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1.
Life Sci ; 266: 118886, 2021 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-33310044

RESUMO

AIMS: Triple negative breast cancer (TNBC) has drawn more and more attention due to its high mitotic indices, high metastatic rate and poor prognosis. Gene therapy, especially RNA interference (RNAi), has become a promising targeted therapy. However, improvement of transfection efficiency and discovery of target genes are major problems for the delivery of small interfering RNAs (siRNA). MATERIALS AND METHODS: In the present study, we developed GALA- and CREKA-modified PEG-SS-PEI to deliver siRNAs targeting on EGFR and BRD4 for TNBC therapy. The PEG-SS-PEI/siRNA complexes were prepared by electrostatic interaction and characterized by dynamic light scattering (DLS) and transmission electron microscope (TEM). The release characteristic, stability, cellular uptake and intracellular localization of the complexes were also studied. The effect of the complexes on cell viability was measured in MDA-MB-231 and HUVEC cells. The in vitro anti-tumor activities of the complexes were analyzed by Transwell invasion assay and wound healing assay. The gene silencing effect was evaluated by quantitative real time-polymerase chain reaction (qRT-PCR) and western blot. KEY FINDINGS: The results revealed that the GALA- and CREKA-modified PEG-SS-PEI/siRNA complexes showed excellent transfection efficiency with redox-sensitive release profile and good biological compatibility. The complexes protected siRNA from the degradation of RNA enzymes. The complexes significantly inhibited the proliferation, invasion and migration of MDA-MB-231 cells via the synergistic inhibition of EGFR/PI3K/Akt and BRD4/c-Myc pathways. SIGNIFICANCE: Taken together, co-delivery of siEGFR and siBRD4 by GALA-PEG-SS-PEI and CREKA-PEG-SS-PEI may provide a more effective strategy for the treatment of TNBC.


Assuntos
Proteínas de Ciclo Celular/administração & dosagem , Peptídeos Penetradores de Células/química , Inativação Gênica , Polietilenoglicóis/química , Polietilenoimina/análogos & derivados , RNA Interferente Pequeno/administração & dosagem , Fatores de Transcrição/administração & dosagem , Neoplasias de Mama Triplo Negativas/terapia , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Proliferação de Células , Receptores ErbB/administração & dosagem , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Feminino , Terapia Genética , Humanos , Polietilenoimina/química , RNA Interferente Pequeno/genética , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Células Tumorais Cultivadas
2.
Biomed Pharmacother ; 134: 111154, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33360931

RESUMO

A promising direction in Biopharmaceuticals is the development of specific peptide-based systems to improve drug delivery. This approach may increase tumor specificity and drug penetration into the target cell. Similar systems have been designed for several antitumor drugs. However, for photodynamic therapy drugs, such studies are not yet enough. Previously, we have developed a method of inclusion of chlorin e6 (Ce6), a photosensitizer used in photodynamic therapy, in phospholipid nanoparticles with a diameter of up to 30 nm, and reported an increase in its effectiveness in the experiments in vivo. In this work, we propose to modify a previously developed delivery system for Ce6 by the addition of cell-penetrating (R7) and/or targeting NGR peptides. The interaction of the compositions developed with HepG2 and MCF-7 tumor cells is shown. The expression of CD13 protein with affinity to NGR on the surface of these cells has been studied using flow cytometry. The expression of this protein on the HepG2 cells and its absence on MCF-7 was demonstrated. After incubation of tumor cells with the resulting Ce6 compositions, we evaluated the cellular accumulation, photoinduced, and dark cytotoxicity of the drugs. After irradiation, the highest level of cytotoxicity was observed when R7 peptide was added to the system, either alone or in combination with NGR. In addition to R7, the NGR-motif peptide increased the internalization of Ce6 in HepG2 cells without affecting its photodynamic activity. In this work we also discuss possible mechanisms of action of the cell-penetrating peptide when attached to phospholipid nanoparticles.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Antígenos CD13/metabolismo , Carcinoma Hepatocelular/tratamento farmacológico , Peptídeos Penetradores de Células/metabolismo , Portadores de Fármacos , Neoplasias Hepáticas/tratamento farmacológico , Nanopartículas , Fosfolipídeos/química , Fotoquimioterapia , Fármacos Fotossensibilizantes/farmacologia , Porfirinas/farmacologia , Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Carcinoma Hepatocelular/enzimologia , Carcinoma Hepatocelular/patologia , Sobrevivência Celular/efeitos dos fármacos , Peptídeos Penetradores de Células/química , Composição de Medicamentos , Células Hep G2 , Humanos , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/fisiopatologia , Células MCF-7 , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/metabolismo , Porfirinas/química , Porfirinas/metabolismo
3.
PLoS One ; 15(8): e0237473, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32813720

RESUMO

Solid phase peptide synthesis (SPPS) has enabled widespread use of synthetic peptides in applications ranging from pharmaceuticals to materials science. The demand for synthetic peptides has driven recent efforts to produce automated SPPS synthesizers which utilize fluid-handling components common to chemistry laboratories to drive costs down to several thousand dollars. Herein, we describe the design and validation of a more 'frugal' SPPS synthesizer that uses inexpensive, consumer-grade fluid-handling components to achieve a prototype price point between US$300 and $600. We demonstrated functionality by preparing and characterizing peptides with a variety of distinct properties including binding functionality, nanoscale self-assembly, and oxidation-induced fluorescence. This system yielded micromoles of peptide at a cost of approximately $1/residue, a cost which may be further reduced by optimization and bulk purchasing.


Assuntos
Peptídeos/síntese química , Técnicas de Síntese em Fase Sólida/métodos , Sequência de Aminoácidos , Automação , Peptídeos Penetradores de Células/síntese química , Peptídeos Penetradores de Células/química , Desenho de Equipamento , Fluorometria , Nanoestruturas/química , Oxirredução , Peptídeos/química , Técnicas de Síntese em Fase Sólida/economia , Técnicas de Síntese em Fase Sólida/instrumentação
4.
Sci Rep ; 10(1): 5809, 2020 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-32242067

RESUMO

Extracellular matrix in solid tumors has emerged as a specific, stable, and abundant target for affinity-guided delivery of anticancer drugs. Here we describe the homing peptide that interacts with the C-isoform of Tenascin-C (TNC-C) upregulated in malignant tissues. TNC-C binding PL3 peptide (amino acid sequence: AGRGRLVR) was identified by in vitro biopanning on recombinant TNC-C. Besides TNC-C, PL3 interacts via its C-end Rule (CendR) motif with cell-and tissue penetration receptor neuropilin-1 (NRP-1). Functionalization of iron oxide nanoworms (NWs) and metallic silver nanoparticles (AgNPs) with PL3 peptide increased tropism of systemic nanoparticles towards glioblastoma (GBM) and prostate carcinoma xenograft lesions in nude mice (eight and five-fold respectively). Treatment of glioma-bearing mice with proapoptotic PL3-guided NWs improved the survival of the mice, whereas treatment with untargeted particles had no effect. PL3-coated nanoparticles were found to accumulate in TNC-C and NRP-1-positive areas in clinical tumor samples, suggesting a translational relevance. The systemic tumor-targeting properties and binding of PL3-NPs to the clinical tumor sections, suggest that the PL3 peptide may have applications as a targeting moiety for the selective delivery of imaging and therapeutic agents to solid tumors.


Assuntos
Antineoplásicos/farmacocinética , Peptídeos Penetradores de Células/farmacocinética , Glioblastoma/metabolismo , Neoplasias da Próstata/metabolismo , Tenascina/metabolismo , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Peptídeos Penetradores de Células/administração & dosagem , Peptídeos Penetradores de Células/química , Feminino , Humanos , Masculino , Nanopartículas Metálicas/química , Camundongos , Camundongos Nus , Neuropilina-1/metabolismo , Células PC-3 , Ligação Proteica , Prata/química , Distribuição Tecidual , Microambiente Tumoral
5.
Int J Nanomedicine ; 15: 1837-1851, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32256063

RESUMO

Introduction: Gold nanorods are highly reactive, have a large surface-to-volume ratio, and can be functionalized with biomolecules. Gold nanorods can absorb infrared electromagnetic radiation, which is subsequently dispersed as local heat. Gold nanoparticles can be used as powerful tools for the diagnosis and therapy of different diseases. To improve the biological barrier permeation of nanoparticles with low cytotoxicity, in this study, we conjugated gold nanorods with cell-penetrating peptides (oligoarginines) and with the amphipathic peptide CLPFFD. Methods: We studied the interaction of the functionalized gold nanorods with biological membrane models (liposomes) by dynamic light scattering, transmission electron microscopy and the Langmuir balance. Furthermore, we evaluated the effects on cell viability and permeability with an MTS assay and TEM. Results and Discussion: The interaction study by DLS, the Langmuir balance and cryo-TEM support that GNR-Arg7CLPFFD enhances the interactions between GNRs and biological membranes. In addition, cells treated with GNR-Arg7CLPFFD internalized 80% more nanoparticles than cells treated with GNR alone and did not induce cell damage. Conclusion: Our results indicate that incorporation of an amphipathic sequence into oligoarginines for the functionalization of gold nanorods enhances biological membrane nanoparticle interactions and nanoparticle cell permeability with respect to nanorods functionalized with oligoarginine. Overall, functionalized gold nanorods with amphipathic arginine rich peptides might be candidates for improving drug delivery by facilitating biological barrier permeation.


Assuntos
Peptídeos Penetradores de Células/química , Lipossomos/farmacocinética , Nanotubos/química , Arginina/química , Linhagem Celular Tumoral , Sobrevivência Celular , Peptídeos Penetradores de Células/farmacocinética , Sistemas de Liberação de Medicamentos , Difusão Dinâmica da Luz , Ouro/química , Humanos , Lipossomos/química , Nanopartículas Metálicas/química , Microscopia Eletrônica de Transmissão , Peptídeos/química
6.
J Nanobiotechnology ; 18(1): 48, 2020 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-32183823

RESUMO

BACKGROUND: Specific targeting ability and good cell penetration are two critical requirements of tumor-targeted delivery systems. In the present work, we developed a novel matrix metalloprotein-triggered, cell-penetrating, peptide-modified, star-shaped nanoparticle (NP) based on a functionalized copolymer (MePEG-Peptide-Tri-CL), with the peptide composed of GPLGIAG (matrix metalloprotein-triggered peptide for targeted delivery) and r9 (cell-penetrating peptide for penetration improvement) to enhance its biological specificity and therapeutic effect. RESULTS: Based on the in vitro release study, a sustained release profile was achieved for curcumin (Cur) release from the Cur-P-NPs at pH 7.4. Furthermore, the release rate of Cur was accelerated in the enzymatic reaction. MTT assay results indicated that the biocompatibility of polymer NPs (P-NPs) was inversely related to the NP concentration, while the efficiency toward tumor cell inhibition was positively related to the Cur-P-NP concentration. In addition, Cur-P-NPs showed higher fluorescence intensity than Cur-NPs in tumor cells, indicating improved penetration of tumor cells. An in vivo biodistribution study further demonstrated that Cur-P-NPs exhibited stronger targeting to A549 xenografts than to normal tissue. Furthermore, the strongest tumor growth inhibition (76.95%) was observed in Cur-P-NP-treated A549 tumor xenograft nude mice, with slight pulmonary toxicity. CONCLUSION: All results demonstrated that Cur-P-NP is a promising drug delivery system that possesses specific enzyme responsiveness for use in anti-tumor therapy.


Assuntos
Peptídeos Penetradores de Células/farmacologia , Sistemas de Liberação de Medicamentos/métodos , Metaloproteínas/farmacologia , Nanopartículas/administração & dosagem , Animais , Linhagem Celular Tumoral , Peptídeos Penetradores de Células/química , Curcumina/farmacologia , Portadores de Fármacos , Liberação Controlada de Fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Nanopartículas/química , Tamanho da Partícula , Polímeros/química , Distribuição Tecidual , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Biochim Biophys Acta Biomembr ; 1862(6): 183262, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32147356

RESUMO

The naturally occurring host defense peptide (HDP), aurein 2.2, secreted by the amphibian Litoria aurea, acts as a moderate antibacterial, affecting Gram positive bacteria such as Staphylococcus aureus by forming selective ion pores. In a quest to find more active analogues of aurein 2.2, peptides 73 and 77 were discovered. These peptides were rich in arginine and tryptophan and found to have MICs of 4 µg/mL. Here we examined what impact the increased charge from +2 to +3 and a slight increase in hydrophobic moment relative to aurein 2.2 had on the mechanism of action of these two analogues. Using a time-kill assay, both peptides 73 and 77 were found to kill bacteria more effectively than the parent peptide. Using solution CD and NMR, the peptides were found to not adopt a continuous α-helical structure, i.e. the analogues were not helical from residue 1-13 like the parent peptide. Results obtained from oriented CD (OCD), DiSC35 and pyranine assays and a gel retardation experiment showed that the peptides did not function by membrane perturbation and further showed that peptide 73 and 77 did not interact with DNA. Overall, the data were consistent with these peptides acting as cell penetrating peptides with intracellular targets, which did not appear to be DNA.


Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Proteínas de Anfíbios/química , Anfíbios , Animais , Peptídeos Catiônicos Antimicrobianos/farmacologia , Peptídeos Penetradores de Células/química , Descoberta de Drogas , Bactérias Gram-Positivas/efeitos dos fármacos , Conformação Proteica , Relação Estrutura-Atividade
8.
Dalton Trans ; 49(7): 2323-2330, 2020 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-32022053

RESUMO

A superoxide dismutase mimic (Mn1) was functionalized with three positively charged-peptides: RRRRRRRRR (Mn1-R9), RRWWWRRWRR (Mn1-RW9) or Fx-r-Fx-K (Mn1-MPP). Characterization of the physico-chemical properties of the complexes show that they share similar binding affinity for Mn2+, apparent reduction potential and intrinsic superoxide dismutase activity. However, their accumulation in cells is different (Mn1-R9 < Mn1-MPP < Mn1-RW9 < Mn1), as well as their subcellular distribution. In addition, the three functionalized-complexes display a better anti-inflammatory activity than Mn1 when assayed at 10 µM. This improvement is due to a combination of an anti-inflammatory effect of the peptidyl moiety itself, and of the SOD mimic for Mn1-RW9 and Mn1-MPP. In contrast, the enhanced anti-inflammatory activity of Mn1-R9 is solely due to the SOD mimic.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Peptídeos Penetradores de Células/farmacologia , Superóxido Dismutase/metabolismo , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/metabolismo , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/metabolismo , Células HT29 , Humanos , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Estrutura Molecular , Superóxido Dismutase/química , Termodinâmica
9.
Drug Dev Ind Pharm ; 46(3): 427-442, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32070151

RESUMO

The combination of nanoparticles (NPs) and cell-penetrating peptide (CPP) represents a new opportunity to develop plasmid DNA (pDNA) delivery systems with desirable properties for lung delivery. In this study, poly(lactide-co-glycolide) (PLGA) NPs containing pDNA were formulated with and without CPP using a double-emulsion technique. NPs were characterized in regards of size, surface charge, release profile, pDNA encapsulation efficiency and pDNA integrity. Cellular uptake, intracellular trafficking, uptake mechanism and pDNA expression were assessed in both A549 and Beas-2B cells. Manufactured PLGA-NPs efficiently encapsulated pDNA with approximately 50% released in the first 24 h of incubation. Addition of CPP was essential to promote NP internalization in both cell lines, with 83.85 ± 1.2% and 96.76 ± 1.7% of Beas-2B and A549 cells, respectively, with internalized NP-DNA-CPP after 3 h of incubation. Internalization appears to occur mainly via clathrin-mediated endocytosis, with other pathways also being used by the different cell lines. An endosomal-escape mechanism seems to happen in both cell lines, and eGFP expression was observed in Beas-2B after 96 h of incubation. In summary, the NP-DNA-CPP delivery system efficiently encapsulated and protected pDNA structure and is being investigated as a promising tool for gene delivery to the lungs.


Assuntos
Peptídeos Penetradores de Células/química , DNA/administração & dosagem , Nanopartículas , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Células A549 , Linhagem Celular , Clatrina/metabolismo , Emulsões , Endocitose , Células Epiteliais , Técnicas de Transferência de Genes , Humanos , Pulmão/citologia , Pulmão/metabolismo , Plasmídeos
10.
Pharmacol Rep ; 72(2): 456-464, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32048262

RESUMO

BACKGROUND: Parkinson's disease (PD) is the second most common neurodegenerative disease of the elderly. Current therapies are only symptomatic, and have no disease-modifying effect. Therefore, disease progresses continuously over time, presenting with both motor and non-motor features. The precise molecular basis for PD is still elusive, but the aggregation of the protein alpha-synuclein (α-syn) is a key pathological hallmark of the disease and is, therefore, a major focus of current research. Considering the intrinsic properties of cell-penetrating peptides (CPPs) for mediating drug delivery of neurotherapeutics across the blood brain barrier (BBB), these might open novel opportunities for the development of new solutions for the treatment of brain-related aspects of PD and other neurodegenerative disorders. METHODS: Here, we synthesized solid-phase CPPs using an amphipathic model peptide (MAP) conjugated with the drug Rasagiline (RAS), which we named RAS-MAP, and evaluated its effect on α-syn inclusion formation in a human cell-based model of synucleinopathy. RESULTS: We found that treatment with RAS-MAP at low concentrations (1-3 µM) reduced α-syn aggregation in cells. CONCLUSIONS: For the first time, we report that conjugation of a current drug used in the therapy of PD with CPP reduces α-syn aggregation, which might prove beneficial in PD and other synucleinopathies.


Assuntos
Peptídeos Penetradores de Células/química , Portadores de Fármacos/química , Indanos/farmacologia , Fármacos Neuroprotetores/farmacologia , Agregação Patológica de Proteínas/prevenção & controle , alfa-Sinucleína/metabolismo , Barreira Hematoencefálica/metabolismo , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Humanos , Indanos/administração & dosagem , Fármacos Neuroprotetores/administração & dosagem , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/metabolismo , Técnicas de Síntese em Fase Sólida
11.
Biomater Sci ; 8(7): 1961-1972, 2020 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-32064471

RESUMO

The development of magnetic resonance imaging (MRI) contrast agents with high sensitivity and good biocompatibility is of great value for the diagnosis of primary hepatocellular carcinoma (HCC). Here, a novel MRI contrast agent based on calcium phosphate (CaP) nanoparticles modified with a liver cancer cell targeting peptide A54 (A54-CaP) was fabricated. The T1-positive contrast agent Gd-DTPA was encapsulated inside the nanoparticles (A54-CaPNPs), with a mean diameter of 30 nm and a high encapsulation efficiency of 92.73%. The A54-CaPNP solution exhibited higher longitudinal relaxivity (6.07 mM-1 s-1) than that of the clinically used MRI contrast agent Gd-DTPA (3.56 mM-1 s-1). A much higher accumulation of the nanoparticles in the liver cells was observed, which was directed by the A54 targeting peptide. Furthermore, the MRI diagnostic efficiency of A54-CaPNPs was systematically investigated in an orthotopic liver cancer model and primary HCC model. In vivo MRI experiments showed that A54-CaPNPs had higher sensitivity in the BEL-7402 orthotopic liver cancer model with a more remarkable contrast enhancement and a longer imaging time compared to those without A54 modification. Moreover, the experiments on primary HCC models suggested that A54-CaPNPs showed greatly enhanced MR imaging performance in comparison with Gd-DTPA. These results suggest that A54-CaPNPs possess great potential to enable the non-invasive early diagnosis of primary HCC for timely surgical resection.


Assuntos
Fosfatos de Cálcio/química , Carcinoma Hepatocelular/diagnóstico por imagem , Peptídeos Penetradores de Células/administração & dosagem , Meios de Contraste/administração & dosagem , Gadolínio/química , Neoplasias Hepáticas/diagnóstico por imagem , Animais , Linhagem Celular Tumoral , Peptídeos Penetradores de Células/química , Meios de Contraste/química , Detecção Precoce de Câncer , Feminino , Células Hep G2 , Humanos , Imagem por Ressonância Magnética , Masculino , Camundongos , Nanopartículas , Transplante de Neoplasias , Tamanho da Partícula
12.
Biochim Biophys Acta Biomembr ; 1862(8): 183212, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32057757

RESUMO

The LAH4 family of amphipathic peptides exhibits pronounced antimicrobial, cell penetrating and nucleic acid transfection activities. Furthermore, variants were designed with potent lentiviral transduction enhancement. When viewed along a helical wheel the four histidines are arranged to form an amphipathic structure. In order to optimize some of these biological activities the number of leucine and alanine residues exposed to the hydrophilic surface was systematically varied which resulted in the design of vectofusin a peptide with strong lentiviral transduction enhancement activities. Here the series of peptides with varying numbers of alanine or leucine residues, respectively, framed by the histidines was tested for their calcein release activity. Interestingly, the membrane pore formation and DNA transfection activities show a clear correlation with the hydrophilic angle. In contrast the membrane partitioning and the propensity to adopt helical conformations was hardly affected as long as the hydrophilic angle did not exceed a limiting value of 150°.


Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , DNA/genética , Histidina/genética , Membranas/efeitos dos fármacos , Alanina/genética , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/genética , Linhagem Celular Tumoral , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/genética , Peptídeos Penetradores de Células/farmacologia , DNA/efeitos dos fármacos , Humanos , Interações Hidrofóbicas e Hidrofílicas/efeitos dos fármacos , Lentivirus/genética , Leucina/genética , Membranas/metabolismo , Porosidade , Transfecção
13.
Nanoscale ; 12(6): 3855-3870, 2020 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-31996884

RESUMO

We describe a novel antibiotic delivery system based on magnetic nanoparticles (NPs) conjugated to a cell-penetrating peptide (CPP). Silica-coated iron oxide NPs were produced via a co-deposition method, and coated by a polyvinyl alcohol (PVA) polymeric network via physicochemical binding. Vancomycin (VAN) was then entrapped into this PVA network. A hexapeptide sequence Gly-Ala-Phe-Pro-His-Arg, was synthesized in the solid phase and then conjugated onto the surface of the magnetic NPs. The drug ratio incorporation into the carrier system and drug release were monitored through precise analysis. Confocal microscopy showed that the NPs could be internalized into Staphylococcus aureus and Escherichia coli bacterial cells. The antimicrobial effects of VAN were significantly enhanced by this system with a low dosage of VAN. Advantages include rapid targeted-drug delivery process, drug dose reduction, and equal effects on both Gram-positive and Gram-negative bacteria.


Assuntos
Antibacterianos/farmacologia , Peptídeos Penetradores de Células/química , Composição de Medicamentos/métodos , Nanopartículas de Magnetita/química , Vancomicina/farmacologia , Antibacterianos/química , Antibacterianos/farmacocinética , Peptídeos Penetradores de Células/farmacocinética , Portadores de Fármacos/química , Escherichia coli/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Staphylococcus aureus/efeitos dos fármacos , Vancomicina/química , Vancomicina/farmacocinética
14.
Colloids Surf B Biointerfaces ; 188: 110772, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31999965

RESUMO

This study aimed to develop sheddable polyethylene glycol (PEG) shells with TAT-modified core cross-linked nanomicelles as drug-delivery carriers of doxorubicin (DOX) to establish a programmed response against the tumor microenvironment, enhanced endocytosis, and lysosomal pH-triggered DOX release. First, poly(L-succinimide) (PSI) underwent a ring-opening reaction with ethylenediamine to generate poly(N-(2-aminoethyl)-l-aspartamide) (P(ae-Asp)). Next, the thiolytic cleavable PEG, 3,4-dihydroxyphenylacetic acid, and TAT were grafted onto P(ae-Asp) to synthesize the amphiphilic graft copolymer of mPEG-SS-g-P(ae-Asp)-MCA-DA-TAT. In aqueous solution, the amphiphilic polymer self-assembled into nanomicelles, encapsulating DOX into the hydrophobic core of micelles. TAT was shielded by the PEG corona during circulation to avoid non-specific transmembrane interaction with normal cells, while the tumor redox environment-responsive shedding of PEG could expose TAT to promote internalization of tumor cells. In order to improve the stability of nanomicelles and achieve pH-triggered drug release, a core cross-linking strategy based on the coordination of catechol and Fe3+ was adopted. In vitro studies demonstrated that core cross-linked nanomicelles maintained the nanostructure in 100 times dilution in pH 7.4 phosphate-buffered saline (PBS). Moreover, DOX release from DOX-loaded core cross-linked nanomicelles (DOX-TAT-CCLMs) was favored at simulated lysosomal conditions over simulated plasma conditions, indicating that these nanomicelles demonstrate characteristics of pH-triggered DOX release. The TAT modification considerably enhanced the mean fluorescence intensity of the nanomicelles endocytosed by MCF-7/ADR cells by 8 times, compared with DOX·HCl after 8 h of incubation. Notably, the IC50 value of nanomicelles (11.61 ±â€¯0.95 µg/mL) was nearly 4 times lower than that of DOX·HCl against MCF-7/ADR cells, implying that the nanomicelles could overcome drug resistance observed in MCF-7/ADR cells. Furthermore, the DOX-TAT-CCLMs reported superior tumor growth suppression in a 4T1 tumor-bearing mouse model. Thus, the redox- and pH- stimuli stepwise-responsive novel nanomicelles fabricated from the mPEG-SS-g-P(ae-Asp)-MCA-DA-TAT graft copolymer exhibited multifunctionality and displayed great potential for drug delivery.


Assuntos
Antibióticos Antineoplásicos/farmacologia , Peptídeos Penetradores de Células/química , Doxorrubicina/farmacologia , Polietilenoglicóis/química , Antibióticos Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Reagentes para Ligações Cruzadas/química , Doxorrubicina/química , Liberação Controlada de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Concentração de Íons de Hidrogênio , Lisossomos/química , Células MCF-7 , Micelas , Nanopartículas/química , Tamanho da Partícula , Propriedades de Superfície
15.
J Nanobiotechnology ; 18(1): 15, 2020 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-31952530

RESUMO

BACKGROUND: The successful deliveries of siRNA depend on their stabilities under physiological conditions because greater in vivo stability enhances cellular uptake and enables endosomal escape. Viral-based systems appears as most efficient approaches for gene delivery but often compromised in terms of biocompatibility, patient safety and high cost scale up process. Here we describe a novel platform of gene delivery by elastin-like polypeptide (ELP) based targeting biopolymers. RESULTS: For better tumor targeting and membrane penetrating characteristics, we designed various chimeric ELP-based carriers containing a cell penetrating peptide (Tat), single or multiple copies of AP1 an IL-4 receptor targeting peptide along with coding sequence of ELP and referred as Tat-A1E28 or Tat-A4V48. These targeted polypeptides were further analyzed for its ability to deliver siRNA (Luciferase gene) in tumor cells in comparison with non-targeted controls (Tat-E28 or E28). The positively charged amino acids of these polypeptides enabled them to readily complex with negatively charged nucleic acids. The complexation of nucleic acid with respective polypeptides facilitated its transfection efficiency as well as stability. The targeted polypeptides (Tat-A1E28 or Tat-A4V48) selectively delivered siRNA into tumor cells in a receptor-specific fashion, achieved endosomal and lysosomal escape, and released gene into cytosol. The target specific delivery of siRNA by Tat-A1E28 or Tat-A4V48 was further validated in murine breast carcinoma 4T1 allograft mice model. CONCLUSION: The designed delivery systems efficiently delivered siRNA to the target site of action thereby inducing significant gene silencing activity. The study shows Tat and AP1 functionalized ELPs constitute a novel gene delivery system with potential therapeutic applications.


Assuntos
Peptídeos Penetradores de Células/química , Elastina/química , Peptídeos/química , RNA Interferente Pequeno/química , Animais , Biopolímeros , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular , Feminino , Técnicas de Transferência de Genes , Terapia Genética , Humanos , Luciferases/genética , Camundongos Endogâmicos BALB C , Transplante de Neoplasias , Imagem Óptica , RNA Interferente Pequeno/administração & dosagem , Receptores de Interleucina-4/metabolismo , Transfecção
16.
Int J Pharm ; 576: 118999, 2020 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-31893541

RESUMO

Colorectal cancer (CRC) is a major cause of cancer-related mortality worldwide. Moreover, metastasis is one of the main causes of death in CRC patients. Nanotechnology-based gene therapy has shown significant therapeutic benefits in recent clinical trials for cancer treatment. Recent studies have shown that pigment epithelium-derived factor (PEDF) protein can inhibit tumor growth and metastasis by anti-angiogenesis and pro-apoptosis. In this study, we prepared a PEDF-DNA-loaded liposome for cancer-targeted gene therapy for metastatic CRC using an iRGD peptide. Our results showed that cancer-targeted PEDF-DNA liposomes (R-LP/PEDF) exhibited enhanced inhibitory effects on invasion, migration, and pro-apoptosis of CRC cells in vitro. In addition, it reduced metastasis tumor nodules in lung and prolonged the survival time in a mouse model of metastatic CRC.


Assuntos
Peptídeos Penetradores de Células/metabolismo , Neoplasias Colorretais/terapia , Proteínas do Olho/genética , Marcação de Genes , Neoplasias Pulmonares/prevenção & controle , Fatores de Crescimento Neural/genética , Oligopeptídeos/metabolismo , Serpinas/genética , Animais , Apoptose , Linhagem Celular Tumoral , Movimento Celular , Peptídeos Penetradores de Células/química , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Proteínas do Olho/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Fatores de Crescimento Neural/metabolismo , Oligopeptídeos/química , Receptores Imunológicos/metabolismo , Receptores de Peptídeos/metabolismo , Serpinas/metabolismo , Carga Tumoral
17.
Int J Pharm ; 576: 119008, 2020 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-31901358

RESUMO

Viral nanoparticles represent potential natural versatile platforms for targeted gene and drug delivery. Improving the efficiency of gene transfer mediated by viral vectors could not only enhance their therapeutic potential, but also contribute to understanding the limitations in interactions of nanoparticles with cells and the development of new therapeutic approaches. In this study, four cell-penetrating peptides (CPPs), cationic octaarginine (R8), histidine-rich peptides (LAH4 and KH27K) and fusogenic peptide (FUSO), are investigated for their effect on infection by mouse polyomavirus (MPyV) or on transduction of reporter genes delivered by MPyV or related viral vectors. Peptides noncovalently associated with viral particles enhance gene transfer (with the exception of FUSO). Removal of cellular heparan sulfates by the heparinase does not significantly change the enhancing potential of CPPs. Instead, CPPs influences the physical state of viral particles: R8 slightly destabilizes the intact virus, KH27K induces its aggregation and LAH4 promotes disassembly and aggregation of the particles that massively and rapidly associate with cells. The findings indicate that peptides acting as transduction-enhancing agents of polyomavirus-based nanoparticles modulate their physical state, which can be an important prerequisite for sensitization of cells and determination of the further fate of viral particles inside cells.


Assuntos
Peptídeos Penetradores de Células/metabolismo , Vetores Genéticos , Polyomavirus/metabolismo , Transdução Genética , Vírion/metabolismo , Animais , Capsídeo/metabolismo , Capsídeo/ultraestrutura , Peptídeos Penetradores de Células/química , Células HEK293 , Humanos , Camundongos , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Polyomavirus/genética , Polyomavirus/ultraestrutura , Vírion/genética , Vírion/ultraestrutura
18.
J Liposome Res ; 30(1): 93-106, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31012367

RESUMO

To investigate the efficacy of cell penetrating peptide TAT-modified liposomes loaded with salvianolic acid B (SAB-TAT-LIP) on the proliferation, migration, survival, and TGF-ß1 expression of human skin fibroblasts (HSF) and to preliminarily evaluate its effect on the prevention and treatment of hypertrophic scars. SAB-TAT-LIP was prepared using the pH gradient reverse-phase evaporation method. The properties of the liposomes including morphology, percent entrapment efficiency, mean diameter, polydispersity index (PDI), zeta potential, in vitro release, in vitro percutaneous absorption, and serum stability were studied. The proliferation of HSF cells was examined by MTT assays. Cell migration was assessed using the transwell and scratch assays. Cell cycle status was assessed by flow cytometry. Apoptotic induction was assessed through Annexin V-FITC/PI staining. The entrapment efficiency of SAB-TAT-LIP was 86.70 ± 0.85% and the salvianolic acid B (SAB) displayed a unimodal size-distribution with a mean diameter of 183.2 nm ± 4.09, a PDI of 0.190, and a zeta potential of -9.25 mV ± 0.92. The in vitro cumulative release of SAB-TAT-LIP was 62.49% after 24 h. The in vitro 32 h cumulative transdermal rate was 17.21% and the dermal retention was 44.39 µg/cm2±6.87. SAB-TAT-LIP inhibited HSF proliferation in a concentration and time-dependent mode and inhibited the migration and invasion of HSF cells. SAB-TAT-LIP also significantly increased apoptotic induction, the number cells in the G0/G1 phase of the cell cycle, and decreased the levels of TGF-ß1 after 48 h of treatment relative to the control group (p < 0.05, p < 0.01). SAB-TAT-LIP offers a promising therapeutic strategy for transdermal delivery during the prevention and treatment of HS.


Assuntos
Benzofuranos/química , Peptídeos Penetradores de Células/química , Cicatriz Hipertrófica/tratamento farmacológico , Cicatriz Hipertrófica/prevenção & controle , Lipossomos/química , Administração Cutânea , Animais , Anexina A5/metabolismo , Apoptose/efeitos dos fármacos , Benzofuranos/administração & dosagem , Linhagem Celular , Permeabilidade da Membrana Celular , Proliferação de Células/efeitos dos fármacos , Composição de Medicamentos , Estabilidade de Medicamentos , Feminino , Fibroblastos/citologia , Humanos , Masculino , Ratos Wistar , Pele , Fator de Crescimento Transformador beta1/metabolismo
19.
Biochim Biophys Acta Biomembr ; 1862(2): 183098, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31676372

RESUMO

Cell-penetrating peptides (CPPs) internalization occurs both by endocytosis and direct translocation through the cell membrane. These different entry routes suggest that molecular partners at the plasma membrane, phospholipids or glycosaminoglycans (GAGs), bind CPPs with different affinity or selectivity. The analysis of sequence-dependent interactions of CPPs with lipids and GAGs should lead to a better understanding of the molecular mechanisms underlying their internalization. CPPs are short sequences generally containing a high number of basic arginines and lysines and sometimes aromatic residues, in particular tryptophans. Tryptophans are crucial residues in membrane-active peptides, because they are important for membrane interaction. Membrane-active peptides often present facial amphiphilicity, which also promote the interaction with lipid bilayers. To study the role of Trp and facial amphiphilicity in cell interaction and penetration of CPPs, a nonapeptide series containing only Arg, Trp or D-Trp residues at different positions was designed. Our quantitative study indicates that to maintain/increase the uptake efficiency, Arg can be advantageously replaced by Trp in the nonapeptides. The presence of Trp in oligoarginines increases the uptake in cells expressing GAGs at their surface, while it compensates for the loss of charge interactions from Arg and maintains similar peptide uptake in GAG-deficient cells. In addition, we show that facial amphiphilicity is not required for efficient uptake of these nonapeptides. Thermodynamic analyses point towards a key role of Trp that highly contributes to the binding enthalpy of complexes formation. Density functional theory (DFT) analysis highlights that salt bridge-π interactions play a crucial role for the GAG-dependent entry mechanisms.


Assuntos
Membrana Celular/metabolismo , Peptídeos Penetradores de Células/química , Sequência de Aminoácidos , Animais , Arginina , Células CHO , Peptídeos Penetradores de Células/farmacocinética , Cricetinae , Cricetulus , Endocitose , Glicosaminoglicanos/metabolismo , Humanos , Transporte Proteico , Termodinâmica , Triptofano
20.
Nanomedicine ; 23: 102112, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31669083

RESUMO

Combination therapy has emerged as an efficient way to deliver chemotherapeutics for treatment of glioblastoma. It provides collaborative approach of targeting cancer cells by acting via multiple mechanisms, thereby reducing drug resistance. However, the presence of impermeable blood brain barrier (BBB) restricts the delivery of chemotherapeutic drugs into the brain. To overcome this limitation, we designed a dual functionalized liposomes by modifying their surface with transferrin (Tf) and a cell penetrating peptide (CPP) for receptor and adsorptive mediated transcytosis, respectively. In this study, we used two different CPPs (based on physicochemical properties) and investigated the influence of insertion of CPP to Tf-liposomes on biocompatibility, cellular uptake, and transport across the BBB both in vitro and in vivo. The biodistribution profile of Tf-CPP liposomes showed more than 10 and 2.7 fold increase in doxorubicin and erlotinib accumulation in mice brain, respectively as compared to free drugs with no signs of toxicity.


Assuntos
Antineoplásicos , Barreira Hematoencefálica/metabolismo , Peptídeos Penetradores de Células , Doxorrubicina , Sistemas de Liberação de Medicamentos , Cloridrato de Erlotinib , Transferrina , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Barreira Hematoencefálica/patologia , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/farmacocinética , Peptídeos Penetradores de Células/farmacologia , Doxorrubicina/química , Doxorrubicina/farmacocinética , Doxorrubicina/farmacologia , Cloridrato de Erlotinib/química , Cloridrato de Erlotinib/farmacocinética , Cloridrato de Erlotinib/farmacologia , Feminino , Lipossomos , Masculino , Camundongos , Camundongos Nus , Transferrina/química , Transferrina/farmacocinética , Transferrina/farmacologia
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