RESUMO
Although patients generally prefer oral drug delivery to injections, low permeability of the gastrointestinal tract makes this method impossible for most biomacromolecules. One potential solution is codelivery of macromolecules, including therapeutic proteins or nucleic acids, with intestinal permeation enhancers; however, enhancer use has been limited clinically by modest efficacy and toxicity concerns surrounding long-term administration. Here, we hypothesized that plant-based foods, which are well tolerated by the gastrointestinal tract, may contain compounds that enable oral macromolecular absorption without causing adverse effects. Upon testing more than 100 fruits, vegetables, and herbs, we identified strawberry and its red pigment, pelargonidin, as potent, well-tolerated enhancers of intestinal permeability. In mice, an oral capsule formulation comprising pelargonidin and a 1 U/kg dose of insulin reduced blood glucose levels for over 4 h, with bioactivity exceeding 100% relative to subcutaneous injection. Effects were reversible within 2 h and associated with actin and tight junction rearrangement. Furthermore, daily dosing of mice with pelargonidin for 1 mo resulted in no detectable side effects, including weight loss, tissue damage, or inflammatory responses. These data suggest that pelargonidin is an exceptionally effective enhancer of oral protein uptake that may be safe for routine pharmaceutical use.
Assuntos
Antocianinas , Fragaria , Absorção Intestinal , Intestinos , Proteínas , Administração Oral , Animais , Antocianinas/química , Antocianinas/farmacologia , Fragaria/química , Insulina/administração & dosagem , Insulina/farmacocinética , Absorção Intestinal/efeitos dos fármacos , Intestinos/efeitos dos fármacos , Intestinos/metabolismo , Camundongos , Permeabilidade , Proteínas/administração & dosagem , Proteínas/farmacocinéticaRESUMO
The clinical translation of messengerRNA (mRNA) drugs has been slowed by a shortage of delivery vehicles that potently and safely shuttle mRNA into target cells. Here, we describe the properties of a particularly potent branched-tail lipid nanoparticle that delivers mRNA to >80% of three major liver cell types. We characterize mRNA delivery spatially, temporally, and as a function of injection type. Following intravenous delivery, our lipid nanoparticle induced greater protein expression than two benchmark lipids, C12-200 and DLin-MC3-DMA, at an mRNA dose of 0.5 mg/kg. Lipid nanoparticles were sufficiently potent to codeliver three distinct mRNAs (firefly luciferase, mCherry, and erythropoietin) and, separately, Cas9 mRNA and single guide RNA (sgRNA) for proof-of-concept nonviral gene editing in mice. Furthermore, our branched-tail lipid nanoparticle was neither immunogenic nor toxic to the liver. Together, these results demonstrate the unique potential of this lipid material to improve the management of diseases rooted in liver dysfunction.
Assuntos
Edição de Genes , Nanopartículas , Animais , Técnicas de Transferência de Genes , Lipídeos , Camundongos , RNA Mensageiro/genéticaRESUMO
PURPOSE: Caco-2 monolayers are the most common model of the intestinal epithelium and are critical to the development of oral drug delivery strategies and gastrointestinal disease treatments. However, current monolayer systems are cost- and/or time-intensive, hampering progress. This study evaluates two separate methods to reduce resource input: FB Essence as a fetal bovine serum (FBS) alternative and a new, 3-day Caco-2 system deemed "thrifty, rapid intestinal monolayers" (TRIM). METHODS: Caco-2 cells were cultured with FB Essence and compared to cells in 10% FBS for proliferation and monolayer formation. TRIM were compared to commonly-used 21-day and Corning® HTS monolayer systems, as well as mouse intestines, for permeability behavior, epithelial gene expression, and tight junction arrangement. RESULTS: No amount of FB Essence maintained Caco-2 cells beyond 10 passages. In contrast, TRIM compared favorably in permeability and gene expression to intestinal tissues. Furthermore, TRIM cost $109 and required 1.3 h of time per 24-well plate, compared to $164 and 3.7 h for 21-day monolayers, and $340 plus 1.0 h for the HTS system. CONCLUSIONS: TRIM offer a new approach to generating Caco-2 monolayers that resemble the intestinal epithelium. They are anticipated to accelerate the pace of in vitro intestinal experiments while easing financial burden.
Assuntos
Mucosa Intestinal/metabolismo , Administração Oral , Animais , Células CACO-2 , Proliferação de Células , Células Cultivadas , Colágeno/química , Dextranos/metabolismo , Liberação Controlada de Fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Permeabilidade , Propriedades de Superfície , Junções Íntimas/metabolismoRESUMO
PURPOSE: A major obstacle preventing oral administration of macromolecular therapeutics is poor absorption across the intestinal epithelium into the bloodstream. One strategy to improve transport across this barrier is the use of chemical permeation enhancers. Several molecular families with permeation enhancing potential have been identified previously, including piperazines. In particular, 1-phenylpiperazine has been shown to enhance transepithelial transport with minimal cytotoxicity compared to similarly effective molecules. To better understand how the chemistry of 1-phenylpiperazine affects its utility as an intestinal permeation enhancer, this study examined a small library of 13 derivatives of 1-phenylpiperazine. METHODS: The efficacy and cytotoxicity of 13 phenylpiperazine compounds were assessed in a Caco-2 model of the intestinal epithelium. Efficacy was measured using the paracellular diffusion marker calcein as well as by immunostaining and confocal imaging of Caco-2 monolayers. RESULTS: Of the 13 derivatives, two enhanced the permeability of the fluorescent marker calcein over 100-fold. It was found that hydroxyl or primary amine substitutions on the phenyl ring significantly increased toxicity, while aliphatic substitutions resulted in efficacy and toxicity profiles comparable to 1-phenylpiperazine. CONCLUSIONS: Several potent derivatives, including 1-methyl-4-phenylpiperazine and 1-(4-methylphenyl)piperazine, displayed lower toxicity than 1-phenylpiperazine, suggesting promise in future applications.
Assuntos
Absorção Intestinal/efeitos dos fármacos , Piperazinas/química , Piperazinas/farmacologia , Administração Oral , Transporte Biológico , Células CACO-2 , Sobrevivência Celular , Corantes Fluorescentes/química , Humanos , Mucosa Intestinal/metabolismo , Estrutura Molecular , Imagem Óptica/métodos , Permeabilidade , Piperazinas/metabolismo , Piperazinas/toxicidade , Relação Estrutura-AtividadeRESUMO
The oral delivery of macromolecular drugs, including proteins and nucleic acids, is one of the greatest unmet needs in modern biomedicine. Although engineering solutions have been used to overcome enzymatic degradation and the low pH in the stomach, poor absorption across the intestinal epithelium into the bloodstream continues to pose the most significant challenge to clinical translation. One common approach to increase the flux of macromolecules across the intestinal epithelium is the use of chemical permeation enhancers. Unfortunately, the vast majority of effective enhancers have been thwarted by toxicity, and the structural and molecular parameters that contribute to this behavior are poorly understood. Previous work has shown that select piperazine-derived molecules favorably affect transepithelial and intracellular delivery outcomes, suggesting that piperazine-derived molecules interface uniquely with cellular barriers. To gain a better understanding of piperazine-mediated permeation enhancement, this work examined piperazine and 13 of its simple, hydrocarbon-substituted derivatives using Caco-2 monolayers as a model of the intestinal epithelium. After evaluating each piperazine for permeation enhancement efficacy and cytotoxicity at three concentrations, it became clear that piperazine derivatives consistently enhance permeability with each derivative resulting in noncytotoxic permeation enhancement at one or more concentrations. In attempting to identify structure-function relationships for the piperazine derivatives, it was found that treatment concentration, structural characteristics, and molecular pKa were not reliable indicators of permeation potential. Interestingly, the pH of the enhancer solution was identified as a controlling parameter even when accounting for the effects from pH change alone. Specifically, piperazine treatments with a pH between 9.2 and 9.6 guaranteed noncytotoxic efficacy. Furthermore, all effective treatments resulted in pH values between 8.7 and 9.6, behavior that was not shared by the other small, noncyclic amines studied. These data have important implications in the design of oral biologic delivery systems that employ permeation enhancers and underscore the need to carefully control the final treatment pH of the local intestinal epithelial environment.
Assuntos
Permeabilidade da Membrana Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Mucosa Intestinal/efeitos dos fármacos , Piperazinas/farmacologia , Administração Oral , Transporte Biológico , Células CACO-2 , Humanos , Concentração de Íons de Hidrogênio , Mucosa Intestinal/citologia , Piperazinas/administração & dosagem , Piperazinas/químicaRESUMO
Drug-carrying nanoparticles are a promising strategy to deliver therapeutics into the brain, but their translation requires better characterization of interactions between nanomaterials and endothelial cells of the blood-brain barrier (BBB). Here, we use a library of 18 layer-by-layer electrostatically assembled nanoparticles (NPs) to independently assess the impact of NP core and surface materials on in vitro uptake, transport, and intracellular trafficking in brain endothelial cells. We demonstrate that NP core stiffness determines the magnitude of transport, while surface chemistry directs intracellular trafficking. Finally, we demonstrate that these factors similarly dictate in vivo BBB transport using intravital imaging through cranial windows in mice. We identify that hyaluronic acid surface chemistry increases transport across the BBB in vivo, and flow conditions are necessary to replicate this finding in vitro. Taken together, these findings highlight the importance of assay geometry, cell biology, and fluid flow in developing nanocarriers for delivery to the brain.
RESUMO
Although protein drugs are powerful biologic therapeutics, they cannot be delivered orally because their large size and hydrophilicity limit their absorption across the intestinal epithelium. One potential solution is the incorporation of permeation enhancers into oral protein formulations; however, few have advanced clinically due to toxicity concerns surrounding chronic use. To better understand these concerns, we conducted a 30-day longitudinal study of daily oral permeation enhancer use in mice and resultant effects on intestinal health. Specifically, we investigated three permeation enhancers: sodium caprate (C10), an industry standard, as well as 1-phenylpiperazine (PPZ) and sodium deoxycholate (SDC). Over 30 days of treatment, all mice gained weight, and none required removal from the study due to poor health. Furthermore, intestinal permeability did not increase following chronic use. We also quantified the gene expression of four tight junction proteins (claudin 2, claudin 3, ZO-1, and JAM-A). Significant differences in gene expression between untreated and permeation enhancer-treated mice were found, but these varied between treatment groups, with most differences resolving after a 1-week washout period. Immunofluorescence microscopy revealed no observable differences in protein localization or villus architecture between treated and untreated mice. Overall, PPZ and SDC performed comparably to C10, one of the most clinically advanced enhancers, and results suggest that the chronic use of some permeation enhancers may be therapeutically viable from a safety standpoint.
RESUMO
Glioblastoma is characterized by diffuse infiltration into surrounding healthy brain tissues, which makes it challenging to treat. Complete surgical resection is often impossible, and systemically delivered drugs cannot achieve adequate tumor exposure to prevent local recurrence. Convection-enhanced delivery (CED) offers a method for administering therapeutics directly into brain tumor tissue, but its impact has been limited by rapid clearance and off-target cellular uptake. Nanoparticle (NP) encapsulation presents a promising strategy for extending the retention time of locally delivered therapies while specifically targeting glioblastoma cells. However, the brain's extracellular structure poses challenges for NP distribution due to its narrow, tortuous pores and a harsh ionic environment. In this study, we investigated the impact of NP surface chemistry using layer-by-layer (LbL) assembly to design drug carriers for broad spatial distribution in brain tissue and specific glioblastoma cell targeting. We found that poly-l-glutamate and hyaluronate were effective surface chemistries for targeting glioblastoma cells in vitro. Coadsorbing either polymer with a small fraction of PEGylated polyelectrolytes improved the colloidal stability without sacrificing cancer cell selectivity. Following CED in vivo, gadolinium-functionalized LbL NPs enabled MRI visualization and exhibited a distribution volume up to three times larger than liposomes and doubled the retention half-time up to 13.5 days. Flow cytometric analysis of CED-treated murine orthotopic brain tumors indicated greater cancer cell uptake and reduced healthy cell uptake for LbL NPs compared to nonfunctionalized liposomes. The distinct cellular outcomes for different colayered LbL NPs provide opportunities to tailor this modular delivery system for various therapeutic applications.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Nanopartículas , Humanos , Camundongos , Animais , Glioblastoma/patologia , Lipossomos/metabolismo , Polímeros/metabolismo , Encéfalo/metabolismo , Nanopartículas/química , Neoplasias Encefálicas/diagnóstico por imagem , Neoplasias Encefálicas/tratamento farmacológico , Sistemas de Liberação de Medicamentos , Linhagem Celular TumoralRESUMO
To accelerate the translation of cancer nanomedicine, we used an integrated genomic approach to improve our understanding of the cellular processes that govern nanoparticle trafficking. We developed a massively parallel screen that leverages barcoded, pooled cancer cell lines annotated with multiomic data to investigate cell association patterns across a nanoparticle library spanning a range of formulations with clinical potential. We identified both materials properties and cell-intrinsic features that mediate nanoparticle-cell association. Using machine learning algorithms, we constructed genomic nanoparticle trafficking networks and identified nanoparticle-specific biomarkers. We validated one such biomarker: gene expression of SLC46A3, which inversely predicts lipid-based nanoparticle uptake in vitro and in vivo. Our work establishes the power of integrated screens for nanoparticle delivery and enables the identification and utilization of biomarkers to rationally design nanoformulations.
Assuntos
Antineoplásicos , Biomarcadores Farmacológicos , Proteínas de Transporte de Cobre , Composição de Medicamentos , Sistemas de Liberação de Fármacos por Nanopartículas , Nanopartículas , Neoplasias , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/metabolismo , Linhagem Celular Tumoral , Proteínas de Transporte de Cobre/genética , Expressão Gênica , Genômica , Humanos , Lipossomos , Camundongos , Nanomedicina , Nanopartículas/administração & dosagem , Nanopartículas/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismoRESUMO
BACKGROUND: Inflammatory bowel disease (IBD) is a family of debilitating disorders that affects more than 1 million people in the United States. Many animal studies of IBD use a dextran sulfate sodium (DSS) mouse model of colitis that induces rapid and severe colitis symptoms. Although the typical seven-day DSS model is appropriate for many studies, it destroys intestinal barrier function and results in intestinal permeability that is substantially higher than what is typically observed in patients. As such, therapies that enhance or restore barrier integrity are difficult or impossible to evaluate. METHODS: We identify administration conditions that result in more physiologically relevant intestinal damage by systematically varying the duration of DSS administration. We administered 3.0% DSS for four to seven days and assessed disease metrics including weight, fecal consistency, intestinal permeability, spleen weight, and colon length. Histology was performed to assess the structural integrity of the intestinal epithelium. RESULTS: Extended exposure (seven days) to DSS resulted in substantial, unrecoverable loss of intestinal structure and intestinal permeability increases of greater than 600-fold. Attenuated DSS administration durations (four days) produced less severe symptoms by all metrics. Intestinal permeability increased only 8-fold compared to healthy mice, better recapitulating the 2-18 fold increases in permeability observed in patients. The attenuated model retains the hallmark properties of colitis against which to compare therapeutic candidates. Our results demonstrate that an attenuated DSS colitis model obtains clinically relevant increases in intestinal permeability, enabling the effective evaluation of therapeutic candidates that promote barrier function.
RESUMO
The oral delivery of bioactive peptides and proteins is prevented by the intestinal epithelial barrier, in which intercellular tight junction complexes block the uptake of macromolecules. Here we show that anionic nanoparticles induce tight junction relaxation, increasing intestinal permeability and enabling the oral delivery of proteins. This permeation-enhancing effect is a function of nanoparticle size and charge, with smaller (≤ 200 nm) and more negative particles (such as silica) conferring enhanced permeability. In healthy mice, silica nanoparticles enabled the oral delivery of insulin and exenatide, with 10 U kg-1 orally delivered insulin sustaining hypoglycaemia for a few hours longer than a 1 U kg-1 dose of subcutaneously injected insulin. In healthy, hyperglycaemic and diabetic mice, the oral delivery of 10 U kg-1 insulin led to a dose-adjusted bioactivity of, respectively, 35%, 29% and 23% that of the subcutaneous injection of 1 U kg-1 insulin. The permeation-enhancing effect of the nanoparticles was reversible, non-toxic, and attributable to the binding to integrins on the surface of epithelial cells.
Assuntos
Permeabilidade da Membrana Celular/efeitos dos fármacos , Insulina/administração & dosagem , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Nanopartículas/administração & dosagem , Junções Íntimas/efeitos dos fármacos , Administração Oral , Animais , Linhagem Celular , Diabetes Mellitus Tipo 1/tratamento farmacológico , Sistemas de Liberação de Medicamentos , Exenatida/administração & dosagem , Camundongos Endogâmicos C57BL , Nanopartículas/química , Tamanho da Partícula , Dióxido de Silício/administração & dosagem , Junções Íntimas/metabolismoRESUMO
A major obstacle for topical and enteral drug delivery is the poor transport of macromolecular drugs through the epithelium. One potential solution is the use of permeation enhancers that alter epithelial structures. Piperazine derivatives are known permeation enhancers that modulate epithelial structures, reduce transepithelial electrical resistance, and augment the absorption of macromolecular drugs. The mechanism by which piperazine derivatives disrupt the structures of epithelial monolayers is not well understood. Here, the effects of 1-phenylpiperazine and 1-methyl-4-phenylpiperazine are modeled in the epithelial cell line NRK-52E. Live-cell imaging reveals a dose-dependent gross reorganization of monolayers at high concentrations, but reorganization differs based on the piperazine molecule. Results show that low concentrations of piperazine derivatives increase myosin force generation within the cells and do not disrupt the cytoskeletal structure. Also, cytoskeletally attached cadherin junctions are disrupted before tight junctions. In summary, piperazines appear to increase myosin-mediated contraction followed by disruption of cell-cell contacts. These results provide new mechanistic insight into how transient epithelial permeation enhancers act and will inform of the development of future generations of transepithelial delivery systems.
Assuntos
Caderinas , Preparações Farmacêuticas , Piperazinas , Células CACO-2 , Caderinas/genética , Células Epiteliais , Humanos , Permeabilidade , Piperazinas/farmacologiaRESUMO
Oral drug delivery is a preferred administration route due to its low cost, high patient compliance and fewer adverse events compared to intravenous administration. However, many pharmaceuticals suffer from poor solubility and low oral bioavailability. One major factor that contributes to low bioavailability are efflux transporters which prevent drug absorption through intestinal epithelial cells. P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP) are two important efflux transporters in the intestine functioning to prevent toxic materials from entering systemic circulation. However, due to its broad substrate specificity, P-gp limits the absorption of many therapeutics, including chemotherapeutics and antibacterial agents. Methods to inhibit P-gp with competitive inhibitors have not been clinically successful. Here, we show that micron scale devices (microdevices) made from a commonly used biomaterial, polyethylene glycol (PEG), inhibit P-gp through a biosimilar mucus in Caco-2 cells and that transporter function is restored when the microdevices are removed. Microdevices were shown to inhibit P-gp mediated transport of calcein AM, doxorubicin, and rhodamine 123 (R123) and BCRP mediated transport of BODIPY-FL-prazosin. When in contact with Caco-2 cells, microdevices decrease the cell surface amount of P-gp without affecting the passive transport. Moreover, there was an increase in mucosal to serosal transport of R123 with microdevices in an ex-vivo mouse model and increased absorption in vivo. This biomaterial-based approach to inhibit efflux transporters can be applied to a range of drug delivery systems and allows for a nonpharmacologic method to increase intestinal drug absorption while limiting toxic effects.