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Accurate analysis of blood concentration and circulation half-life is an important consideration for any intravenously administered agent in preclinical development or for therapeutic application. However, the currently available tools to measure these parameters are laborious, expensive, and inefficient for handling multiple samples from complex multivariable experiments. Here we describe a robust high-throughput quantitative microscopy-based method to measure the blood concentration and circulation half-life of any fluorescently labeled agent using only a small (2 µL) amount of blood volume, enabling additional end-point measurements to be assessed in the same subject. To validate this method, we demonstrate its use to measure the circulation half-life in mice of two types of fluorescently labeled polymeric nanoparticles of different sizes and surface chemistries and of a much smaller fluorescently labeled monoclonal antibody. Furthermore, we demonstrate the improved accuracy of this method compared to previously described methods.
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Monitoramento de Medicamentos/métodos , Ensaios de Triagem em Larga Escala/métodos , Microscopia/métodos , Preparações Farmacêuticas/administração & dosagem , Preparações Farmacêuticas/química , Animais , Feminino , Meia-Vida , Humanos , Injeções Intravenosas , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Nanopartículas/químicaRESUMO
This Review examines the state-of-the-art in the delivery of nucleic acid therapies that are directed to the vascular endothelium. First, we review the most important homeostatic functions and properties of the vascular endothelium and summarize the nucleic acid tools that are currently available for gene therapy and nucleic acid delivery. Second, we consider the opportunities available with the endothelium as a therapeutic target and the experimental models that exist to evaluate the potential of those opportunities. Finally, we review the progress to date from investigations that are directly targeting the vascular endothelium: for vascular disease, for peri-transplant therapy, for angiogenic therapies, for pulmonary endothelial disease, and for the blood-brain barrier, ending with a summary of the future outlook in this field.
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Ácidos Nucleicos , Ácidos Nucleicos/genética , Endotélio Vascular , Barreira Hematoencefálica , Terapia Genética , Transporte BiológicoRESUMO
Gene therapy promises to treat diseases that arise from genetic abnormalities by correcting the underlying cause of the disease rather than treating the associated symptoms. Successful transfer of nucleic acids into cells requires efficient delivery vehicles that protect the cargo and can penetrate the appropriate cellular barriers before releasing their contents. Many viral vectors and synthetic polycationic vectors for nucleic acid delivery do not translate well from in vitro to in vivo applications due to their instability and toxicity. We synthesized and characterized a library of biocompatible low charge density polymers from a family of poly(amine- co-ester) (PACE) terpolymers produced via enzyme catalyzed polymerization. PACE polymers are highly customizable; we found that the terpolymer composition can be optimized to produce efficient transfection of various nucleic acids-including DNA plasmids, mRNA, and siRNA-in specific cell types with low toxicity. Our findings suggest that the unique tunability of PACEs offers new tools for gene therapy and other biomedical applications.
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Técnicas de Transferência de Genes , Nanopartículas/química , 3,4-Metilenodioxianfetamina/análogos & derivados , 3,4-Metilenodioxianfetamina/química , Células 3T3 , Animais , Ácidos Decanoicos/química , Ácidos Dicarboxílicos/química , Ésteres/química , Células HEK293 , Humanos , Macrolídeos/química , Camundongos , Poliaminas/química , PolimerizaçãoRESUMO
Aligned fibers of extracellular matrix (ECM) affect the direction, efficiency, and persistence of migrating cells. To uncover the mechanisms by which multicellular tissues align their surrounding ECM before migration, we used an engineered three-dimensional culture model to investigate the dynamics of ECM alignment around tissues of defined geometry. Analysis of ECM alignment over time revealed that tissues rapidly reorganize their surrounding matrix, with a characteristic time that depends on the type of cell and the initial tissue geometry. We found that matrix metalloproteinase activity is not required for matrix alignment before cell migration. Instead, alignment is driven by Rho-mediated cytoskeletal contractility and accelerated by propagation of tension through intercellular adhesions. Our data suggest that multicellular tissues align their surrounding matrix by pulling collectively to exert strain, which is primarily a physical process. Consistently, the pattern of matrix alignment depends on tissue geometry and the resulting distribution of mechanical strain, with asymmetric tissues generating a higher degree of matrix alignment along their longest axes. The rapid ability of multicellular tissues to physically remodel their matrix enables their constituent cells to migrate efficiently along aligned fibers and to quickly change their direction according to other microenvironmental cues, which is important for both normal and disease processes.
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Matriz Extracelular/metabolismo , Modelos Biológicos , Animais , Linhagem Celular Tumoral , Movimento Celular , Citoesqueleto/metabolismo , Metaloproteinases da Matriz/metabolismo , Camundongos , Invasividade NeoplásicaRESUMO
BACKGROUND: Cystic Fibrosis (CF) is an autosomal recessive genetic disease caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein for which there is no cure. One approach to cure CF is to correct the underlying mutations in the CFTR gene. We have used triplex-forming peptide nucleic acids (PNAs) loaded into biodegradable nanoparticles (NPs) in combination with donor DNAs as reagents for correcting mutations associated with genetic diseases including CF. Previously, we demonstrated that PNAs induce recombination between a donor DNA and the CFTR gene, correcting the F508del CFTR mutation in human cystic fibrosis bronchial epithelial cells (CFBE cells) and in a CF murine model leading to improved CFTR function with low off-target effects, however the level of correction was still below the threshold for therapeutic cure. METHODS: Here, we report the use of next generation, chemically modified gamma PNAs (γPNAs) containing a diethylene glycol substitution at the gamma position for enhanced DNA binding. These modified γPNAs yield enhanced gene correction of F508del mutation in human bronchial epithelial cells (CFBE cells) and in primary nasal epithelial cells from CF mice (NECF cells). RESULTS: Treatment of CFBE cells and NECF cells grown at air-liquid interface (ALI) by NPs containing γtcPNAs and donor DNA resulted in increased CFTR function measured by short circuit current and improved gene editing (up to 32 %) on analysis of genomic DNA. CONCLUSIONS: These findings provide the basis for further development of PNA and NP technology for editing of the CFTR gene.
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The in vivo efficacy of polymeric nanoparticles (NPs) is dependent on their pharmacokinetics, including time in circulation and tissue tropism. Here we explore the structure-function relationships guiding physiological fate of a library of poly(amine-co-ester) (PACE) NPs with different compositions and surface properties. We find that circulation half-life as well as tissue and cell-type tropism is dependent on polymer chemistry, vehicle characteristics, dosing, and strategic co-administration of distribution modifiers, suggesting that physiological fate can be optimized by adjusting these parameters. Our high-throughput quantitative microscopy-based platform to measure the concentration of nanomedicines in the blood combined with detailed biodistribution assessments and pharmacokinetic modeling provides valuable insight into the dynamic in vivo behavior of these polymer NPs. Our results suggest that PACE NPs-and perhaps other NPs-can be designed with tunable properties to achieve desired tissue tropism for the in vivo delivery of nucleic acid therapeutics. These findings can guide the rational design of more effective nucleic acid delivery vehicles for in vivo applications.
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Macrófagos , Nanopartículas , Polímeros , Animais , Nanopartículas/química , Distribuição Tecidual , Camundongos , Polímeros/química , Macrófagos/metabolismo , Humanos , Feminino , Sistemas de Liberação de Medicamentos , Camundongos Endogâmicos C57BLRESUMO
In utero gene editing has the potential to modify disease causing genes in multiple developing tissues before birth, possibly allowing for normal organ development, disease improvement, and conceivably, cure. In cystic fibrosis (CF), a disease that arises from mutations in the cystic fibrosis transmembrane conductance regulator ( CFTR ) gene, there are signs of multiorgan disease affecting the function of the respiratory, gastrointestinal, and reproductive systems already present at birth. Thus, treating CF patients early is crucial for preventing or delaying irreversible organ damage. Here we demonstrate proof-of-concept of multiorgan mutation correction in CF using peptide nucleic acids (PNAs) encapsulated in polymeric nanoparticles and delivered systemically in utero. In utero editing was associated with sustained postnatal CFTR activity, at a level similar to that of wild-type mice, in both respiratory and gastrointestinal tissue, without detection of off-target mutations in partially homologous loci. This work suggests that systemic in utero gene editing represents a viable strategy for treating monogenic diseases before birth that impact multiple tissue types.
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Polymeric nanoparticles are highly tunable drug delivery systems that show promise in targeting therapeutics to specific sites within the body. Rational nanoparticle design can make use of mathematical models to organize and extend experimental data, allowing for optimization of nanoparticles for particular drug delivery applications. While rational nanoparticle design is attractive from the standpoint of improving therapy and reducing unnecessary experiments, it has yet to be fully realized. The difficulty lies in the complexity of nanoparticle structure and behavior, which is added to the complexity of the physiological mechanisms involved in nanoparticle distribution throughout the body. In this review, we discuss the most important aspects of rational design of polymeric nanoparticles. Ultimately, we conclude that many experimental datasets are required to fully model polymeric nanoparticle behavior at multiple scales. Further, we suggest ways to consider the limitations and uncertainty of experimental data in creating nanoparticle design optimization schema, which we call quantitative nanoparticle design frameworks.
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Modelos Teóricos , Nanopartículas , Sistemas de Liberação de Medicamentos , PolímerosRESUMO
An inhalable platform for messenger RNA (mRNA) therapeutics would enable minimally invasive and lung-targeted delivery for a host of pulmonary diseases. Development of lung-targeted mRNA therapeutics has been limited by poor transfection efficiency and risk of vehicle-induced pathology. Here, we report an inhalable polymer-based vehicle for delivery of therapeutic mRNAs to the lung. We optimized biodegradable poly(amine-co-ester) (PACE) polyplexes for mRNA delivery using end-group modifications and polyethylene glycol. These polyplexes achieved high transfection of mRNA throughout the lung, particularly in epithelial and antigen-presenting cells. We applied this technology to develop a mucosal vaccine for severe acute respiratory syndrome coronavirus 2 and found that intranasal vaccination with spike protein-encoding mRNA polyplexes induced potent cellular and humoral adaptive immunity and protected susceptible mice from lethal viral challenge. Together, these results demonstrate the translational potential of PACE polyplexes for therapeutic delivery of mRNA to the lungs.
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COVID-19 , Nanopartículas , Animais , Camundongos , Polímeros , RNA Mensageiro/genética , COVID-19/prevenção & controle , Pulmão , VacinaçãoRESUMO
Structural fetal diseases, such as congenital diaphragmatic hernia (CDH) can be diagnosed prenatally. Neonates with CDH are healthy in utero as gas exchange is managed by the placenta, but impaired lung function results in critical illness from the time a baby takes its first breath. MicroRNA (miR) 200b and its downstream targets in the TGF-ß pathway are critically involved in lung branching morphogenesis. Here, we characterize the expression of miR200b and the TGF-ß pathway at different gestational times using a rat model of CDH. Fetal rats with CDH are deficient in miR200b at gestational day 18. We demonstrate that novel polymeric nanoparticles loaded with miR200b, delivered in utero via vitelline vein injection to fetal rats with CDH results in changes in the TGF-ß pathway as measured by qRT-PCR; these epigenetic changes improve lung size and lung morphology, and lead to favorable pulmonary vascular remodeling on histology. This is the first demonstration of in utero epigenetic therapy to improve lung growth and development in a pre-clinical model. With refinement, this technique could be applied to fetal cases of CDH or other forms of impaired lung development in a minimally invasive fashion.
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Poly(ethylene glycol) (PEG) is widely employed for passivating nanoparticle (NP) surfaces to prolong blood circulation and enhance localization of NPs to target tissue. However, the immune response of PEGylated NPs-including anti-PEG antibody generation, accelerated blood clearance (ABC), and loss of delivery efficacy-is of some concern, especially for treatments that require repeat administrations. Although polyglycerol (PG), which has the same ethylene oxide backbone as PEG, has received attention as an alternative to PEG for NP coatings, the pharmacokinetic and immunogenic impact of PG has not been studied systematically. Here, linear PG, hyperbranched PG (hPG), and PEG-coated polylactide (PLA) NPs with varying surface densities were studied in parallel to determine the pharmacokinetics and immunogenicity of PG and hPG grafting, in comparison with PEG. We found that linear PG imparted the NPs a stealth property comparable to PEG, while hPG-grafted NPs needed a higher surface density to achieve the same pharmacokinetic impact. While linear PG-grafted NPs induced anti-PEG antibody production in mice, they exhibited minimal accelerated blood clearance (ABC) effects due to the poor interaction with anti-PEG immunoglobulin M (IgM). Further, we observed no anti-polymer IgM responses or ABC effects for hPG-grafted NPs.
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An inhalable platform for mRNA therapeutics would enable minimally invasive and lung targeted delivery for a host of pulmonary diseases. Development of lung targeted mRNA therapeutics has been limited by poor transfection efficiency and risk of vehicle-induced pathology. Here we report an inhalable polymer-based vehicle for delivery of therapeutic mRNAs to the lung. We optimized biodegradable poly(amine-co-ester) polyplexes for mRNA delivery using end group modifications and polyethylene glycol. Our polyplexes achieved high transfection of mRNA throughout the lung, particularly in epithelial and antigen-presenting cells. We applied this technology to develop a mucosal vaccine for SARS-CoV-2. Intranasal vaccination with spike protein mRNA polyplexes induced potent cellular and humoral adaptive immunity and protected K18-hACE2 mice from lethal viral challenge. One-sentence summary: Inhaled polymer nanoparticles (NPs) achieve high mRNA expression in the lung and induce protective immunity against SARS-CoV-2.
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Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. We sought to correct the multiple organ dysfunction of the F508del CF-causing mutation using systemic delivery of peptide nucleic acid gene editing technology mediated by biocompatible polymeric nanoparticles. We confirmed phenotypic and genotypic modification in vitro in primary nasal epithelial cells from F508del mice grown at air-liquid interface and in vivo in F508del mice following intravenous delivery. In vivo treatment resulted in a partial gain of CFTR function in epithelia as measured by in situ potential differences and Ussing chamber assays and correction of CFTR in both airway and GI tissues with no off-target effects above background. Our studies demonstrate that systemic gene editing is possible, and more specifically that intravenous delivery of PNA NPs designed to correct CF-causing mutations is a viable option to ameliorate CF in multiple affected organs.
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Non-viral vehicles hold therapeutic promise in advancing the delivery of a variety of cargos in vitro and in vivo, including small molecule drugs, biologics, and especially nucleic acids. However, their efficacy at the cellular level is limited by several delivery barriers, with endolysosomal degradation being most significant. The entrapment of vehicles and their cargo in the acidified endosome prevents access to the cytosol, nucleus, and other subcellular compartments. Understanding the factors that contribute to uptake and intracellular trafficking, especially endosomal entrapment and release, is key to overcoming delivery obstacles within cells. In this review, we summarize and compare experimental techniques for assessing the extent of endosomal escape of a variety of non-viral vehicles and describe proposed escape mechanisms for different classes of lipid-, polymer-, and peptide-based delivery agents. Based on this evaluation, we present forward-looking strategies utilizing information gained from mechanistic studies to inform the rational design of efficient delivery vehicles.
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Endossomos , Polímeros , Citosol , Lisossomos , PeptídeosRESUMO
There is growing interest in PEGylation of cationic polymeric vehicles for gene delivery in order to improve vehicle stability and reduce toxicity, but little is known about the effects of PEG coatings on transfection. We used a polymer from the poly(amine-co-ester) (PACE) family blended with PEG-conjugated PACE at different ratios in order to explore the effects of polyplex PEGylation on the transfection efficiency of plasmid DNA, mRNA, and siRNA in vitro and mRNA in vivo. We discovered that concentrations of PACE-PEG as low as 0.25% by weight improved polyplex stability but also inhibited transfection in vitro. In vivo, the effect of PACE-PEG incorporation on mRNA transfection varied by delivery route; the addition of PACE-PEG improved local delivery to the lung, but PEGylation had little effect on intravenous systemic delivery. By both delivery routes, transfection was inhibited at concentrations higher than 5 wt% PACE-PEG. These results demonstrate that excess PEGylation can be detrimental to vehicle function, and suggest that PEGylation of cationic vehicles must be optimized by PEG content, cargo type, and delivery route.
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Ésteres , Polietilenoglicóis , Aminas , Técnicas de Transferência de Genes , Tamanho da Partícula , TransfecçãoRESUMO
Fetal treatment of congenital lung disease, such as cystic fibrosis, surfactant protein syndromes, and congenital diaphragmatic hernia, has been made possible by improvements in prenatal diagnostic and interventional technology. Delivery of therapeutic agents to fetal lungs in nanoparticles improves cellular uptake. The efficacy and safety of nanoparticle-based fetal lung therapy depends on targeting of necessary cell populations. This study aimed to determine the relative distribution of nanoparticles of a variety of compositions and sizes in the lungs of fetal mice delivered through intravenous and intra-amniotic routes. Intravenous delivery of particles was more effective than intra-amniotic delivery for epithelial, endothelial and hematopoietic cells in the fetal lung. The most effective targeting of lung tissue was with 250nm Poly-Amine-co-Ester (PACE) particles accumulating in 50% and 44% of epithelial and endothelial cells. This study demonstrated that route of delivery and particle composition impacts relative cellular uptake in fetal lung, which will inform future studies in particle-based fetal therapy.
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Hérnias Diafragmáticas Congênitas , Nanopartículas , Surfactantes Pulmonares , Animais , Células Endoteliais , Feminino , Pulmão , Camundongos , GravidezRESUMO
In patients with glioblastoma, resistance to the chemotherapeutic temozolomide (TMZ) limits any survival benefits conferred by the drug. Here we show that the convection-enhanced delivery of nanoparticles containing disulfide bonds (which are cleaved in the reductive environment of the tumour) and encapsulating an oxaliplatin prodrug and a cationic DNA intercalator inhibit the growth of TMZ-resistant cells from patient-derived xenografts, and hinder the progression of TMZ-resistant human glioblastoma tumours in mice without causing any detectable toxicity. Genome-wide RNA profiling and metabolomic analyses of a glioma cell line treated with the cationic intercalator or with TMZ showed substantial differences in the signalling and metabolic pathways altered by each drug. Our findings suggest that the combination of anticancer drugs with distinct mechanisms of action with selective drug release and convection-enhanced delivery may represent a translational strategy for the treatment of TMZ-resistant gliomas.
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Neoplasias Encefálicas , Glioma , Nanopartículas , Animais , Neoplasias Encefálicas/tratamento farmacológico , Linhagem Celular Tumoral , Convecção , DNA , Glioma/tratamento farmacológico , Humanos , Substâncias Intercalantes , Camundongos , Temozolomida , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Polymeric vehicles are versatile tools for therapeutic gene delivery. Many polymers-when assembled with nucleic acids into vehicles-can protect the cargo from degradation and clearance in vivo, and facilitate its transport into intracellular compartments. Design options in polymer synthesis yield a comprehensive range of molecules and resulting vehicle formulations. These properties can be manipulated to achieve stronger association with nucleic acid cargo and cells, improved endosomal escape, or sustained delivery depending on the application. Here, we describe current approaches for polymer use and related strategies for gene delivery in preclinical and clinical applications. Polymer vehicles delivering genetic material have already achieved significant therapeutic endpoints in vitro and in animal models. From our perspective, with preclincal assays that better mimic the in vivo environment, improved strategies for target specificity, and scalable techniques for polymer synthesis, the impact of this therapeutic approach will continue to expand.
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Técnicas de Transferência de Genes , Ácidos Nucleicos/administração & dosagem , Polímeros/administração & dosagem , Animais , HumanosRESUMO
Many important biological applications of peptide nucleic acids (PNAs) target nucleic acid binding in eukaryotic cells, which requires PNA translocation across at least one membrane barrier. The delivery challenge is further exacerbated for applications in whole organisms, where clearance mechanisms rapidly deplete and/or deactivate exogenous agents. We have demonstrated that nanoparticles (NPs) composed of biodegradable polymers can encapsulate and release PNAs (alone or with co-reagents) in amounts sufficient to mediate desired effects in vitro and in vivo without deleterious reactions in the recipient cell or organism. For example, poly(lactic-co-glycolic acid) (PLGA) NPs can encapsulate and deliver PNAs and accompanying reagents to mediate gene editing outcomes in cells and animals, or PNAs alone to target oncogenic drivers in cells and correct cancer phenotypes in animal models. In this chapter, we provide a primer on PNA-induced gene editing and microRNA targeting-the two PNA-based biotechnological applications where NPs have enhanced and/or enabled in vivo demonstrations-as well as an introduction to the PLGA material and detailed protocols for formulation and robust characterization of PNA/DNA-laden PLGA NPs.
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Nanopartículas/química , Ácidos Nucleicos Peptídicos , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , DNA/genética , Portadores de Fármacos , Sistemas de Liberação de Medicamentos , Edição de Genes , Ácidos Nucleicos Peptídicos/administração & dosagem , Ácidos Nucleicos Peptídicos/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Interferência de RNARESUMO
Degradable poly(amine-co-ester) (PACE) terpolymers hold tremendous promise for siRNA delivery because these materials can be formulated into delivery vehicles with highly efficient siRNA encapsulation, providing effective knockdown with low toxicity. Here, we demonstrate that PACE nanoparticles (NPs) provide substantial protein knockdown in human embryonic kidney cells (HEK293) and hard-to-transfect primary human umbilical vein endothelial cells (HUVECs). After intravenous administration, NPs of solid PACE (sPACE)-synthesized with high monomer content of a hydrophobic lactone-accumulated in the liver and, to a lesser extent, in other tissues. Within the liver, a substantial fraction of sPACE NPs were phagocytosed by liver macrophages, while a smaller fraction of NPs accumulated in hepatic stellate cells and liver sinusoidal endothelial cells, suggesting that sPACE NPs could deliver siRNA to diverse cell populations within the liver. To test this hypothesis, we loaded sPACE NPs with siRNA designed to knockdown Nogo-B, a protein that has been implicated in the progression of alcoholic liver disease and liver fibrosis. These sPACE:siRNA NPs produced up to 60% Nogo-B protein suppression in the liver after systemic administration. We demonstrate that sPACE NPs can effectively deliver siRNA therapeutics to the liver to mediate protein knockdown in vivo.