RESUMO
Controlling the end-groups of biocompatible polymers is crucial for enabling polymer-based therapeutics and nanomedicine. Typically, end-group diversification is a challenging and time-consuming endeavor, especially for polymers prepared via ionic polymerization mechanisms with limited functional group tolerance. In this study, we present a facile end-group diversification approach for poly(2-oxazoline)s (POx), enabling quick and reliable production of heterotelechelic polymers to facilitate POxylation. The approach relies on the careful tuning of reaction parameters to establish differential reactivity of a pentafluorobenzyl initiator fragment and the living oxazolinium chain-end, allowing the selective introduction of N-, S-, O-nucleophiles via the termination of the polymerization, and a consecutive nucleophilic para-fluoro substitution. The value of this approach for the accelerated development of nanomedicine is demonstrated through the synthesis of well-defined lipid-polymer conjugates and POx-polypeptide block-copolymers, which are well-suited for drug and gene delivery. Furthermore, we investigated the application of a lipid-POx conjugate for the formulation and delivery of mRNA-loaded lipid nanoparticles for immunization against the SARS-COV-2 virus, underscoring the value of POx as a biocompatible polymer platform.
Assuntos
Nanomedicina , Oxazóis , Oxazóis/química , Nanomedicina/métodos , Humanos , SARS-CoV-2 , Polímeros/química , Polímeros/síntese química , Nanopartículas/química , Polimerização , AnimaisRESUMO
This study demonstrates the multiple carbon morphology forming abilities of two dissimilar polyion complex (PIC)-based double hydrophilic block copolymers (DHBC) along with three different phenol concentrations when subjecting the blend in aqueous media via a hydrothermal-assisted carbonization strategy. The morphological transition from worm-like to spherical along with granular is found for the blend of oppositely charged poly(ethylene glycol) (PEG)-conjugated poly(amino acid) block copolymers, PEG-poly(l-lysine) (PEG-PLys) and PEG-poly(glutamic acid) (PEG-PGlu), along with three different concentrations of phenol. In contrast, after mixing the combination of PEG-PLys and PEG-poly(aspartic acid) (PEG-PAsp) separately with three different phenol contents, elliptical to irregular to spherical structural transition occurred. Fourier transform infrared and circular dichroism spectroscopic studies indicated that the formation of worm-like hybrid micellar structures is attributed to the presence of the ß-sheet structure, whereas spherical-shaped hybrid micellar structures are formed due to the existence of α-helix and random coil structures. We discuss the mechanism for the secondary structure-induced morphology formation based on the theory related to the packing parameter, which is commonly used for analyzing the shape of the micellar structures. Secondary structures of the PIC-based DHBC system are responsible for forming multiple carbon morphologies, whereas these structures are absent in the case of the amphiphilic block copolymer (ABC) system. Furthermore, ABC-based template methods require organic solvent, ultrasonication, and a prolonged solvent evaporation process to obtain multiple carbon morphologies. Scanning electron microscopy observations suggested there is no significant morphological change even after subjecting the hybrid micelles to carbonization at elevated temperatures. Raman scattering studies revealed that the degree of graphitization and the graphitic crystallite domain size of the carbonized sample depend on the phenol content. Carbon materials exhibited the highest specific surface area of 579 m2 g-1 along with a pore volume of 0.398 cc g-1, and this observation suggests that the prepared carbons are porous. Our findings illustrate the facile and effective strategy to fabricate the multiple carbon morphologies that can be used as potential candidates for energy storage applications.
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Current strategies to direct therapy-loaded nanoparticles to the brain rely on functionalizing nanoparticles with ligands which bind target proteins associated with the blood-brain barrier (BBB). However, such strategies have significant brain-specificity limitations, as target proteins are not exclusively expressed at the brain microvasculature. Therefore, novel strategies which exploit alternative characteristics of the BBB are required to overcome nonspecific nanoparticle targeting to the periphery, thereby increasing drug efficacy and reducing detrimental peripheral side effects. Here, we present a simple, yet counterintuitive, brain-targeting strategy which exploits the higher impermeability of the BBB to selectively label the brain endothelium. This is achieved by harnessing the lower endocytic rate of brain endothelial cells (a key feature of the high BBB impermeability) to promote selective retention of free, unconjugated protein-binding ligands on the surface of brain endothelial cells compared to peripheral endothelial cells. Nanoparticles capable of efficiently binding to the displayed ligands (i.e., labeled endothelium) are consequently targeted specifically to the brain microvasculature with minimal "off-target" accumulation in peripheral organs. This approach therefore revolutionizes brain-targeting strategies by implementing a two-step targeting method which exploits the physiology of the BBB to generate the required brain specificity for nanoparticle delivery, paving the way to overcome targeting limitations and achieve clinical translation of neurological therapies. In addition, this work demonstrates that protein targets for brain delivery may be identified based not on differential tissue expression, but on differential endocytic rates between the brain and periphery.
Assuntos
Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Portadores de Fármacos/metabolismo , Sistemas de Liberação de Medicamentos/instrumentação , Células Endoteliais/metabolismo , Nanopartículas/metabolismo , Animais , Transporte Biológico , Encéfalo/irrigação sanguínea , Endotélio/metabolismo , Humanos , Ratos , Ratos Sprague-DawleyRESUMO
For efficient delivery of messenger (m)RNA, delivery carriers need two major functions: protecting mRNA from nucleases and translocating mRNA from endolysosomes to the cytoplasm. Herein, these two complementary functionalities are integrated into a single polyplex by fine-tuning the catiomer chemical structure and incorporating the endosomal escape modality. The effect of the methylene spacer length on the catiomer side chain is evaluated by comparing poly(l-lysine) (PLL) with a tetramethylene spacer and poly(L-ornithine) (PLO) with a trimethylene spacer. Noteworthily, the nuclease stability of the mRNA/catiomer polyplexes is largely affected by the difference in one methylene group, with PLO/mRNA polyplex showing enhanced stability compared to PLL/mRNA polyplex. To introduce the endosomal escape function, the PLO/mRNA polyplex is wrapped with a charge-conversion polymer (CCP), which is negatively charged at extracellular pH but turns positive at endosomal acidic pH to disrupt the endosomal membrane. Compared to the parent PLO/mRNA polyplex, CCP facilitated the endosomal escape of the polyplex in cultured cells to improve the protein expression efficiency from mRNA by approximately 80-fold. Collectively, this system synergizes the protective effect of PLO against nucleases and the endosomal escape capability of CCP in mRNA delivery.
Assuntos
Endossomos , Polímeros , Endossomos/química , Endossomos/metabolismo , Ornitina/análise , Ornitina/metabolismo , Polímeros/química , RNA Mensageiro , TransfecçãoRESUMO
A critical role of polyethylene glycol (PEG) crowding in the packaging of plasmid DNA (pDNA) into polyplex micelles (PMs) was investigated using a series of PEG-b-poly(l-lysine) (PEG-PLys) block copolymers with varying molecular weights of both PEG and PLys segments. Rod-shaped PMs preferentially formed when the tethered PEG chains covering pDNA in a precondensed state were dense enough to overlap one another (reduced tethering density (RTD) > 1), whereas globular PMs were obtained when they were not overlapped (RTD < 1). These results submitted a scheme that steric repulsive effect of PEG regulated packaging pathways of pDNA either through folding into rod-shape or collapsing into globular depending on whether the PEG chains are overlapped or not. The rod-shaped PMs gave significantly higher gene expression efficacies in a cell-free system compared to the globular PMs, demonstrating the practical relevance of regulating packaging structure of pDNA for developing efficient gene delivery systems.
Assuntos
DNA/química , DNA/genética , Expressão Gênica , Micelas , Plasmídeos/química , Plasmídeos/genética , Polietilenoglicóis/química , Sistema Livre de Células , Técnicas de Transferência de Genes , Humanos , Polímeros/química , TransfecçãoRESUMO
Achieving precise control over the morphology and function of polymeric nanostructures during self-assembly remains a challenge in materials as well as biomedical science, especially when independent control over particle properties is desired. Herein, we report on nanostructures derived from amphiphilic block copolypept(o)ides by secondary-structure-directed self-assembly, presenting a strategy to adjust core polarity and function separately from particle preparation in a bioreversible manner. The peptide-inherent process of secondary-structure formation allows for the synthesis of spherical and worm-like core-cross-linked architectures from the same block copolymer, introducing a simple yet powerful approach to versatile peptide-based core-shell nanostructures.
RESUMO
Polymeric nanoreactors (NRs) have distinct advantages to improve chemical reaction efficiency, but the inâ vivo applications are limited by lack of tissue-specificity. Herein, novel glucose oxidase (GOD)-loaded therapeutic vesicular NRs (theraNR) are constructed based on a diblock copolymer containing poly(ethylene glycol) (PEG) and copolymerized phenylboronic ester or piperidine-functionalized methacrylate (P(PBEM-co-PEM)). Upon systemic injection, theraNR are inactive in normal tissues. At a tumor site, theraNR are specifically activated by the tumor acidity via improved permeability of the membranes. Hydrogen peroxide (H2 O2 ) production by the catalysis of GOD in theraNR increases tumor oxidative stress significantly. Meanwhile, high levels of H2 O2 induce self-destruction of theraNR releasing quinone methide (QM) to deplete glutathione and suppress the antioxidant ability of cancer cells. Finally, theraNR efficiently kill cancer cells and ablate tumors via the synergistic effect.
Assuntos
Antineoplásicos/uso terapêutico , Morte Celular/efeitos dos fármacos , Portadores de Fármacos , Nanoestruturas , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Polietilenoglicóis/química , Antineoplásicos/administração & dosagem , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Antioxidantes/metabolismo , Ácidos Borônicos/química , Permeabilidade da Membrana Celular , Ésteres , Glucose Oxidase/metabolismo , Glutationa/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Concentração de Íons de Hidrogênio , Indolquinonas/química , Metacrilatos/química , Microscopia Eletrônica de Transmissão , Neoplasias/metabolismo , Piperidinas/química , Estudo de Prova de ConceitoRESUMO
The role of poly(ethylene-glycol) (PEG) in rod-shaped polyplex micelle structures, having a characteristic core of folded plasmid DNA (pDNA) and a shell of tethered PEG chains, is investigated using PEG-detachable polyplex micelles. Rod shapes undergo change to compacted globule shapes by removal of PEG from polyplex micelles prepared from block copolymer with acid-labile linkage between PEG and poly(l-lysine) (PLys) through exposure to acidic milieu. This structural change supports the previous investigation on the rod shapes that PEG shell prevents the DNA structure from being globule shaped as the most favored structure in minimizing surface area. Noteworthy, despite the PEG is continuously depleted, the structural change does not occur in gradual shortening manner but the rod shapes keep their length unchanged and abruptly transform into globule shapes. Analysis of PEG density reveals the transition occurred when tethered PEG of rod shapes has decreased to a critical crowdedness, i.e., discontacted with neighboring PEG, which eventually illuminates another contribution, rigidity of DNA packaged as bundle in the rod shapes, in addition to the steric repulsion of PEG, in sustaining rod shapes. This investigation affirms significant role of PEG and also DNA rigidity as bundle in the formation of rod-shaped structures enduring the quest of compaction of charge-neutralized DNA in the polyplex micelles.
Assuntos
DNA/química , Micelas , Plasmídeos/química , Polietilenoglicóis/química , Polilisina/química , Concentração de Íons de Hidrogênio , Polietilenoglicóis/síntese químicaRESUMO
Improving the stability of polyplex micelles under physiological conditions is a critical issue for promoting gene transfection efficiencies. To this end, hydrophobic palisade was installed between the inner core of packaged plasmid DNA (pDNA) and the hydrophilic shell of polyplex micelles using a triblock copolymer consisting of hydrophilic poly(2-ethyl-2-oxazoline), thermoswitchable amphiphilic poly(2-n-propyl-2-oxazoline) (PnPrOx) and cationic poly(L-lysine). The two-step preparation procedure, mixing the triblock copolymer with pDNA below the lower critical solution temperature (LCST) of PnPrOx, followed by incubation above the LCST to form a hydrophobic palisade of the collapsed PnPrOx segment, induced the formation of spatially aligned hydrophilic-hydrophobic double-protected polyplex micelles. The prepared polyplex micelles exhibited significant tolerance against attacks from nuclease and polyanions compared to those without hydrophobic palisades, thereby promoting gene transfection. These results corroborated the utility of amphiphilic poly(oxazoline) as a molecular thermal switch to improve the stability of polyplex gene carriers relevant for physiological applications.
Assuntos
DNA/química , Micelas , Plasmídeos/genética , Poliaminas/química , Polilisina/química , Transfecção/métodos , Interações Hidrofóbicas e Hidrofílicas , Microscopia Eletrônica de Transmissão , Polietilenoglicóis/químicaRESUMO
Targeted nanomedicines are a promising technology for treatment of disease; however, preparation and characterization of well-defined protein-nanoparticle systems remain challenging. Here, we describe a platform technology to prepare antibody binding fragment (Fab)-bearing nanoparticles and an accompanying real-time cell-based assay to determine their cellular uptake compared to monoclonal antibodies (mAbs) and Fabs. The nanoparticle platform was composed of core-cross-linked polyion complex (PIC) micelles prepared from azide-functionalized PEG-b-poly(amino acids), that is, azido-PEG-b-poly(l-lysine) [N3-PEG-b-PLL] and azido-PEG-b-poly(aspartic acid) [N3-PEG-b-PAsp]. These PIC micelles were 30 nm in size and contained approximately 10 polymers per construct. Fabs were derived from an antibody binding the EphA2 receptor expressed on cancer cells and further engineered to contain a reactive cysteine for site-specific attachment and a cleavable His tag for purification from cell culture expression systems. Azide-functionalized micelles and thiol-containing Fab were linked using a heterobifunctional cross-linker (FPM-PEG4-DBCO) that contained a fluorophenyl-maleimide for stable conjugation to Fabs thiols and a strained alkyne (DBCO) group for coupling to micelle azide groups. Analysis of Fab-PIC micelle conjugates by fluorescence correlation spectroscopy, size exclusion chromatography, and UV-vis absorbance determined that each nanoparticle contained 2-3 Fabs. Evaluation of cellular uptake in receptor positive cancer cells by real-time fluorescence microscopy revealed that targeted Fab-PIC micelles achieved higher cell uptake than mAbs and Fabs, demonstrating the utility of this approach to identify targeted nanoparticle constructs with unique cellular internalization properties.
Assuntos
Anticorpos Monoclonais/química , Reagentes de Ligações Cruzadas/química , Fragmentos Fab das Imunoglobulinas/química , Nanopartículas/química , Polímeros/química , Neoplasias da Próstata/metabolismo , Receptor EphA2/metabolismo , Anticorpos Monoclonais/metabolismo , Humanos , Fragmentos Fab das Imunoglobulinas/metabolismo , Masculino , Micelas , Polímeros/metabolismo , Células Tumorais CultivadasRESUMO
Selectively spooling single plasmid DNA (pDNA), as a giant polyelectrolyte, into a nanosized toroidal structure or folding it into a rod-like structure has been accomplished by polyion complexation with block catiomers to form polymeric micelles in varying NaCl concentrations. The interactive potency between the pDNA and block catiomers was determined to play a critical role in defining the ultimate structure of the pDNA; the formation of toroidal or rod-like structures was achieved by complexation in 600 or 0 mM NaCl solutions, respectively. Compared with the rod-like structure, the toroidal structure possessed superior biological functions capable not only of elevating in vitro transcription but also of elevating in vivo gene transduction efficiency. This demonstrated the great utility of the toroidal pDNA packaging as a distinct structured gene carrier. Furthermore, the fact that the NaCl concentration at which the toroidal structure was specifically formed corresponds to seawater stimulates interest in this ordered nanostructure as a possible inherent structure for DNA.
Assuntos
Expressão Gênica , Micelas , Músculo Esquelético/metabolismo , Plasmídeos , Transcrição Gênica , Transdução Genética/métodos , Animais , Camundongos , Plasmídeos/química , Plasmídeos/farmacologiaRESUMO
A distinct tadpole-shaped nanostructure characterized by a spherical head and an extended shaft was identified in a single plasmid DNA (pDNA)-based polymeric micelle. The tadpole-shaped structure was constructed by adding anionic chondroitin sulfate (CS) to the rod-shaped polyplex micelle containing a single pDNA molecule packaged by the PEG-polycation block copolymer through their electrostatic self-assembly. The complex consequently developed a novel structure composed of segregated domains of the CS-rich inflated head and CS-poor folded DNA tail. Hence, this tadpole structure can be regarded as evidence that distinct phase segregation occurred in a single polymeric micelle containing pDNA.
Assuntos
Micelas , Plasmídeos/metabolismo , Polímeros/química , Sulfatos de Condroitina/química , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Técnicas de Transferência de Genes , Nanoestruturas/química , Plasmídeos/genética , Poliaminas/química , Polieletrólitos , Polietilenoglicóis/química , Espectrometria por Raios XRESUMO
Cell-penetrating peptides (CPPs), for example, arginine (Arg) rich peptides, are used for the intracellular delivery of nucleic acids. In this study, oligosarcosine-conjugated Arg-rich peptides were designed as plasmid DNA (pDNA) carriers, and the physicochemical parameters and transfection efficiency of the peptide/pDNA complexes were evaluated. Oligosarcosine with different lengths were conjugated to a base sequence composed of arginine and α-aminoisobutyric acid (Aib) [(Aib-Arg-Arg)3]. Oligosarcosine conjugation inhibited the aggregation of the complexes after mixing with pDNA, shielded the positive charge of the complexes, and provided efficient pDNA transfection in cultured cells. The efficiency of the pDNA transfection was improved by varying the length of the oligosarcosine moiety (10-15 units were optimal). The cellular uptake efficiency and intracellular distribution of pDNA were the same regardless of oligosarcosine conjugation. These results implied that intracellular processes, including the decondensation of pDNA, contributed to the efficiency of the protein expression from pDNA. This study demonstrated the advantages of oligosarcosine conjugation to Arg-rich CPPs and provided valuable insight into the future design of CPPs.
Assuntos
Arginina , Peptídeos Penetradores de Células , Arginina/genética , DNA/química , DNA/genética , Plasmídeos/genética , Transfecção , Peptídeos Penetradores de Células/farmacologia , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/genéticaRESUMO
The stealth effect plays a central role on capacitating nanomaterials for drug delivery applications through improving the pharmacokinetics such as blood circulation, biodistribution, and tissue targeting. Here based on a practical analysis of stealth efficiency and a theoretical discussion of relevant factors, we provide an integrated material and biological perspective in terms of engineering stealth nanomaterials. The analysis surprisingly shows that more than 85% of the reported stealth nanomaterials encounter a rapid drop of blood concentration to half of the administered dose within 1 h post administration although a relatively long ß-phase is observed. A term, pseudo-stealth effect, is used to delineate this common pharmacokinetics behavior of nanomaterials, that is, dose-dependent nonlinear pharmacokinetics because of saturating or depressing bio-clearance of reticuloendothelial system (RES). We further propose structural holism can be a watershed to improve the stealth effect; that is, the whole surface structure and geometry play important roles, rather than solely relying on a single factor such as maximizing repulsion force through polymer-based steric stabilization (e.g., PEGylation) or inhibiting immune attack through a bio-inspired component. Consequently, engineering delicate structural hierarchies to minimize attractive binding sites, that is, minimal charges/dipole and hydrophobic domain, becomes crucial. In parallel, the pragmatic implementation of the pseudo-stealth effect and dynamic modulation of the stealth effect are discussed for future development.
Assuntos
Sistemas de Liberação de Medicamentos , Polietilenoglicóis , Humanos , Distribuição Tecidual , Cinética , Polietilenoglicóis/químicaRESUMO
The success of gene therapy relies on gene nanocarriers to achieve therapeutic effects in vivo. Surface shielding of poly(ethylene glycol) (PEG), known as PEGylation, onto gene delivery carriers is a predominant strategy for extending blood circulation and improving therapeutic outcomes in vivo. Nevertheless, PEGylation frequently compromises the transfection efficiency by decreasing the interactions with the cellular membrane of the targeted cells, thereby preventing the cellular uptake and the subsequent endosomal escape. Herein, we developed a stepwise pH-responsive polyplex micelle for the plasmid DNA delivery with the surface covered by ethylenediamine-based polycarboxybetaines. This polyplex micelle switched its surface charge from neutral at pH 7.4 to positive at tumorous and endo-/lysosomal pH (i.e., pH 6.5 and 5.5, respectively), thus enhancing the cellular uptake and facilitating the endosomal escape toward efficient gene transfection. Additionally, the polyplex micelle demonstrated prolonged blood circulation as well as enhanced tumor accumulation, leading to highly effective tumor growth suppression by delivering an antiangiogenic gene. These results suggest the usefulness of a pH-responsive charge-switchable shell polymer on the surface of the polyplex micelle for the efficient nucleic acid delivery.
Assuntos
Micelas , Neoplasias , Humanos , DNA , Polímeros , Polietilenoglicóis , Transfecção , Neoplasias/tratamento farmacológico , Concentração de Íons de HidrogênioRESUMO
Polyplex for messenger RNA (mRNA) delivery requires strong yet reversible association between mRNA and polycation for extracellular robustness and selective intracellular disintegration. Herein, RNA oligonucleotide (OligoRNA) derivatives that bridge mRNA and polycation are developed to stabilize polyplex micelles (PMs). A set of the OligoRNAs introduced with a polyol moiety in their 5' end is designed to hybridize to fixed positions along mRNA strand. After PM preparation from the hybridized mRNA and poly(ethylene glycol)-polycation block copolymer derived with phenylboronic acid (PBA) moieties in its cationic segment, PBA moieties form reversible phenylboronate ester linkages with a polyol moiety at 5' end of OligoRNAs and a diol moiety at their 3' end ribose, in the PM core. The OligoRNAs work as a node to bridge ionically complexed mRNA and polycation, thereby improving PM stability against polyion exchange reaction and ribonuclease attack in extracellular environment. After cellular uptake, intracellular high concentration of adenosine triphosphate triggers the cleavage of phenylboronate ester linkages, resulting in mRNA release from PM. Ultimately, the PM provides efficient mRNA introduction in cultured cells and mouse lungs after intratracheal administration, demonstrating the potential of the bridging strategy in polyplex-based mRNA delivery.
Assuntos
Micelas , RNA , Animais , Ésteres , Camundongos , Oligonucleotídeos , Polieletrólitos , Polietilenoglicóis , RNA Mensageiro/genética , TransfecçãoRESUMO
Intracellular protein delivery is a powerful strategy for developing innovative therapeutics. Nanocarriers present great potential to deliver proteins inside cells by promoting cellular uptake and overcoming entrapment and degradation in acidic endo/lysosomal compartments. Thus, because cytosolic access is essential for eliciting the function of proteins, significant efforts have been dedicated to engineering nanocarriers with maximal endosomal escape regardless of the cell type. On the other hand, controlling the ability of nanocarriers to escape from the endo/lysosomal compartments of particular cells may offer the opportunity for enhancing delivery precision. To test this hypothesis, we developed pH-sensitive polymeric nanocarriers with adjustable endosomal escape potency for selectively reaching the cytosol of defined cancer cells with dysregulated endo/lysosomal acidification. By loading antibodies against nuclear pore complex in the nanocarriers, we demonstrated the selective delivery into the cytosol and subsequent nucleus targeting of cancer cells rather than non-cancerous cells both in vitro and in vivo. Systemically injected nanocarriers loading anti-c-MYC antibodies suppressed c-MYC in solid tumors and inhibit tumor growth without side effects, confirming the therapeutic potential of our approach. These results indicated that regulating the ability of nanocarriers to escape from endo/lysosomal compartments in particular cells is a practical approach for gaining delivery specificity.
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Nanopartículas , Neoplasias , Citosol/metabolismo , Portadores de Fármacos/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Endossomos/metabolismo , Humanos , Lisossomos/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Polímeros/metabolismoRESUMO
Carriers for messenger RNA (mRNA) delivery require propensities to protect the mRNA from enzymatic degradation and to selectively release mRNA in the cytosol for smooth mRNA translation. To meet these requirements, we designed mRNA-loaded polyplex micelles (PMs) with ATP-responsive crosslinking in the inner core by complexing mRNA with poly(ethylene glycol)-polycation block copolymers derivatized with phenylboronic acid and polyol groups, which form crosslinking structures via spontaneous phenylboronate ester formation. PMs thus prepared are tolerable against enzymatic attack and, in turn, disintegrate in the cytosol to release mRNA when triggered by the cleavage of phenylboronate ester linkages in response to elevated ATP concentration. Two structural factors of the PM, including (i) the introduction ratios of phenylboronate ester crosslinkers and (ii) the structure and protonation degree of amino groups in the polycation segment, are critical for maximizing protein expression in cultured cells due to the optimized balance between the robustness in the biological milieu and the ATP-responsive mRNA release in the cytosol. The optimal PM formulation was further stabilized by installing cholesterol moieties into both the mRNA and ω-end of the block copolymer to elicit longevity in blood circulation after intravenous injection.
Assuntos
Ésteres , Micelas , Trifosfato de Adenosina , Ácidos Borônicos , Portadores de Fármacos , Polietilenoglicóis , RNA MensageiroRESUMO
Genome editing using CRISPR/Cas9 has attracted considerable attention for the treatment of genetic disorders and viral infections. Co-delivery of Cas9 mRNA and single guide (sg)RNA is a promising strategy to efficiently edit the genome of various cell types, including non-dividing cells, with minimal safety concerns. However, co-delivery of two RNA species with significantly different sizes, such as Cas9 mRNA (4.5 kb) and sgRNA (0.1 kb), is still challenging, especially in vivo. Here, we addressed this issue by using a PEGylated polyplex micelle (PM) condensing the RNA in its core. PM loading sgRNA alone released sgRNA at minimal dilution in buffer, while PM loading Cas9 mRNA alone was stable even at higher dilutions. Interestingly, co-encapsulating sgRNA with Cas9 mRNA in a single PM prevented sgRNA release upon dilution, which led to the enhanced tolerability of sgRNA against enzymatic degradation. Subsequently, PM with co-encapsulated RNA widely induced genome editing in parenchymal cells in the mouse brain, including neurons, astrocytes, and microglia, following intraparenchymal injection, at higher efficiency than that by co-delivery of PMs loaded with either Cas9 mRNA or sgRNA separately. To the best of our knowledge, this is the first report demonstrating the utility of RNA-based delivery of CRISPR/Cas9 in inducing genome editing in the brain parenchymal cells. Furthermore, the efficiency of genome editing using PMs was higher than using a non-PEGylated polyplex, due to the enhanced diffusion of PMs in the brain tissue. The results reported herein demonstrate the potential of using PMs to co-encapsulate Cas9 mRNA and sgRNA for in vivo genome editing.
Assuntos
Edição de Genes , RNA Guia de Cinetoplastídeos , Animais , Encéfalo , Sistemas CRISPR-Cas , Camundongos , Micelas , RNA Guia de Cinetoplastídeos/genética , RNA Mensageiro/genéticaRESUMO
Prevention of metastatic and local-regional recurrence of cancer after surgery remains difficult. Targeting postsurgical premetastatic niche and microresiduals presents an excellent prospective opportunity but is often challenged by poor therapeutic delivery into minimal residual tumors. Here, an enzymatically transformable polymer-based nanotherapeutic approach is presented that exploits matrix metalloproteinase (MMP) overactivation in tumor-associated tissues to guide the codelivery of colchicine (microtubule-disrupting and anti-inflammatory agent) and marimastat (MMP inhibitor). The dePEGylation of polymersomes catalyzed by MMPs not only exposes the guanidine moiety to improve tissue/cell-targeting/retention to increase bioavailability, but also differentially releases marimastat and colchicine to engage their extracellular (MMPs) and intracellular (microtubules) targets of action, respectively. In primary tumors/overt metastases, the vasculature-specific targeting of nanotherapeutics can function synchronously with the enhanced permeability and retention effect to deter malignant progression of metastatic breast cancer. After the surgical removal of large primary tumors, nanotherapeutic agents are localized in the premetastatic niche and at the site of the postsurgical wound, disrupting the premetastatic microenvironment and eliminating microresiduals, which radically reduces metastatic and local-regional recurrence. The findings suggest that nanotherapeutics can safely widen the therapeutic window to resuscitate colchicine and MMP inhibitors for other inflammatory disorders.