RESUMO
The development of new strategies for enhancing drug delivery to the brain represents a major challenge in treating cerebral diseases. In this paper, we report on the synthesis and structural characterization of a biocompatible nanoparticle (NP) made up of poly(lactic-co-glycolic acid) (PLGA)-polyethylene glycol (PEG) co-polymer (namely PELGA) functionalized with the membranotropic peptide gH625 (gH) and the iron-mimicking peptide CRTIGPSVC (CRT) for transport across the blood-brain barrier (BBB). gH possesses a high translocation potency of the cell membrane. Conversely, CRT selectively recognizes the brain endothelium, which interacts with transferrin (Tf) and its receptor (TfR) through a non-canonical ligand-directed mechanism. We hypothesize that the delivery across the BBB of PELGA NPs should be efficiently enhanced by the NP functionalization with both gH and CRT. Synthesis of peptides and their conjugation to the PLGA as well as NP physical-chemical characterization are performed. Moreover, NP uptake, co-localization, adhesion under dynamic conditions, and permeation across in vitro BBB model are evaluated as a function of gH/CRT functionalization ratio. Results establish that the cooperative effect of CRT and gH may change the intra-cellular distribution of NPs and strengthen NP delivery across the BBB at the functionalization ratio 33% gHâ»66% CRT.
Assuntos
Cerebelo/citologia , Portadores de Fármacos/química , Endotélio/química , Nanopartículas/química , Peptídeos/química , Polímeros/síntese química , Animais , Materiais Biocompatíveis/química , Barreira Hematoencefálica/química , Barreira Hematoencefálica/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Células Cultivadas , Cerebelo/química , Cerebelo/metabolismo , Desenho de Fármacos , Endotélio/citologia , Endotélio/metabolismo , Lactatos/química , Camundongos , Peptídeos/metabolismo , Polietilenoglicóis/química , Ácido Poliglicólico/química , Polímeros/química , Receptores da Transferrina/metabolismo , Transferrina/metabolismoRESUMO
The blood brain barrier (BBB) represents a challenge in the development of new nano-delivery systems able to reach the central nervous system (CNS). In order to test the efficacy of these nanocarriers, it is fundamental to use in vitro models that resemble the in vivo cell culture conditions. Here, we demonstrate for the first time the ability of a membranotropic peptide, namely gH625, to transport a cargo-acting as a shuttle-across the BBB layer under flow conditions that mimic the blood flow rate. To this aim, a BBB microfluidic device was designed based on a transparent polyester porous membrane sandwiched between a top and a bottom overlying channel made of poly(methyl methacrylate) (PMMA). Our data clearly indicate that this microfluidic system allows the growth of brain endothelial bEnd.3 cells and the formation of a confluent layer at 7 days of culture that hinders the passage of nanoparticles compared to porous membrane alone. The device was validated at a 5 µL/min working flow rate, where the capability of the model to remain intact after nanoparticle passage was shown. Very interestingly, the decoration with the gH625 peptide enhances the adhesion of nanoparticles to the endothelial layer and the BBB crossing in flow conditions, thus confirming the efficacy of the gH625 as a delivery platform to the brain. Biotechnol. Bioeng. 2017;114: 1087-1095. © 2016 Wiley Periodicals, Inc.
Assuntos
Barreira Hematoencefálica/metabolismo , Modelos Biológicos , Nanopartículas/metabolismo , Peptídeos/metabolismo , Animais , Transporte Biológico , Linhagem Celular , Desenho de Equipamento , Camundongos , Técnicas Analíticas Microfluídicas/instrumentação , Peptídeos/químicaRESUMO
Nature has provided a valuable source of inspiration for developing high performance multifunctional materials. Particularly, catechol-containing amino acid l-3,4-dihydroxyphenylalanine (l-DOPA) has aroused the interest to design hybrid multifunctional materials with superior adhesive ability. DOPA oxidative polymerization mediated by either melanogenic enzymes or an alkaline environment involving catechol intermolecular cross-linking, ultimately leads to melanin oligomers. Recently, relevant studies disclosed the ability of Ti-based nanostructures to tune melanin's supramolecular structure during its formation, starting from melanogenic precursors, thus improving both antioxidant and antimicrobial properties. In this work, we propose a novel biomimetic approach to design hybrid DOPA melanin-like nanostructures through a hydrothermal synthesis opportunely modified by using citric acid to control hydrolysis and condensation reactions of titanium alkoxide precursors. UV-Vis and Electron paramagnetic resonance (EPR) spectroscopic evidences highlighted the key role of citrate-Ti(IV) and DOPA-Ti(IV) complexes in controlling DOPA polymerization, which specifically occurred during the hydrothermal step, mediating and tuning its conversion to melanin-like oligomers. Trasmission electron microscopy (TEM) images proved the efficacy of the proposed synthesis approach in tuning the formation of nanosized globular nanostructures, with high biocide performances. The obtained findings could provide strategic guidelines to set up biomimetic processes, exploiting the catechol-metal complex to obtain hybrid melanin-like nanosystems with optimized multifunctional behavior.
RESUMO
Delivery of therapeutic agents inside the cytosol, avoiding the confinement in endo-lysosomal compartments and their degradative environment, is one of the key targets of nanomedicine to gain the maximum remedial effects. Current approaches based on cell penetrating peptides (CPPs), despite improving the cellular uptake efficiency of nanocarriers, have shown controversial results in terms of intracellular localization. To elucidate the delivery potential of CPPs, in this work we analyzed the role of the particle size in influencing the ability of a membranotropic peptide, namely gH625, to escape the endo-lysosomal pathway and deliver the particles in the cytosol. To this aim, we carried out a systematic assessment of the cellular uptake and distribution of monodisperse platinum nanoparticles (PtNPs), having different diameters (2.5, 5 and 20 nm) and citrate capping or gH625 peptide functionalization. The presence of gH625 significantly increased the amount of internalized NPs in human cervix epithelioid carcinoma cells, as a function of particle size. However, scanning transmission electron microscopy (STEM) and electron tomography (ET) revealed a prevalent confinement of PtNPs within vesicular structures, regardless of the particle size and surface functionalization. Only in the case of the smallest 2.5 nm particles, the membranotropic peptide was able to partly maintain its functionality, enabling cytosolic delivery of a small fraction of internalized PtNPs, though particle agglomeration in culture medium limited single-particle transport across the cell membrane. Interestingly, membrane crossing by 2.5 nm functionalized-PtNPs seemed to occur by diffusion through the lipid bilayer, with no apparent membrane damage. For larger particle sizes (≥5 nm), their hindrance likely blocked the membranotropic mechanism. Combining the enhanced uptake and partial cytosolic delivery promoted by gH625, we were able to achieve a strong improvement of the antioxidant nanozyme function of 2.5 nm PtNPs, decreasing both the endogenous ROS level and its overproduction following an external oxidative insult.
Assuntos
Peptídeos Penetradores de Células/química , Portadores de Fármacos/química , Nanopartículas Metálicas , Tamanho da Partícula , Platina , Citosol , Células HeLa , HumanosRESUMO
Therapeutic approaches based on nanomedicine have garnered great attention in cancer research. In vitro biological models that better mimic in vivo conditions are crucial tools to more accurately predict their therapeutic efficacy in vivo. In this work, a new 3D breast cancer microtissue has been developed to recapitulate the complexity of the tumor microenvironment and to test its efficacy as screening platform for drug delivery systems. The proposed 3D cancer model presents human breast adenocarcinoma cells and cancer-associated fibroblasts embedded in their own ECM, thus showing several features of an in vivo tumor, such as overexpression of metallo-proteinases (MMPs). After demonstrating at molecular and protein level the MMP2 overexpression in such tumor microtissues, we used them to test a recently validated formulation of endogenous MMP2-responsive nanoparticles (NP). The presence of the MMP2-sensitive linker allows doxorubicin release from NP only upon specific enzymatic cleavage of the peptide. The same NP without the MMP-sensitive linker and healthy breast microtissues were also produced to demonstrate NP specificity and selectivity. Cell viability after NP treatment confirmed that controlled drug delivery is achieved only in 3D tumor microtissues suggesting that the validation of therapeutic strategies in such 3D tumor model could predict human response. STATEMENT OF SIGNIFICANCE: A major issue of modern cancer research is the development of accurate and predictive experimental models of human tumors consistent with tumor microenvironment and applicable as screening platforms for novel therapeutic strategies. In this work, we developed and validated a new 3D microtissue model of human breast tumor as a testing platform of anti-cancer drug delivery systems. To this aim, biodegradable nanoparticles responsive to physiological changes specifically occurring in tumor microenvironment were used. Our findings clearly demonstrate that the breast tumor microtissue well recapitulates in vivo physiological features of tumor tissue and elicits a specific response to microenvironmentally-responsive nanoparticles compared to healthy tissue. We believe this study is of particular interest for cancer research and paves the way to exploit tumor microtissues for several testing purposes.