considered to increase permeability of the cell membrane and enhance the uptake of large molecules. Moreover, the moderate enzyme treatment partially degraded the cell wall thereby facilitating the increase of the molecular introduction efficiency.

Interface-Enrichment-Induced Instability and Drug-Loading-Enhanced Stability in Inhalable Delivery of Supramolecular Filaments.

Filamentous microorganisms traveling in aerosol particles display enhanced deposition and retention in the lungs. Inspired by this shape-related biological effect, we report here on the use of supramolecular filaments as potential inhalable drug carriers within aerosols via jet nebulization. We found that the peptide design and supramolecular stability play a crucial role in the interfacial stability and aerosolization properties of the supramolecular filaments. Monomeric units with a positively charged C-terminus produced filaments with reduced aerosol stability, promoting morphological changes after nebulization. Conversely, having a neutral or negatively charged terminus yielded filaments with enhanced stability, where supramolecular integrity is maintained with only reduced length. Our results suggest that molecular enrichment at the air-liquid interface during nebulization is the primary factor to deplete the monomeric peptide amphiphiles in solution, accounting for the observed morphological disruption/transitions. Importantly, encapsulation of drugs and dyes within filaments notably stabilize their supramolecular structure during nebulization, and the loaded filaments exhibit a linear release profile from a nebulizer device. We envision the use of this supramolecular carrier system as an effective platform for the inhalation-based treatment of many lung diseases.

Nanostructured supramolecular hydrogels: Towards the topical treatment of Psoriasis and other skin diseases.

Supramolecular hydrogels were synthesized using a bis-imidazolium based amphiphile, and incorporating chemically diverse drugs, such as the cytostatics gemcitabine hydrochloride and methotrexate sodium salt, the immunosuppressive drug tacrolimus, as well as the corticoid drugs betamethasone 17-valerate and triamcinolone acetonide, and their potential as drug delivery agents in the dermal treatment of Psoriasis was evaluated. The rheological behavior of gels was studied, showing in all cases suitable viscoelastic properties for topical drug delivery. Scanning electron microscopy (SEM) shows that the drugs included have a great influence on the gel morphology at the microscopic level, as the incorporation of gemcitabine hydrochloride leads to slightly thicker fibers, the incorporation of tacrolimus induces flocculation and spherical precipitates, and the incorporation of methotrexate forms curled fibers. 1H NMR spectroscopy experiments show that these drugs not only remain dissolved at the interstitial space, but up to 72% of either gemcitabine or methotrexate, and up to 38% of tacrolimus, is retained within the gel fibers in gels formed with a 1:1 gelator:drug molar ratio. This unique fiber incorporation not only protects the drug from degradation, but also importantly induces a Two Phase Exponential drug release, where the first phase corresponds to the drug dissolved in the interstitial space, while the second phase corresponds to the drug exiting from the gel fibers, and where the speed in each phase is in accordance with the physicochemical properties of the drugs, opening perspectives for controlled delivery. Skin permeation ex vivo tests show how these gels successfully promote the drug permeation and retention inside the skin for reaching their therapeutic target, while in vivo experiments demonstrate that they decrease the hyperplasia and reduce the macroscopic tissue damage typically observed in psoriatic skin, significantly more than the drugs in solution. All these characteristics, beside the spontaneous and easy preparation (room temperature and soft stirring), make these gels a good alternative to other routes of administration for Psoriasis treatment, increasing the drug concentration at the target tissue, and minimizing side effects.