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1.
Molecules ; 29(9)2024 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-38731625

RESUMO

Upon a variety of environmental stresses, eukaryotic cells usually recruit translational stalled mRNAs and RNA-binding proteins to form cytoplasmic condensates known as stress granules (SGs), which minimize stress-induced damage and promote stress adaptation and cell survival. SGs are hijacked by cancer cells to promote cell survival and are consequently involved in the development of anticancer drug resistance. However, the design and application of chemical compounds targeting SGs to improve anticancer drug efficacy have rarely been studied. Here, we developed two types of SG inhibitory peptides (SIPs) derived from SG core proteins Caprin1 and USP10 and fused with cell-penetrating peptides to generate TAT-SIP-C1/2 and SIP-U1-Antp, respectively. We obtained 11 SG-inducing anticancer compounds from cell-based screens and explored the potential application of SIPs in overcoming resistance to the SG-inducing anticancer drug sorafenib. We found that SIPs increased the sensitivity of HeLa cells to sorafenib via the disruption of SGs. Therefore, anticancer drugs which are competent to induce SGs could be combined with SIPs to sensitize cancer cells, which might provide a novel therapeutic strategy to alleviate anticancer drug resistance.


Assuntos
Antineoplásicos , Sorafenibe , Grânulos de Estresse , Humanos , Sorafenibe/farmacologia , Antineoplásicos/farmacologia , Antineoplásicos/química , Grânulos de Estresse/metabolismo , Células HeLa , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Peptídeos/farmacologia , Peptídeos/química , Sobrevivência Celular/efeitos dos fármacos , Ubiquitina Tiolesterase/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Linhagem Celular Tumoral , Peptídeos Penetradores de Células/farmacologia , Peptídeos Penetradores de Células/química
2.
Biomed Pharmacother ; 174: 116610, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38642503

RESUMO

Depression ranks as the fourth most prevalent global disease, with suicide incidents occurring at a younger age. Sulpiride (SUL), an atypical antidepressant drug acting as a dopamine D2 receptor antagonist and possessing anti-inflammatory properties, exhibits limited ability to penetrate the blood brain barrier (BBB). This weak penetration hampers its inhibitory effect on prolactin release in the pituitary gland, consequently leading to hyperprolactinemia. In order to enhance the central nervous system efficacy of sulpiride and reduce serum prolactin levels, we covalently linked sulpiride to VPALR derived from the nuclear DNA repair protein ku70. In vivo study on depressive mice using intraperitoneal injection of VPALR-SUL demonstrated a significant increase in struggle time and total distance compared to those treated with only sulpiride while also reducing serum prolactin concentration. The pharmacokinetic study results showed that VPALR-SUL prolonged half-life and increased bioavailability. In conclusion, VPALR-SUL exhibited potential for enhancing sulpiride transport across the BBB, augmenting its antidepressant effects, and reducing serum prolactin levels. This study laid a foundation for improving sulpiride delivery and developing novel antidepressants.


Assuntos
Antidepressivos , Peptídeos Penetradores de Células , Prolactina , Sulpirida , Animais , Prolactina/sangue , Sulpirida/farmacologia , Antidepressivos/farmacologia , Camundongos , Masculino , Peptídeos Penetradores de Células/farmacologia , Depressão/tratamento farmacológico , Depressão/sangue , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/efeitos dos fármacos , Disponibilidade Biológica
4.
BMC Biol ; 22(1): 86, 2024 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-38637801

RESUMO

BACKGROUND: The blood-brain barrier serves as a critical interface between the bloodstream and brain tissue, mainly composed of pericytes, neurons, endothelial cells, and tightly connected basal membranes. It plays a pivotal role in safeguarding brain from harmful substances, thus protecting the integrity of the nervous system and preserving overall brain homeostasis. However, this remarkable selective transmission also poses a formidable challenge in the realm of central nervous system diseases treatment, hindering the delivery of large-molecule drugs into the brain. In response to this challenge, many researchers have devoted themselves to developing drug delivery systems capable of breaching the blood-brain barrier. Among these, blood-brain barrier penetrating peptides have emerged as promising candidates. These peptides had the advantages of high biosafety, ease of synthesis, and exceptional penetration efficiency, making them an effective drug delivery solution. While previous studies have developed a few prediction models for blood-brain barrier penetrating peptides, their performance has often been hampered by issue of limited positive data. RESULTS: In this study, we present Augur, a novel prediction model using borderline-SMOTE-based data augmentation and machine learning. we extract highly interpretable physicochemical properties of blood-brain barrier penetrating peptides while solving the issues of small sample size and imbalance of positive and negative samples. Experimental results demonstrate the superior prediction performance of Augur with an AUC value of 0.932 on the training set and 0.931 on the independent test set. CONCLUSIONS: This newly developed Augur model demonstrates superior performance in predicting blood-brain barrier penetrating peptides, offering valuable insights for drug development targeting neurological disorders. This breakthrough may enhance the efficiency of peptide-based drug discovery and pave the way for innovative treatment strategies for central nervous system diseases.


Assuntos
Peptídeos Penetradores de Células , Doenças do Sistema Nervoso Central , Humanos , Barreira Hematoencefálica/química , Células Endoteliais , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/farmacologia , Peptídeos Penetradores de Células/uso terapêutico , Encéfalo , Doenças do Sistema Nervoso Central/tratamento farmacológico
5.
ACS Appl Mater Interfaces ; 16(15): 18422-18433, 2024 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-38573069

RESUMO

DNA nanopores have emerged as powerful tools for molecular sensing, but the efficient insertion of large DNA nanopores into lipid membranes remains challenging. In this study, we investigate the potential of cell-penetrating peptides (CPPs), specifically SynB1 and GALA, to enhance the insertion efficiency of large DNA nanopores. We constructed SynB1- or GALA-functionalized DNA nanopores with an 11 nm inner diameter and visualized and quantified their membrane insertion using a TIRF microscopy-based single-liposome assay. The results demonstrated that incorporating an increasing number of SynB1 or GALA peptides into the DNA nanopore significantly enhanced the membrane perforation. Kinetic analysis revealed that the DNA nanopore scaffold played a role in prearranging the CPPs, which facilitated membrane interaction and pore formation. Notably, the use of pH-responsive GALA peptides allowed highly efficient and pH-controlled insertion of large DNA pores. Furthermore, single-channel recording elucidated that the insertion process of single GALA-modified nanopores into planar lipid bilayers was dynamic, likely forming transient large toroidal pores. Overall, our study highlights the potential of CPPs as insertion enhancers for DNA nanopores, which opens avenues for improved molecule sensing and the controlled release of cargo molecules.


Assuntos
Peptídeos Penetradores de Células , Nanoporos , Cinética , DNA/química , Bicamadas Lipídicas/química
6.
Int J Pharm ; 656: 124092, 2024 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-38583820

RESUMO

Corneal collagen crosslinking (CXL) is an effective method to halt the disease progression of keratoconus, a progressive corneal dystrophy leading to cone shaped cornea. Despite the efficacy of standard protocol, the concerning step of this procedure is epithelial debridement performed to facilitate the entry of riboflavin drug. Riboflavin, a key molecule in CXL protocol, is a sparsely permeable hydrophilic drug in corneal tissues. The present study has employed cell penetrating peptide (CPP), Tat2, to enhance the penetration of riboflavin molecule, and thereby improve currently followed CXL protocol. This study demonstrates approximately two-fold enhanced uptake of CPP riboflavin conjugate, Tat2riboflavin-5'Phosphate (RiTe conjugate), both in vitro and in vivo. Two different CXL protocols (Epi ON and Epi OFF) have been introduced and implemented in rabbit corneas using RiTe conjugate in the present study. The standard and RiTe conjugate mediated CXL procedures exhibited an equivalent extent of crosslinking in both the methods. Reduced keratocyte loss and no endothelial damage in RiTe conjugate mediated CXL further ascertains the safety of the proposed CXL protocols. Therefore, RiTe conjugate mediated CXL protocols present as potential alternatives to the standard keratoconus treatment in providing equally effective, less invasive and patient compliant treatment modality.


Assuntos
Colágeno , Córnea , Reagentes de Ligações Cruzadas , Ceratocone , Riboflavina , Ceratocone/tratamento farmacológico , Ceratocone/metabolismo , Animais , Coelhos , Colágeno/metabolismo , Riboflavina/farmacologia , Reagentes de Ligações Cruzadas/química , Córnea/metabolismo , Córnea/efeitos dos fármacos , Peptídeos Penetradores de Células , Fármacos Fotossensibilizantes/farmacologia , Fármacos Fotossensibilizantes/uso terapêutico
7.
ACS Appl Mater Interfaces ; 16(14): 17069-17079, 2024 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-38563247

RESUMO

Extracellular vesicles (EVs), including exosomes and microvesicles (MVs), transfer bioactive molecules from donor to recipient cells in various pathophysiological settings, thereby mediating intercellular communication. Despite their significant roles in extracellular signaling, the cellular uptake mechanisms of different EV subpopulations remain unknown. In particular, plasma membrane-derived MVs are larger vesicles (100 nm to 1 µm in diameter) and may serve as efficient molecular delivery systems due to their large capacity; however, because of size limitations, receptor-mediated endocytosis is considered an inefficient means for cellular MV uptake. This study demonstrated that macropinocytosis (lamellipodia formation and plasma membrane ruffling, causing the engulfment of large fluid volumes outside cells) can enhance cellular MV uptake. We developed experimental techniques to induce macropinocytosis-mediated MV uptake by modifying MV membranes with arginine-rich cell-penetrating peptides for the intracellular delivery of therapeutic molecules.


Assuntos
Micropartículas Derivadas de Células , Peptídeos Penetradores de Células , Vesículas Extracelulares , Arginina , Pinocitose , Vesículas Extracelulares/metabolismo , Peptídeos Penetradores de Células/química
8.
Int J Biol Macromol ; 267(Pt 2): 130915, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38561118

RESUMO

BACKGROUND: Chemotherapy resistance of osteosarcoma (OS) is still the crux of poor clinical curative effect.E3 ubiquitin-protein ligase Rad18 (Rad18) contributed to doxorubicin resistance in OS, which ultimately mediated DNA damage tolerance and led to a poor prognosis and chemotherapy response in patients. METHODS: In this study, doxorubicin was loaded in the process of Fe2+ and siRad18 forming nanoparticles(FSD) through coordination, chitosan modified with cell penetrating peptide (H6R6) was synthesized and coated on the surface of the NPs(FSD-CHR). FSD-CHR was then dispersed in thermosensitive hydrogel(PPP) for peritumoral injection of osteosarcoma in situ. Subsequently, the physicochemical properties and molecular biological characteristics of the drug delivery system were characterized. Finally, an osteosarcoma model was established to study the anti-tumor effects of multifunctional nanoparticles and the immunotherapy effect combined with αPD-L1. RESULTS: FSD-CHR has enhanced tumor tissue permeability, siRad18 can significantly reduce Dox-mediated DNA damage tolerance and enhance anti-tumor effects, and iron-based NPs show enhanced ROS upregulation. FSD-CHR@PPP showed significant inhibition of osteosarcoma growth in vivo and a reduced incidence of lung metastasis. In addition, siRad18 was unexpectedly found to enhance Dox-mediated immunogenic cell death (ICD).FSD-CHR@PPP combined with PD-L1 blocking significantly enhanced anti-tumor effects due to decreased PD-L1 enrichment. CONCLUSION: Hydrogel encapsulation of permeable nanoparticles provides an effective strategy for doxorubicin-resistant OS, showing that gene therapy blocking DNA damage tolerance can enhance treatment response to chemotherapy and appears to enhance the effect of ICD inducers to activate the immune system.


Assuntos
Peptídeos Penetradores de Células , Quitosana , Doxorrubicina , Terapia Genética , Hidrogéis , Nanopartículas , Osteossarcoma , Osteossarcoma/tratamento farmacológico , Osteossarcoma/patologia , Osteossarcoma/genética , Quitosana/química , Hidrogéis/química , Doxorrubicina/farmacologia , Doxorrubicina/química , Animais , Humanos , Camundongos , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/farmacologia , Nanopartículas/química , Linhagem Celular Tumoral , Terapia Genética/métodos , Permeabilidade , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/patologia , Neoplasias Ósseas/genética , Neoplasias Ósseas/terapia , Temperatura
9.
BMC Biotechnol ; 24(1): 24, 2024 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-38685061

RESUMO

BACKGROUND: Treatment with tumor-targeted toxins attempts to overcome the disadvantages of conventional cancer therapies by directing a drug's cytotoxic effect specifically towards cancer cells. However, success with targeted toxins has been hampered as the constructs commonly remain bound to the outside of the cell or, after receptor-mediated endocytosis, are either transported back to the cell surface or undergo degradation in lysosomes. Hence, solutions to ensure endosomal escape are an urgent need in treatment with targeted toxins. In this work, a molecular adapter that consists of a cell penetrating peptide and two cleavable peptides was inserted into a targeted toxin between the ribosome-inactivating protein dianthin and the epidermal growth factor. Applying cell viability assays, this study examined whether the addition of the adapter further augments the endosomal escape enhancement of the glycosylated triterpenoid SO1861, which has shown up to more than 1000-fold enhancement in the past. RESULTS: Introducing the peptide adapter into the targeted toxin led to an about 12-fold enhancement in the cytotoxicity on target cells while SO1861 caused a 430-fold increase. However, the combination of adapter and glycosylated triterpenoid resulted in a more than 4300-fold enhancement and in addition to a 51-fold gain in specificity. CONCLUSIONS: Our results demonstrated that the cleavable peptide augments the endosomal escape mediated by glycosylated triterpenoids while maintaining specificity. Thus, the adapter is a promising addition to glycosylated triterpenoids to further increase the efficacy and therapeutic window of targeted toxins.


Assuntos
Endossomos , Humanos , Endossomos/metabolismo , Endossomos/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/farmacologia , Triterpenos/farmacologia , Triterpenos/química , Linhagem Celular Tumoral , Fator de Crescimento Epidérmico/metabolismo , Fator de Crescimento Epidérmico/farmacologia
11.
Molecules ; 29(6)2024 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-38542884

RESUMO

Cell-penetrating peptides (CPPs) are invaluable tools for delivering various substances into cells by crossing biological membranes. However, the effects of cell-penetrating peptide fusion proteins on the biological activity of antibodies remain to be fully understood. Here, we engineered a recombinant protein, LP-scFv, which combines the single-chain variable region of anti-human epidermal growth factor receptor-2 with a novel and non-oxic cell-penetrating peptide as a leader peptide. The introduction of this leader peptide led to a more than twofold increase in the internalization efficiency of the single-chain antibody, as confirmed using microscopic analysis and flow cytometry. The effects of the single-chain antibodies and LP-scFv on cell viability were evaluated using the MTT assay. Both the single-chain antibodies and LP-scFv reduced the viability of BT474 and NCI-N87 cells in a dose-dependent manner while exhibiting minimal toxicity towards MCF-7 and MCF-10A cells. Further investigation into LP-scFv's mechanism revealed that the induced leader peptide does not alter the MAPK-ERK1/2 and PI3K/AKT pathways of single-chain antibodies. An enhanced antitumor activity was also confirmed in an NCI-N87 tumor xenograft model in mice with a reduction of 45.2% in tumor growth inhibition (vs. 23.1% for scFv) with a 50 mg/kg dose after orthotopic injection administration, which was equivalent to that of trastuzumab (vs. 55.7% for trastuzumab). Overall, these results indicate that LP-scFv exhibits significant permeation activity in HER2-positive cells to enhance the intracellular dose effect on antitumor activity in vitro and in vivo. This research lays the foundation for designing novel antibody-based therapies for cancer.


Assuntos
Neoplasias da Mama , Peptídeos Penetradores de Células , Anticorpos de Cadeia Única , Humanos , Animais , Camundongos , Feminino , Neoplasias da Mama/patologia , Anticorpos de Cadeia Única/farmacologia , Peptídeos Penetradores de Células/farmacologia , Peptídeos Penetradores de Células/uso terapêutico , Fosfatidilinositol 3-Quinases/metabolismo , Linhagem Celular Tumoral , Trastuzumab/uso terapêutico , Receptor ErbB-2/metabolismo , Sinais Direcionadores de Proteínas , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Acc Chem Res ; 57(8): 1098-1110, 2024 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-38530194

RESUMO

ConspectusFor the delivery of drugs, different nanosized drug carriers (e.g., liposomes, lipid nanoparticles, and micelles) have been developed in order to treat diseases that afflict society. Frequently, these vehicles are formed by the self-assembly of small molecules to encapsulate the therapeutic cargo of interest. Over decades, nanoparticles have been optimized to make them more efficient and specific to fulfill tailor-made tasks, such as specific cell targeting or enhanced cellular uptake. In recent years, lipid-based nanoparticles in particular have taken center stage; however, off-targeting side effects and poor endosomal escape remain major challenges since therapies require high efficacy and acceptable toxicity.To overcome these issues, many different approaches have been explored to make drug delivery more specific, resulting in reduced side effects, to achieve an optimal therapeutic effect and a lower required dose. The fate of nanoparticles is largely dependent on size, shape, and surface charge. A common approach to designing drug carriers with targeting capability is surface modification. Different approaches to functionalize nanoparticles have been investigated since the attachment of targeting moieties plays a significant role in whether they can later interact with surface-exposed receptors of cells. To this end, various strategies have been used involving different classes of biomolecules, such as small molecules, nucleic acids, antibodies, aptamers, and peptides.Peptides in particular are often used since there are many receptors overexpressed in different specific cell types. Furthermore, peptides can be produced and modified at a low cost, enabling high therapeutic screening. Cell-penetrating peptides (CPPs) and cell-targeting peptides (CTPs) are frequently used for this purpose. Less studied in this context are fusogenic coiled-coil peptides. Lipid-based nanoparticles functionalized with these peptides are able to avoid the endolysosomal pathway; instead such particles can be taken up by membrane fusion, resulting in increased delivery of payload. Furthermore, they can be used for targeting cells/organs but are not directed at surface-exposed receptors. Instead, they recognize complementary peptide sequences, facilitating their uptake into cells.In this Account, we will discuss peptides as moieties for enhanced cytosolic delivery, targeted uptake, and how they can be attached to lipid-based nanoparticles to alter their properties. We will discuss the properties imparted to the particles by peptides, surface modification approaches, and recent examples showing the power of peptides for in vitro and in vivo drug delivery. The main focus will be on the functionalization of lipid-based nanoparticles by fusogenic coiled-coil peptides, highlighting the relevance of this concept for the development of future therapeutics.


Assuntos
Peptídeos Penetradores de Células , Nanopartículas , Lipossomos/química , Sistemas de Liberação de Medicamentos , Nanopartículas/química , Portadores de Fármacos , Peptídeos Penetradores de Células/química , Lipídeos/química
13.
Bioconjug Chem ; 35(3): 419-431, 2024 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-38450606

RESUMO

The design of a potent amyloid-ß protein (Aß) inhibitor plays a pivotal role in the prevention and treatment of Alzheimer's disease (AD). Despite endogenous transthyretin (TTR) being recognized as an Aß inhibitor, the weak inhibitory and blood brain barrier (BBB) crossing capabilities hinder it for Aß aggregation inhibition and transport. Therefore, we have herein designed a recombinant TTR by conjugating a cationic cell penetrating peptide (penetratin, Pen), which not only enabled the fusion protein, TTR-Pen (TP), to present high BBB penetration but also greatly enhanced the potency of Aß inhibition. Namely, the protein fusion made TP positively charged, leading to a potent suppression of Aß40 fibrillization at a low concentration (1.5 µM), while a TTR concentration as high as 12.5 µM was required to gain a similar function. Moreover, TP could mitigate Aß-induced neuronal death, increase cultured cell viability from 72% to 92% at 2.5 µM, and extend the lifespan of AD nematodes from 14 to 18 d. Thermodynamic studies revealed that TP, enriched in positive charges, presented extensive electrostatic interactions with Aß40. Importantly, TP showed excellent BBB penetration performance, with a 10 times higher BBB permeability than TTR, which would allow TP to enter the brain of AD patients and participate in the transport of Aß species out of the brain. Thus, it is expected that the fusion protein has great potential for drug development in AD treatment.


Assuntos
Doença de Alzheimer , Peptídeos Penetradores de Células , Humanos , Barreira Hematoencefálica/metabolismo , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Peptídeos Penetradores de Células/farmacologia , Peptídeos Penetradores de Células/metabolismo , Pré-Albumina/metabolismo , Pré-Albumina/uso terapêutico , Peptídeos beta-Amiloides/metabolismo , Proteínas Recombinantes/uso terapêutico
14.
Protein Sci ; 33(4): e4944, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38501479

RESUMO

Antibody (Ab)-based drugs have been widely used in targeted therapies and immunotherapies, leading to significant improvements in tumor therapy. However, the failure of Ab therapy due to the loss of target antigens or Ab modifications that affect its function limits its application. In this study, we expanded the application of antibodies (Abs) by constructing a fusion protein as a versatile tool for Ab-based target cell detection, delivery, and therapy. We first constructed a SpaC Catcher (SpaCC for short) fusion protein that included the C domains of Staphylococcal protein A (SpaC) and the SpyCatcher. SpaCC conjugated with SpyTag-X (S-X) to form the SpaCC-S-X complex, which binds non-covalently to an Ab to form the Ab-SpaCC-S-X protein complex. The "X" can be a variety of small molecules such as fluoresceins, cell-penetrating peptide TAT, Monomethyl auristatin E (MMAE), and DNA. We found that Ab-SpaCC-S-FITC(-TAT) could be used for target cell detection and delivery. Besides, we synthesized the Ab-SpaCC-SN3-MMAE complex by linking Ab with MMAE by SpaCC, which improved the cytotoxicity of small molecule toxins. Moreover, we constructed an Ab-DNA complex by conjugating SpaCC with the aptamer (Ap) and found that Ab-SpaCC-SN3-Ap boosted the tumor-killing function of T-cells by retargeting tumor cells. Thus, we developed a multifunctional tool that could be used for targeted therapies and immunotherapies, providing a cheap and convenient novel drug development strategy.


Assuntos
Peptídeos Penetradores de Células , Neoplasias , Humanos , Neoplasias/genética , Neoplasias/terapia , Imunoterapia , Anticorpos , DNA , Linhagem Celular Tumoral
15.
ACS Chem Biol ; 19(4): 908-915, 2024 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-38525961

RESUMO

The efficient cytosolic delivery of proteins is critical for advancing novel therapeutic strategies. Current delivery methods are severely limited by endosomal entrapment, and detection methods lack sophistication in tracking the fate of delivered protein cargo. HaloTag, a commonly used protein in chemical biology and a challenging delivery target, is an exceptional model system for understanding and exploiting cellular delivery. Here, we employed a combinatorial strategy to direct HaloTag to the cytosol. We established the use of Virginia Orange, a pH-sensitive fluorophore, and Janelia Fluor 585, a similar but pH-agnostic fluorophore, in a fluorogenic assay to ascertain protein localization within human cells. Using this assay, we investigated HaloTag delivery upon modification with cell-penetrating peptides, carboxyl group esterification, and cotreatment with an endosomolytic agent. We found efficacious cytosolic entry with two distinct delivery methods. This study expands the toolkit for detecting the cytosolic access of proteins and highlights that multiple intracellular delivery strategies can be used synergistically to effect cytosolic access. Moreover, HaloTag is poised to serve as a platform for the delivery of varied cargo into human cells.


Assuntos
Peptídeos Penetradores de Células , Corantes Fluorescentes , Hidrolases , Humanos , Transporte Biológico , Peptídeos Penetradores de Células/metabolismo , Citosol/metabolismo , Endossomos/metabolismo , Concentração de Íons de Hidrogênio , Corantes Fluorescentes/química
16.
Biomater Sci ; 12(9): 2321-2330, 2024 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-38488841

RESUMO

Nanotherapies, valued for their high efficacy and low toxicity, frequently serve as antitumor treatments, but do not readily penetrate deep into tumor tissues and cells. Here we developed an improved tumor-penetrating peptide (TPP)-based drug delivery system. Briefly, the established TPP iNGR was modified to generate a linear NGR peptide capable of transporting nanotherapeutic drugs into tumors through a CendR pathway-dependent, neuropilin-1 receptor-mediated process. Although TPPs have been reported to reach intended tumor targets, they often fail to penetrate cell membranes to deliver tumoricidal drugs to intracellular targets. We addressed this issue by harnessing cell penetrating peptide technology to develop a liposome-based multibarrier-penetrating delivery system (mbPDS) with improved synergistic drug penetration into deep tumor tissues and cells. The system incorporated doxorubicin-loaded liposomes coated with nona-arginine (R9) CPP and cyclic iNGR (CRNGRGPDC) molecules, yielding Lip-mbPDS. Lip-mbPDS tumor-targeting, tumor cell/tissue-penetrating and antitumor capabilities were assessed using CD13-positive human fibrosarcoma-derived cell (HT1080)-based in vitro and in vivo tumor models. Lip-mbPDS evaluation included three-dimensional layer-by-layer confocal laser scanning microscopy, cell internalization/toxicity assays, three-dimensional tumor spheroid-based penetration assays and antitumor efficacy assays conducted in an animal model. Lip-mbPDS provided enhanced synergistic drug penetration of multiple biointerfaces for potentially deep tumor therapeutic outcomes.


Assuntos
Peptídeos Penetradores de Células , Doxorrubicina , Sistemas de Liberação de Medicamentos , Lipossomos , Humanos , Animais , Doxorrubicina/química , Doxorrubicina/administração & dosagem , Doxorrubicina/farmacologia , Peptídeos Penetradores de Células/química , Linhagem Celular Tumoral , Lipossomos/química , Camundongos , Portadores de Fármacos/química , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Antineoplásicos/farmacologia , Camundongos Nus , Peptídeos Cíclicos/química , Peptídeos Cíclicos/administração & dosagem
17.
Biophys J ; 123(7): 901-908, 2024 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-38449310

RESUMO

A cell-penetrating peptide (CPP) is a short amino-acid sequence capable of efficiently translocating across the cellular membrane of mammalian cells. However, the potential of CPPs as a delivery vector is hampered by the strong reduction of its translocation efficiency when it bears an attached molecular cargo. To overcome this problem, we used previously developed diblock copolymers of elastin-like polypeptides (ELPBCs), which we end functionalized with TAT (transactivator of transcription), an archetypal CPP built from a positively charged amino acid sequence of the HIV-1 virus. These ELPBCs self-assemble into micelles at a specific temperature and present the TAT peptide on their corona. These micelles can recover the lost membrane affinity of TAT and can trigger interactions with the membrane despite the presence of a molecular cargo. Herein, we study the influence of membrane surface charge on the adsorption of TAT-functionalized ELP micelles onto giant unilamellar vesicles (GUVs). We show that the TAT-ELPBC micelles show an increased binding constant toward negatively charged membranes compared to neutral membranes, but no translocation is observed. The affinity of the TAT-ELPBC micelles for the GUVs displays a stepwise dependence on the lipid charge of the GUV, which, to our knowledge, has not been reported previously for interactions between peptides and lipid membranes. By unveiling the key steps controlling the interaction of an archetypal CPP with lipid membranes, through regulation of the charge of the lipid bilayer, our results pave the way for a better design of delivery vectors based on CPPs.


Assuntos
Peptídeos Penetradores de Células , Micelas , Animais , Polipeptídeos Semelhantes à Elastina , Adsorção , Bicamadas Lipídicas/química , Peptídeos/química , Lipossomas Unilamelares/química , Peptídeos Penetradores de Células/química , Mamíferos/metabolismo
18.
Molecules ; 29(6)2024 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-38543041

RESUMO

Design of amyloid ß-protein (Aß) inhibitors is considered an effective strategy for the prevention and treatment of Alzheimer's disease (AD). However, the limited blood-brain barrier (BBB) penetration and poor Aß-targeting capability restricts the therapeutic efficiency of candidate drugs. Herein, we have proposed to engineer transthyretin (TTR) by fusion of the Aß-targeting peptide KLVFF and cell-penetrating peptide Penetratin to TTR, and derived a fusion protein, KLVFF-TTR-Penetratin (KTP). Moreover, to introduce the scavenging activity for reactive oxygen species (ROS), a nanocomposite of KTP and manganese dioxide nanoclusters (KTP@MnO2) was fabricated by biomineralization. Results revealed that KTP@MnO2 demonstrated significantly enhanced inhibition on Aß aggregation as compared to TTR. The inhibitory effect was increased from 18%, 33%, and 49% (10, 25, and 50 µg/mL TTR, respectively) to 52%, 81%, and 100% (10, 25, and 50 µg/mL KTP@MnO2). In addition, KTP@MnO2 could penetrate the BBB and target amyloid plaques. Moreover, multiple ROS, including hydroxyl radicals, superoxide radicals, hydrogen peroxide, and Aß-induced-ROS, which cannot be scavenged by TTR, were scavenged by KTP@MnO2, thus resulting in the mitigation of cellular oxidative damages. More importantly, cell culture and in vivo experiments with AD nematodes indicated that KTP@MnO2 at 50 µg/mL increased the viability of Aß-treated cells from 66% to more than 95%, and completely cleared amyloid plaques in AD nematodes and extended their lifespan by 7 d. Overall, despite critical aspects such as the stability, metabolic distribution, long-term biotoxicity, and immunogenicity of the nanocomposites in mammalian models remaining to be investigated, this work has demonstrated the multifunctionality of KTP@MnO2 for targeting Aß in vivo, and provided new insights into the design of multifunctional nanocomposites of protein-metal clusters against AD.


Assuntos
Doença de Alzheimer , Peptídeos Penetradores de Células , Fragmentos de Peptídeos , Animais , Humanos , Peptídeos beta-Amiloides/metabolismo , Doença de Alzheimer/metabolismo , Compostos de Manganês/farmacologia , Óxidos/farmacologia , Pré-Albumina/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Placa Amiloide/metabolismo , Mamíferos/metabolismo
19.
Mol Pharm ; 21(5): 2097-2117, 2024 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-38440998

RESUMO

Currently, one of the most significant and rapidly growing unmet medical challenges is the treatment of neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). This challenge encompasses the imperative development of efficacious therapeutic agents and overcoming the intricacies of the blood-brain barrier for successful drug delivery. Here we focus on the delivery aspect with particular emphasis on cell-penetrating peptides (CPPs), widely used in basic and translational research as they enhance drug delivery to challenging targets such as tissue and cellular compartments and thus increase therapeutic efficacy. The combination of CPPs with nanomaterials such as nanoparticles (NPs) improves the performance, accuracy, and stability of drug delivery and enables higher drug loads. Our review presents and discusses research that utilizes CPPs, either alone or in conjugation with NPs, to mitigate the pathogenic effects of neurodegenerative diseases with particular reference to AD and PD.


Assuntos
Barreira Hematoencefálica , Peptídeos Penetradores de Células , Sistemas de Liberação de Medicamentos , Nanopartículas , Doenças Neurodegenerativas , Doença de Parkinson , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/administração & dosagem , Humanos , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/efeitos dos fármacos , Sistemas de Liberação de Medicamentos/métodos , Nanopartículas/química , Doenças Neurodegenerativas/tratamento farmacológico , Animais , Doença de Parkinson/tratamento farmacológico , Doença de Alzheimer/tratamento farmacológico
20.
ACS Appl Mater Interfaces ; 16(9): 11159-11171, 2024 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-38385360

RESUMO

For the improved delivery of cancer therapeutics and imaging agents, the conjugation of cell-penetrating peptides (CPPs) increases the cellular uptake and water solubility of agents. Among the various CPPs, arginine-rich peptides have been the most widely used. Combining CPPs with enzyme-responsive peptides presents an innovative strategy to target specific intracellular enzymes in cancer cells and when combined with the appropriate click chemistry can enhance theranostic drug delivery through the formation of intracellular self-assembled nanostructures. However, one drawback of CPPs is their high positive charge which can cause nonspecific binding, leading to off-target accumulation and potential toxicity. Hence, balancing cell-specific penetration, toxicity, and biocompatibility is essential for future clinical efficacy. We synthesized six cancer-specific, legumain-responsive RnAANCK peptides containing one to six arginine residues, with legumain being an asparaginyl endopeptidase that is overexpressed in aggressive prostate tumors. When conjugated to Alexa Fluor 488, R1-R6AANCK peptides exhibited a concentration- and time-dependent cell penetration in prostate cancer cells, which was higher for peptides with higher R values, reaching a plateau after approximately 120 min. Highly aggressive DU145 prostate tumor cells, but not less aggressive LNCaP cells, self-assembled nanoparticles in the cytosol after the cleavage of the legumain-specific peptide. The in vivo biocompatibility was assessed in mice after the intravenous injection of R1-R6AANCK peptides, with concentrations ranging from 0.0125 to 0.4 mmol/kg. The higher arginine content in R4-6 peptides showed blood and urine indicators for the impairment of bone marrow, liver, and kidney function in a dose-dependent manner, with instant hemolysis and morbidity in extreme cases. These findings underscore the importance of designing peptides with the optimal arginine residue length for a proper balance of cell-specific penetration, toxicity, and in vivo biocompatibility.


Assuntos
Peptídeos Penetradores de Células , Neoplasias , Animais , Camundongos , Arginina/química , Peptídeos Penetradores de Células/química , Neoplasias/tratamento farmacológico
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