RESUMO
Achieving therapeutic efficacy in protein replacement therapies requires sustaining pharmacokinetic (PK) profiles, while maintaining the bioactivity of circulating proteins. This is often achieved via PEGylation in protein-based therapies, but it remains challenging for proteins produced in vivo in mRNA-based therapies due to the lack of a suitable post-translational modification method. To address this issue, we integrated a genetically encoded zwitterionic polypeptide, EKP, into mRNA constructs to enhance the PK properties of product proteins. Composed of alternating glutamic acid (E), lysine (K), and proline (P), EKP exhibits unique superhydrophilic properties and low immunogenicity. Our results demonstrate that EKP fusion significantly extends the circulation half-life of proteins expressed from mRNA while preserving their bioactivity using human interferon alpha and Neoleukin-2/15 as examples. This EKP fusion technology offers a new approach to overcoming the current limitations in mRNA therapeutics and has the potential to significantly advance the development of mRNA-based protein replacement therapy.
Assuntos
Peptídeos , RNA Mensageiro , Humanos , RNA Mensageiro/genética , RNA Mensageiro/química , Peptídeos/química , Peptídeos/farmacocinética , Animais , Interferon-alfa/farmacocinética , Interferon-alfa/química , Interferon-alfa/genética , CamundongosRESUMO
Living microbial therapies have been proposed as a course of action for a variety of diseases. However, problematic interactions between the host immune system and the microbial organism present significant clinical concerns. Previously, we developed a genetically encoded superhydrophilic zwitterionic peptide, termed EKP, to mimic low-immunogenic zwitterionic materials, which have been used for the chemical modification of biologics such as protein and nucleic acid drugs to increase their in vivo circulation time and reduce their immunogenicity. Herein, we demonstrate the protective effects of the EKP polypeptide genetically cloaking the surface of Saccharomyces cerevisiae as a model microbe in both in vitro and in vivo systems. First, we show that EKP peptide cloaking suppresses the interactions between yeast cells and their specific antibodies, thereby illustrating its cloaking behavior. Then, we examine the in vitro interactions between EKP peptide surface cloaked yeast cells and murine macrophage cells, which exhibit phagocytotic behavior in the presence of foreign microbes. Our results indicate that EKP cloaking suppresses macrophage interactions and thus reduces phagocytosis. Furthermore, EKP cloaked yeast cells demonstrate a prolonged circulation time in mice in vivo.
Assuntos
Peptídeos , Saccharomyces cerevisiae , Animais , Camundongos , Peptídeos/química , Peptídeos/farmacologia , Fagocitose/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/imunologiaRESUMO
The term "zwitterionic polymers" refers to polymers that bear a pair of oppositely charged groups in their repeating units. When these oppositely charged groups are equally distributed at the molecular level, the molecules exhibit an overall neutral charge with a strong hydration effect via ionic solvation. The strong hydration effect constitutes the foundation of a series of exceptional properties of zwitterionic materials, including resistance to protein adsorption, lubrication at interfaces, promotion of protein stabilities, antifreezing in solutions, etc. As a result, zwitterionic materials have drawn great attention in biomedical and engineering applications in recent years. In this review, we give a comprehensive and panoramic overview of zwitterionic materials, covering the fundamentals of hydration and nonfouling behaviors, different types of zwitterionic surfaces and polymers, and their biomedical applications.
Assuntos
Materiais Biocompatíveis , Polímeros , Adsorção , ProteínasRESUMO
Secondary lymphoid organs (SLOs) are an important target for mRNA delivery in various applications. While the current delivery method relies on the drainage of nanoparticles to lymph nodes by intramuscular (IM) or subcutaneous (SC) injections, an efficient mRNA delivery carrier for SLOs-targeting delivery by systemic administration (IV) is highly desirable but yet to be available. In this study, we developed an efficient SLOs-targeting carrier using phosphatidylserine (PS), a well-known signaling molecule that promotes the endocytic activity of phagocytes and cellular entry of enveloped viruses. We adopted these biomimetic strategies and added PS into the standard four-component MC3-based LNP formulation (PS-LNP) to facilitate the cellular uptake of immune cells beyond the charge-driven targeting principle commonly used today. As a result, PS-LNP performed efficient protein expression in both lymph nodes and the spleen after IV administration. In vitro and in vivo characterizations on PS-LNP demonstrated a monocyte/macrophage-mediated SLOs-targeting delivery mechanism.
Assuntos
Nanopartículas , Fosfatidilserinas , Nanopartículas/química , RNA Interferente Pequeno/genética , RNA Mensageiro/genéticaRESUMO
Although recombinant adeno-associated viruses (AAVs) are considered low immunogenic and safe for gene delivery, the immunogenicity of capsids still represents a major obstacle to the readministration of AAV vectors. Here, we design an immunosuppressive zwitterionic phosphoserine (PS)-containing polypeptide to induce AAV-specific immune tolerance and eradicate the immunological response. AAVs modified with the zwitterionic PS polypeptide maintain their transduction activity and tissue tropism but suppress the induction of AAV-specific antibodies. In a hemophilia A mouse model (FVIII knockout mice), the readministration of zwitterionic PS polypeptide-modified AAV8-FVIII vectors successfully evades immunological response, corrects blood FVIII levels, and stops blood loss in tail-bleeding experiments. This potent and safe technology mimics the natural tolerance of apoptotic cells and controls the immunosuppressive, zwitterionic, and degradable polypeptide precisely, reducing the concern of toxicities upon readministrations. This work presents a new concept and a platform of engineered viral vectors by chemically linking immunosuppressive materials to AAV vectors, enabling the readministration of AAV vectors while maintaining their transduction efficiency to a considerable degree.
Assuntos
Dependovirus , Terapia Genética , Animais , Camundongos , Fosfosserina , Dependovirus/genética , Vetores Genéticos , Camundongos Knockout , Peptídeos/genéticaRESUMO
The resolution, line edge roughness, and sensitivity (RLS) trade-off has fundamentally limited the lithographic performance of chemically amplified resists. Production of next-generation transistors using extreme ultraviolet (EUV) lithography depends on a solution to this problem. A resist that simultaneously increases the effective reaction radius of its photogenerated acids while limiting their diffusion radius should provide an elegant solution to the RLS barrier. Here, we describe a generalized synthetic approach to phthalaldehyde derivatives using sulfur(VI) fluoride exchange click chemistry that dramatically expands usable chemical space by enabling virtually any non-ionic photoacid generator (PAG) to be tethered to phthalaldehyde. The resulting polymers represent the first ever PAG-tethered self-immolative resists in an architecture that simultaneously displays high contrast, extraordinary sensitivity, and low roughness under EUV exposure. We believe this class of resists will ultimately enable researchers to overcome the RLS trade-off.
Assuntos
Fluoretos , Polímeros , Polímeros/química , Ácidos/química , Difusão , EnxofreRESUMO
Therapeutic proteins frequently need to be modified with high-molecular-weight molecules to improve their pharmacokinetic properties. The genetic linkage of therapeutic proteins to a high-molecular-weight zwitterionic peptide, termed EKP, offers a promising approach. As with any protein modification, EKP could impact the structural behavior and receptor binding properties of the linked therapeutic protein, thereby altering its bioactivity. To evaluate the effects of EKP on therapeutic proteins, we study the receptor binding properties of high-molecular-weight EKP linked to the growth colony-stimulating factor (GCSF) using the genetically based yeast display platform. We find that yeast-displayed EKP-GCSF and GCSF exhibits similar binding to its receptor GCSF-R, suggesting that EKP does not hinder receptor binding. Furthermore, yeast-displayed EKP-GCSF demonstrates protection against thermal denaturation compared to GCSF. Similarly, to study the structural effects of EKP on GCSF, we employ in silico modeling using alphaFold2 in conjunction with molecular dynamics (MD) simulations. Likewise, in silico modeling reveals that EKP does not alter the structural behavior of GCSF. Finally, we demonstrate the functional benefits of EKP, by which the EKP-GCSF fusion protein produced in Escherichia coli exhibits improved pharmacokinetics and prolonged bioactivity in vivo.
Assuntos
Fator Estimulador de Colônias de Granulócitos , Saccharomyces cerevisiae , Escherichia coli/genética , Fator Estimulador de Colônias de Granulócitos/farmacologia , Peptídeos/metabolismo , Peptídeos/farmacologia , Ligação Proteica , Saccharomyces cerevisiae/metabolismoRESUMO
Zwitterionic polymers exhibit excellent nonfouling performance due to their strong surface hydrations. However, salt molecules may severely reduce the surface hydrations of typical zwitterionic polymers, making the application of these polymers in real biological and marine environments challenging. Recently, a new zwitterionic polymer brush based on the protein stabilizer trimethylamine N-oxide (TMAO) was developed as an outstanding nonfouling material. Using surface-sensitive sum frequency generation (SFG) vibrational spectroscopy, we investigated the surface hydration of TMAO polymer brushes (pTMAO) and the effects of salts and proteins on such surface hydration. It was discovered that exposure to highly concentrated salt solutions such as seawater only moderately reduced surface hydration. This superior resistance to salt effects compared to other zwitterionic polymers is due to the shorter distance between the positively and negatively charged groups, thus a smaller dipole in pTMAO and strong hydration around TMAO zwitterion. This results in strong bonding interactions between the O- in pTMAO and water, and weaker interaction between O- and metal cations due to the strong repulsion from the N+ and hydration water. Computer simulations at quantum and atomistic scales were performed to support SFG analyses. In addition to the salt effect, it was discovered that exposure to proteins in seawater exerted minimal influence on the pTMAO surface hydration, indicating complete exclusion of protein attachment. The excellent nonfouling performance of pTMAO originates from its extremely strong surface hydration that exhibits effective resistance to disruptions induced by salts and proteins.
Assuntos
PolímerosRESUMO
The lymphatic system provides a major route for the dissemination of many diseases such as tumor metastasis and virus infection. At present, treating these diseases remains a knotty task due to the difficulty of delivering sufficient drugs into lymphatics. After subcutaneous (SC) injection, the transferring of drugs to lymphatic vessels is significantly attenuated by physiological barriers in the interstitial space. Moreover, SC injection represents a highly challenging administration route for biological drugs, as it increases the risk of undesirable immune responses. Here, we demonstrate a simple and effective strategy to address this dilemma by conjugating protein therapeutics with zwitterionic poly(carboxy betaine) (PCB) polymers. PCB conjugation to l-asparaginase (ASP), a highly immunogenic enzyme drug, manifests to significantly promote the diffusion of ASP into the lymphatic system while mitigating its immunogenicity. This platform will facilitate the development of new therapies against diverse lymph-related diseases by enabling safe and efficient lymphatic drug delivery.
Assuntos
Sistemas de Liberação de Medicamentos , Vasos Linfáticos , Nanoconjugados , Preparações Farmacêuticas , Sistema LinfáticoRESUMO
Glucagon-like peptide-1 (GLP-1) is of particular interest for treating type 2 diabetes mellitus (T2DM), as it induces insulin secretion in a glucose-dependent fashion and has the potential to facilitate weight control. However, native GLP-1 is a short incretin peptide that is susceptible to fast proteolytic inactivation and rapid clearance from the circulation. Various GLP-1 analogs and bioconjugation of GLP-1 analogs have been developed to counter these issues, but these modifications are frequently accompanied by the sacrifice of potency and the induction of immunogenicity. Here, we demonstrated that with the conjugation of a zwitterionic polymer, poly(carboxybetaine) (pCB), the pharmacokinetic properties of native GLP-1 were greatly enhanced without serious negative effects on its potency and secondary structure. The pCB conjugated GLP-1 further provided glycemic control for up to 6 days in a mouse study. These results illustrate that the conjugation of pCB could realize the potential of using native GLP-1 for prolonged glycemic control in treating T2DM.
Assuntos
Diabetes Mellitus Tipo 2/sangue , Peptídeo 1 Semelhante ao Glucagon/química , Controle Glicêmico/métodos , Hipoglicemiantes/uso terapêutico , Polímeros/química , Animais , Diabetes Mellitus Tipo 2/tratamento farmacológico , Modelos Animais de Doenças , Peptídeo 1 Semelhante ao Glucagon/farmacocinética , Peptídeo 1 Semelhante ao Glucagon/uso terapêutico , Meia-Vida , Hipoglicemiantes/farmacocinética , Camundongos , Estrutura Secundária de ProteínaRESUMO
Albumin molecules are extensively used as biocompatible coatings, and poly(ethylene glycol) (PEG) materials are widely used for antifouling. PEG materials have excellent antifouling property because of their strong surface hydration. Our previous research indicates that hydration at the PEG/bovine serum albumin solution interface is stronger than that at the PEG/water interface. This research shows that this observation is general for different types of albumin molecules. Different albumins including bovine, porcine, rat, rabbit, and sheep serum albumins were studied in this research. It was found that the hydration at the PEG methacrylate (pOEGMA)/albumin solution interface is always stronger than that at the pOEGMA/water interface. Here, we define "strong interfacial hydration" as "ordered strongly hydrogen-bonded interfacial water". We believe that such a strong hydration is because of the strong hydration on the albumin surface, leading to its biocompatible property. All of the albumin molecules demonstrated stronger hydration on the pOEGMA surface compared to other protein molecules such as lysozyme and fibrinogen. The strong hydration on albumin molecules is related to the high surface coverage of glutamic acid and lysine with similar amounts.
Assuntos
Polietilenoglicóis , Soroalbumina Bovina , Adsorção , Animais , Bovinos , Metacrilatos , Muramidase , Coelhos , Ratos , Ovinos , Propriedades de Superfície , Suínos , ÁguaRESUMO
The therapeutic potential of protein drugs has been hindered by difficulties with long-term stability and rapid clearance from the body. Recombinant fusion proteins provide a scalable platform for engineered biologics, whereby a polypeptide domain is appended to alter the physical characteristics of a therapeutic protein and enhance its pharmaceutical viability. Two simple design principles for recombinant fusion proteins, based on the physical properties of the polypeptide domain, have been separately applied to address issues with the stability and delivery of biologics. "Conformationally disordered" peptides, exemplified by the homo amino acid peptide polyG, have been shown to increase the circulation half-life and bioactivity of protein therapeutics in vivo. Superhydrophilic peptides, exemplified by the alternating-charge peptide poly(EK), have been shown to increase the thermostability of proteins in vitro. The combination of superhydrophilicity and conformational disorder in a single fusion peptide could simultaneously address concerns regarding the stability and therapeutic lifetime of biologics. In the current work, we use enhanced sampling molecular dynamics (MD) simulations to investigate the conformational ensemble of poly(EK) and glycine-substituted poly(EK) variants and validate our structural predictions with circular dichroism (CD). We find the (EK)15 peptide exhibits a high propensity for forming antiparallel ß-strand secondary structures, which are stabilized by extensive salt bridging of the positive and negative side chains. MD simulations predict that limited glycine substitutions effectively disrupt the secondary structure and promote disordered conformations at physiologically relevant temperatures. We conclude that the conformational disorder of alternating-charge peptides should be taken into account to improve their suitability for drug delivery applications. We also contribute a computational approach to quantify conformational disorder in polypeptides, which should facilitate the de novo design of effective fusion proteins.
Assuntos
Peptídeos/química , Engenharia de Proteínas/métodos , Dicroísmo Circular , Glicina/química , Simulação de Dinâmica Molecular , Mutação , Peptídeos/genética , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , SoluçõesRESUMO
Inspired by the amino acid composition of natural protein surfaces, we developed a zwitterionic cloak containing multi-layers of short alternating glutamic acid and lysine (EK) peptides as a facile, highly effective and low-immunogenicity approach for the protection and delivery of biotherapeutics. Each EK layer grafted to proteins provides multiple times of new lysine reaction sites for the growth of subsequent EK layers. This unique design allows EK peptides to achieve high coating density on proteins, overcoming the limitation of traditional conjugation strategies that rely on the number of innate lysine groups. A triple-layer EK cloak manifests to successfully eliminate the specific and non-specific interactions of protected asparaginase with biological media while prolong the drug circulation time and significantly mitigate its immunogenicity in vivo, suggesting an EK peptide cloak as a promising approach to improve the safety and efficacy of biotherapeutics.
Assuntos
Ácido Glutâmico/química , Lisina/química , Peptídeos/química , Proteínas/química , Propriedades de SuperfícieRESUMO
Nonfouling zwitterionic polymers have wide applications ranging from the naval industry to biomedical engineering. Strong hydration at polymer surfaces has been proven to be crucial to their nonfouling property, but the absolute orientations of water molecules on the polymers and the competition between water and salt binding have not been elucidated. In this work, the absolute orientations of water molecules on two zwitterionic polymer brushes, poly(carboxybetaine methacrylate) (pCBMA) and poly(sulfobetaine methacrylate) (pSBMA), were measured using regular and phase-sensitive sum frequency generation (SFG) vibrational spectroscopy. The pH-dependent studies in a pH range from 2 to 12 showed that at a pH of 7, the water absolute orientations are different on the pCBMS and pSBMA surfaces. Phase-sensitive SFG studies confirmed the results obtained from the pH-dependent measurements. Salt effects on the hydration of zwitterionic polymers were examined as a function of time, which indicated that the pCBMA surface and the associated interfacial water exhibit a slow restructuring process after salt binding (likely due to the strong binding of pCBMA with water), whereas the surface of pSBMA and the associated water have a fast change after salt binding.
RESUMO
The effect of surface coatings on the performance of antifouling activity under flow can be influenced by the flow/coating interactions. This study evaluates the effect of surface coatings on antifouling activity under different flows for the analyses of coating stability. This was done by exposing DOPA-PCB-300/dopamine coated polydimethylsiloxane (PDMS) to physiological shear stresses using a recirculation system which consisted of dual chamber acrylic flow cells, tygon tubing, flow probe and meter, and perfusion pumps. The effect of shear stress induced by phosphate buffered saline flow on coating stability was characterized with differences in fibrinogen adsorption between control (coated PDMS not loaded with shear stress) and coated samples loaded with various shear stresses. Fibrinogen adsorption data showed that relative adsorption on coated PDMS that were not exposed to shear (5.73% ± 1.97%) was significantly lower than uncoated PDMS (100%, p < 0.001). Furthermore, this fouling level, although lower, was not significantly different from coated PDMS membranes that were exposed to 1 dyn/cm2 (9.55% ± 0.09%, p = 0.23), 6 dyn/cm2 (15.92% ± 10.88%, p = 0.14), and 10 dyn/cm2 (21.62% ± 13.68%, p = 0.08). Our results show that DOPA-PCB-300/dopamine coatings are stable, with minimal erosion, under shear stresses tested. The techniques from this fundamental study may be used to determine the limits of stability of coatings in long-term experiments.
Assuntos
Betaína/análogos & derivados , Incrustação Biológica/prevenção & controle , Materiais Revestidos Biocompatíveis/química , Dimetilpolisiloxanos/química , Dopamina/química , Adsorção , Fibrinogênio/química , Estresse MecânicoRESUMO
Horseradish peroxidase (HRP) holds great potential in wastewater treatment. However, its instability in harsh environments remains a major issue. Various immobilization technologies were developed to retain enzyme stability at the cost of its effectiveness. We demonstrate that zwitterionic encapsulation of HRP retained both protein stability and activity to a large degree. In a water treatment study, encapsulating HRP into a zwitterionic nanogel resulted in a three-fold increase in the catalytic oxidation efficiency of phenol molecules. In addition, zwitterionic nanocapsules exhibited the best performance when compared with nanocapsules made from other hydrophilic polymers. These results indicated that zwitterionic HRP nanocapsules hold great potential in the decontamination of organic pollutants from wastewater.
Assuntos
Peroxidase do Rábano Silvestre/química , Nanogéis/química , Fenol/química , Águas Residuárias/química , Poluentes Químicos da Água/química , Acrilamidas/síntese química , Acrilamidas/química , Armoracia/enzimologia , Estabilidade Enzimática , Peróxido de Hidrogênio/química , Oxirredução , Polímeros/síntese química , Polímeros/química , Purificação da Água/métodosRESUMO
Here, we report a simple yet effective surface-modification approach to imparting hydrophobic surfaces with superhydrophilicity using ultralow fouling/functionalizable carboxybetaine (CB) copolymers via a dip-coating technique. A new series of CB random copolymers with varying amphiphilicities were synthesized and coated on hydrophobic polypropylene (PP) and polystyrene (PS) surfaces. The nonfouling capability of each coating was screened by an enzyme-linked immunosorbent assay (ELISA) and further comprehensively assessed against 100% human serum by a Micro BCA protein assay kit. The random copolymer containing â¼30 mol % CB units showed superhydrophilicity with the highest air contact angle of more than 165° in DI water and the best nonfouling capability against 100% human blood serum. Surfaces of a 96-well plate coated with the optimal CB random copolymer had a significantly better nonfouling capability than those of a commercial 96-well plate with an ultralow attachment surface. The adhesion of mouse embryonic fibroblast cells (NIH3T3) was completely inhibited on surfaces coated with CB random copolymers. Furthermore, the optimal nonfouling CB copolymer surface was functionalized with an antigen via covalent bonding where its specific interactions with its antibody were verified. Thus, this CB random copolymer is capable of imparting both ultralow fouling and functionalizable capabilities to hydrophobic surfaces for blood-contacting devices.
Assuntos
Resinas Acrílicas/química , Incrustação Biológica/prevenção & controle , Compostos de Amônio Quaternário/química , Resinas Acrílicas/síntese química , Resinas Acrílicas/metabolismo , Adsorção , Animais , Proteínas Sanguíneas/metabolismo , Humanos , Camundongos , Células NIH 3T3 , Polipropilenos/química , Poliestirenos/química , Ligação Proteica , Compostos de Amônio Quaternário/síntese química , Compostos de Amônio Quaternário/metabolismoRESUMO
We report the synthesis of a zwitterionic carboxybetaine disulfide cross-linker (CBX-SS) and biodegradable poly(carboxybetaine) (PCB) hydrogels and nanocages (NCs) made using this cross-linker. The structure of CBX-SS combines zwitterionic carboxybetaine to confer nonfouling properties and a disulfide linkage to facilitate degradation. The physical, mechanical, and fouling characteristics of PCB hydrogels cross-linked with CBX-SS were investigated. Then, the degradation characteristics of CBX-SS-cross-linked hydrogels were evaluated through their weight loss and release of an encapsulated protein in a reducing environment. Furthermore, CBX-SS was applied to prepare degradable PCB NCs. Results show that encapsulating the highly immunogenic enzyme uricase in degradable PCB NCs eliminates or prevents an in vivo immune response to both the protein and polymer.
RESUMO
Although PEGylation reduces the immunogenicity of protein drugs to some extent, its limitations for highly immunogenic biotherapeutics have been demonstrated. Herein, a proactive strategy to alleviate the development of anti-drug antibodies (ADAs) against protein drugs by immunomodulatory bioconjugation is reported. Rapamycin was conjugated to a PEGylated protein therapeutic via a cleavable disulfide linker. The conjugated rapamycin can be released from the bioconjugate and prevent immune responses once the bioconjugate is uptaken by antigen-presenting cells. The immunomodulatory bioconjugate significantly reduced the titers of ADAs compared with a PEGylated protein. The inhibition of immune responses was specific to the conjugated antigen, avoiding systemic immune suppression and the risk of increased susceptibility to infections. The reported approach breaks the limitations of PEGylation by the proactive prevention of ADAs.
Assuntos
Anticorpos/imunologia , Imunomodulação , Anticorpos/química , Reações Antígeno-Anticorpo , Antígenos/imunologia , Humanos , Modelos Moleculares , Conformação Molecular , OxirreduçãoRESUMO
Organophosphate hydrolase (OPH) is a bacterial paraoxonase that demonstrates wide substrate affinity against a wide range of organophosphate (OP) compounds. OPH is expressed as a stable dimeric protein in prokaryotic hosts. We demonstrate, to the best of our knowledge, the first example of a stable OPH monomeric unit by expressing a fusion protein containing alternating glutamic acid and lysine sequences (EK) at the C-terminus. This method was able to disrupt formation of the dimer interface found in OPH due to the highly hydrated and nonfouling properties of EK. This OPH-EK fusion protein demonstrated a 70% increase in catalytic activity per active site and increased substrate affinity by reducing Km by approximately 70%. In addition, stability conferred by EK was able to overcome the stability loss caused by the elimination of the dimer interface. This strategy can potentially be used to aid in expressing prokaryotic proteins in eukaryotic hosts.