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The differential enzymatic activity in the endo/lysosomes of particular cells could trigger targeted endosomal escape functions, enabling selective intracellular protein delivery. However, this strategy may be jeopardized due to protein degradation during endosomal trafficking. Herein, using custom made fluorescent probes to assess the endosomal activity of cathepsin B (CTSB) and protein degradation, we found that certain cancer cells with hyperacidified endosomes grant a spatiotemporal window where CTSB activity surpass protein digestion. This inspired the engineering of antibody-loaded polymeric nanocarriers having CTSB-activatable endosomal escape ability. The nanocarriers selectively escaped from the endo/lysosomes in the cells with high endosomal CTSB activity and delivered active antibodies to intracellular targets. This study provides a viable strategy for cell-specific protein delivery using stimuli-responsive nanocarriers with controlled endosomal escape.
Assuntos
Endossomos , Neoplasias , Endossomos/metabolismo , Anticorpos/metabolismo , Polímeros/metabolismo , Lisossomos/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismoRESUMO
The precise mechanism of resistance to anti-cancer drugs such as platinum drugs is not fully revealed. To reveal the mechanism of drug resistance, the molecular networks of anti-cancer drugs such as cisplatin, carboplatin, oxaliplatin, and arsenic trioxide were analyzed in several types of cancers. Since diffuse-type stomach adenocarcinoma, which has epithelial-mesenchymal transition (EMT)-like characteristics, is more malignant than intestinal-type stomach adenocarcinoma, the gene expression and molecular networks in diffuse- and intestinal-type stomach adenocarcinomas were analyzed. Analysis of carboplatin revealed the causal network in diffuse large B-cell lymphoma. The upstream regulators of the molecular networks of cisplatin-treated lung adenocarcinoma included the anti-cancer drug trichostatin A (TSA), a histone deacetylase inhibitor. The upstream regulator analysis of cisplatin revealed an increase in FAS, BTG2, SESN1, and CDKN1A, and the involvement of the tumor microenvironment pathway. The molecular networks were predicted to interact with several microRNAs, which may contribute to the identification of new drug targets for drug-resistant cancer. Analysis of oxaliplatin, a platinum drug, revealed that the SPINK1 pancreatic cancer pathway is inactivated in ischemic cardiomyopathy. The study showed the importance of the molecular networks of anti-cancer drugs and tumor microenvironment in the treatment of cancer resistant to anti-cancer drugs.
Assuntos
Adenocarcinoma , Antineoplásicos , Proteínas Imediatamente Precoces , MicroRNAs , Humanos , Cisplatino , Carboplatina/farmacologia , Platina/farmacologia , Platina/uso terapêutico , Oxaliplatina/uso terapêutico , MicroRNAs/genética , MicroRNAs/uso terapêutico , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Microambiente Tumoral , Inibidor da Tripsina Pancreática de Kazal , Proteínas Supressoras de TumorRESUMO
Treatment of immunologically cold tumors is a major challenge for immune checkpoint inhibitors (ICIs). Interleukin 12 (IL-12) can invigorate ICIs against cold tumors by establishing a robust antitumor immunity. However, its toxicity and systemic induction of counteracting immunosuppressive signals have hindered translation. Here, IL-12 activity is spatiotemporally controlled for safely boosting efficacy without the stimulation of interfering immune responses by generating a nanocytokine that remains inactive at physiological pH, but unleashes its full activity at acidic tumor pH. The IL-12-based nanocytokine (Nano-IL-12) accumulate and release IL-12 in tumor tissues, eliciting localized antitumoral inflammation, while preventing systemic immune response, counteractive immune reactions, and adverse toxicities even after repeated intravenous administration. The Nano-IL-12-mediated spatiotemporal control of inflammation prompt superior anticancer efficacy, and synergize with ICIs to profoundly inflame the tumor microenvironment and completely eradicate ICI-resistant primary and metastatic tumors. The strategy could be a promising approach toward safer and more effective immunotherapies.
Assuntos
Interleucina-12 , Neoplasias , Humanos , Neoplasias/terapia , Inflamação/patologia , Imunoterapia , Microambiente TumoralRESUMO
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.
Assuntos
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
Messenger RNA (mRNA) is emerging as a promising therapeutic modality for a variety of diseases. Because of the fragility and limited intracellular access of mRNA, the development of delivery technologies is essential for promoting the applicability of mRNA-based treatments. Among effective nanocarriers, polymeric micelles loading mRNA by polyion complex (PIC) formation with block catiomers have the potential to meet the delivery needs. Since PICs are relatively unstable in in vivo settings, herein, we constructed mRNA-loaded micelles having pH-responsive cross-linked cores by complexing mRNA with cis-aconitic anhydride-modified poly(ethylene glycol)-poly(l-lysine) (PEG-pLL(CAA)) block copolymers. The micelles were stable at physiological pH (pH 7.4) but achieved the complete release of the mRNA at endosomal pH (pH 5.5-4.5). The cross-linking also enhanced the stability of the micelles against disassembly from polyanions and protected the loaded mRNA from degradation by nucleases. Thus, the cross-linked micelles increased the delivery of mRNA to cancer cells, promoting protein expression both in vitro and in vivo. Our results highlight the potential of PEG-pLL(CAA)-based micelles for mRNA delivery.
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Phosphatidylserine (PtdSer), one of the phospholipids that the apoptotic cell exposes, has emerged for anti-inflammatory therapy via polarizing inflammatory microglia (Mi1) to anti-inflammatory phenotype (Mi2). In this study, we report microglia polarization effect of PtdSer-exposing polymeric particles (PSPs). PSPs upregulated Mi2 microglia and suppressed Mi1 microglia through peroxisome proliferator-activated receptor gamma upregulation in vitro and in vivo. This study highlights the potential of PSPs for anti-inflammatory therapy.
Assuntos
Microglia , PPAR gama , Anti-Inflamatórios/farmacologia , PPAR gama/genética , Fosfatidilserinas/farmacologiaRESUMO
RNA interference (RNAi) by small interfering RNAs (siRNAs) is a promising therapeutic approach. Because siRNA has limited intracellular access and is rapidly cleared in vivo, the success of RNAi depends on efficient delivery technologies. Particularly, polyion complexation between block catiomers and siRNA is a versatile approach for constructing effective carriers, such as unit polyion complexes (uPIC), core-shell polyion complex (PIC) micelles and vesicular siRNAsomes, by engineering the structure of block catiomers. In this regard, the flexibility of block catiomers could be an important parameter in the formation of PIC nanostructures with siRNA, though its effect remains unknown. Here, we studied the influence of block catiomer flexibility on the assembly of PIC structures with siRNA using a complementary polymeric system, i.e. poly(ethylene glycol)-poly(L-lysine) (PEG-PLL) and PEG-poly(glycidylbutylamine) (PEG-PGBA), which has a relatively more flexible polycation segment than PEG-PLL. Mixing PEG-PGBA with siRNA at molar ratios of primary amines in polymer to phosphates in the siRNA (N/P ratios) higher than 1.5 promoted the multimolecular association of uPICs, whereas PEG-PLL formed uPIC at all N/P ratios higher than 1. Moreover, uPICs from PEG-PGBA were more stable against counter polyanion exchange than uPICs from PEG-PLL, probably due to a favorable complexation process, as suggested by computational studies of siRNA/block catiomer binding. In in vitro experiments, PEG-PGBA uPICs promoted effective intracellular delivery of siRNA and efficient gene knockdown. Our results indicate the significance of polycation flexibility on assembling PIC structures with siRNA, and its potential for developing innovative delivery systems.
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Dynamic bursting in tumor vasculature has recently sparked interest as a novel particle transportation route for drug delivery. These bursts facilitate the transport of sub-100 nm nanoparticles into tumors, though their contribution on the access of other blood-borne particles remains unknown. To evaluate the versatility of this phenomenon, the in vivo kinetics of a variety of intravenously injected particles and their penetration in tumor xenografts and allografts are compared. Dextran, polymeric micelles, liposomes, and polymeric vesicles with diameters ranging from 32 to 302 nm are found to colocalize in virtually all vascular bursts. By mathematical modeling, the burst vent size is estimated to be 625 nm or larger, indicating the dynamic and stochastic formation of large permeation routes in tumor vasculature. Furthermore, some burst vents are found to be µm-sized, allowing the transport of 1 µm microspheres. Moreover, antibody drugs and platelets are capable of utilizing vascular burst transportation, demonstrating the application of this phenomenon to other types of therapeutics and cellular components. These findings indicate the vast potential of vascular bursts, extending the biological and therapeutic significance of this phenomenon to a wide range of blood-borne particles and cells.
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Nanopartículas , Neoplasias , Sistemas de Liberação de Medicamentos , Humanos , Lipossomos , Micelas , Neoplasias/tratamento farmacológico , Tamanho da PartículaRESUMO
Phosphorylcholine (PC) has been used to improve the water solubility and biocompatibility of biomaterials. Here, we show that PC can also work as a ligand for targeting cancer cells based on their increased phospholipid metabolism. PC-installed multiarm poly(ethylene glycol)s and polymeric micelles achieved high and rapid internalization in pancreatic cancer cells. This enhanced cellular uptake was drastically reduced when the cells were incubated with excess free PC or at 4 °C, as well as by inhibiting the phospholipid transfer protein (PLTP) on the surface of cancer cells, indicating an energy dependent active transport mediated by PLTP.
Assuntos
Neoplasias Pancreáticas , Fosforilcolina , Humanos , Micelas , Neoplasias Pancreáticas/tratamento farmacológico , Proteínas de Transferência de Fosfolipídeos , PolietilenoglicóisRESUMO
RNA nanotechnology has promise for developing mRNA carriers with enhanced physicochemical and functional properties. However, the potential synergy for mRNA delivery of RNA nanotechnology in cooperation with established carrier systems remains unknown. This study proposes a combinational system of RNA nanotechnology and mRNA polyplexes, by focusing on mRNA steric structure inside the polyplexes. Firstly, several mRNA strands are bundled through hybridization with RNA oligonucleotide crosslinkers to obtain tight mRNA structure, and then the bundled mRNA is mixed with poly(ethylene glycol) (PEG)-polycation block copolymers to prepare PEG-coated polyplex micelles (PMs). mRNA bundling results in highly condensed mRNA packaging inside PM core with dense PEG chains on the surface, thereby, improving PM stability against polyion exchange reaction and ribonuclease (RNase) attack. Importantly, such stabilization effects are attributed to bundled structure of mRNA rather than the increase in total mRNA amount encapsulated in the PMs, as encapsulation of long mRNA strands without bundling fails to improve PM stability. Consequently, PMs loading bundled mRNA exhibit enhanced stability in mouse blood circulation, and induce efficient protein expression in cultured cells and mouse brain.
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Micelas , Polietilenoglicóis , Animais , Camundongos , Polímeros , RNA Mensageiro/genética , TransfecçãoRESUMO
Messenger RNA (mRNA) shows high therapeutic potential, though effective delivery systems are still needed for boosting its application. Nanocarriers loading mRNA via polyion complexation with block catiomers into core-shell micellar structures are promising systems for enhancing mRNA delivery. Engineering the interaction between mRNA and catiomers through polymer design can promote the development of mRNA-loaded micelles (mRNA/m) with increased delivery efficiency. Particularly, the polycation chain rigidity may critically affect the mRNA-catiomer interplay to yield potent nanocarriers, yet its effect remains unknown. Herein, the influence of polycation stiffness on the performance of mRNA/m by developing block complementary catiomers having polycation segments with different flexibility, that is, poly(ethylene glycol)-poly(glycidylbutylamine) (PEG-PGBA) and PEG-poly(L-lysine) (PEG-PLL) is studied. PEG-PGBA allows more than 50-fold stronger binding to mRNA than the relatively more rigid PEG-PLL, resulting in mRNA/m with enhanced protection against enzymatic attack and polyanions. mRNA/m from PEG-PGBA significantly enhances mRNA in vivo bioavailability and increased protein translation, indicating the importance of controlling polycation flexibility for forming stable polyion complexes with mRNA toward improved delivery.
Assuntos
Micelas , Polímeros , Lisina , Polietilenoglicóis , RNA Mensageiro/genéticaRESUMO
Protein drugs have great potential as targeted therapies, yet their application suffers from several drawbacks, such as instability, short half-life, and adverse immune responses. Thus, protein delivery approaches based on stimuli-responsive nanocarriers can provide effective strategies for selectively enhancing the availability and activation of proteins in targeted tissues. Herein, polymeric micelles with the ability of encapsulating proteins are developed via concurrent ion complexation and pH-cleavable covalent bonding between proteins and block copolymers directed to pH-triggered release of the protein payload. Carboxydimethylmaleic anhydride (CDM) is selected as the pH-sensitive moiety, since the CDMamide bond is stable at physiological pH (pH 7.4), while it cleaves at pH 6.5, that is, the pathophysiological pH of tumors and inflammatory tissues. By using poly(ethylene glycol)-poly(l-lysine) block copolymers having 45% CDM addition, different proteins with various sizes and isoelectric points are loaded successfully. By using myoglobin-loaded micelles (myo/m) as a model, the stability of the micelles in physiological conditions and the dissociation and release of functional myoglobin at pH 6.5 are successfully confirmed. Moreover, myo/m shows extended half-life in blood compared to free myoglobin and micelles assembled solely by polyion complex, indicating the potential of this system for in vivo delivery of proteins.
Assuntos
Micelas , Mioglobina , Polietilenoglicóis , Polilisina , Animais , Preparações de Ação Retardada/síntese química , Preparações de Ação Retardada/química , Preparações de Ação Retardada/farmacocinética , Preparações de Ação Retardada/farmacologia , Feminino , Células HEK293 , Meia-Vida , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Camundongos Endogâmicos BALB C , Mioglobina/química , Mioglobina/farmacocinética , Mioglobina/farmacologia , Polietilenoglicóis/química , Polietilenoglicóis/farmacocinética , Polietilenoglicóis/farmacologia , Polilisina/química , Polilisina/farmacocinética , Polilisina/farmacologiaRESUMO
Indoleamine 2,3-dioxygenase (IDO) is an immunomodulating enzyme that is overexpressed in many cancers with poor prognosis. IDO suppresses T cell immunity by catabolizing tryptophan into kynurenine (KYN), which induces apoptosis in T effector cells and enhances T regulatory cells, providing a powerful immunosuppressive mechanism in tumors. Thus, major efforts for developing IDO inhibitors have been undertaken. Among them, 1-Methyl-l-Tryptophan (MLT) and 1-Methyl-d-Tryptophan (MDT) effectively inhibit IDO in preclinical tumor models and the latter is under clinical evaluation. However, both MLT and MDT present poor pharmacokinetics, with the maximum serum concentration being below their 50% inhibitory concentration value. Herein, we have developed polymeric IDO inhibitors based on MLT, which can release active MLT after enzymatic degradation, toward establishing superior antitumor immunotherapies. These polymers were prepared by ring opening polymerization of an N-phenyl carbamate (NPC) derivative of MLT that was synthesized by carbamylation with diphenyl carbonate. By using ω-amino-poly(ethylene glycol) (PEG-NH2) as the macroinitiator, we prepared amphiphilic PEG-poly(MLT) block copolymers, which self-assembled into polymeric micelles in aqueous conditions. The PEG-poly(MLT) block copolymers could be readily degraded by chymotrypsin and the micelles were able to reduce the levels of KYN in activated macrophages. These results provide a strong rationale for pursuing MLT-based polymeric micelles as tumor-targeted prodrug systems.
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Viscosity of protein solution is one of the most troublesome issues for the high-concentration formulation of protein drugs. In this review, we summarize the practical methods that suppress the viscosity of protein solution using small molecular additives. The small amount of salts decreases the viscosity that results from electrostatic repulsion and attraction. The chaotrope suppresses the hydrophobic attraction and cluster formation, which can lower the solution viscosity. Arginine hydrochloride (ArgHCl) also suppresses the solution viscosity due to the hydrophobic and aromatic interactions between protein molecules. The small molecular additives are the simplest resolution of the high viscosity of protein solution as well as understanding of the primary cause in complex phenomena of protein interactions.
Assuntos
Proteínas/química , Anticorpos Monoclonais/química , Química Farmacêutica , Humanos , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Agregados Proteicos , Conformação Proteica , Sais/química , Albumina Sérica/química , Soluções , Eletricidade Estática , ViscosidadeRESUMO
The control of aggregation and solubilization of hen egg white protein (HEWP) is an important issue for industrial applications of one of the most familiar food protein sources. Here, we investigated the effects of edible amino acids on heat-induced aggregation of HEWP. The addition of 0.6M arginine (Arg) completely suppressed the formation of insoluble aggregates of 1mgmL-1 HEWP following heat treatment, even at 90°C for 20min. In contrast, lysine (Lys), glycine (Gly), and sodium chloride (NaCl) did little to suppress the aggregation of HEWP under the same conditions. SDS-PAGE indicated that Arg suppresses the thermal aggregation of almost all types of HEWP at 1mgmL-1. However, Arg did not suppress the thermal aggregation of HEWP at concentrations ≥10mgmL-1 and prompted the formation of aggregates. Transmission electron micrographs revealed a high-density structure of unfolded proteins in the presence of Arg. These results indicate that Arg exerts a greater suppressive effect on a protein mixture, such as HEWP, than on a single model protein. These observations may propose Arg as a safe and reasonable additive to HEWP for the elimination of microorganisms by allowing an increase in sterilization temperature.