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1.
Sci Rep ; 14(1): 6343, 2024 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-38491195

RESUMO

The in vivo diagnosis and monitoring of pulmonary disorders (caused for example by emphysema, Covid-19, immature lung tissue in infants) could be effectively supported by the non-invasive sensing of the lung through light. With this purpose, we investigated the feasibility of probing the lung by means of time-resolved diffuse optics, leveraging the increased depth (a few centimeters) attained by photons collected after prolonged propagation time (a few nanoseconds). We present an initial study that includes measurements performed on 5 healthy volunteers during a breathing protocol, using a time-resolved broadband diffuse optical spectroscopy system. Those measurements were carried out across the spectral range of 600-1100 nm at a source-detector distance of 3 cm, and at 820 nm over a longer distance (7-9 cm). The preliminary analysis of the in vivo data with a simplified homogeneous model revealed a maximum probing depth of 2.6-3.9 cm, suitable for reaching the lung. Furthermore, we observed variations in signal associated with respiration, particularly evident at long photon propagation times. However, challenges stemming from both intra- and inter-subject variability, along with inconsistencies potentially arising from conflicting scattering and absorption effects on the collected signal, hindered a clear interpretation. Aspects that require further investigation for a more comprehensive understanding are outlined.


Assuntos
Óptica e Fotônica , Fótons , Humanos , Análise Espectral/métodos , Pulmão/diagnóstico por imagem
2.
J Biomed Opt ; 27(7)2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35701869

RESUMO

SIGNIFICANCE: Multi-laboratory initiatives are essential in performance assessment and standardization-crucial for bringing biophotonics to mature clinical use-to establish protocols and develop reference tissue phantoms that all will allow universal instrument comparison. AIM: The largest multi-laboratory comparison of performance assessment in near-infrared diffuse optics is presented, involving 28 instruments and 12 institutions on a total of eight experiments based on three consolidated protocols (BIP, MEDPHOT, and NEUROPT) as implemented on three kits of tissue phantoms. A total of 20 synthetic indicators were extracted from the dataset, some of them defined here anew. APPROACH: The exercise stems from the Innovative Training Network BitMap funded by the European Commission and expanded to include other European laboratories. A large variety of diffuse optics instruments were considered, based on different approaches (time domain/frequency domain/continuous wave), at various stages of maturity and designed for different applications (e.g., oximetry, spectroscopy, and imaging). RESULTS: This study highlights a substantial difference in hardware performances (e.g., nine decades in responsivity, four decades in dark count rate, and one decade in temporal resolution). Agreement in the estimates of homogeneous optical properties was within 12% of the median value for half of the systems, with a temporal stability of <5 % over 1 h, and day-to-day reproducibility of <3 % . Other tests encompassed linearity, crosstalk, uncertainty, and detection of optical inhomogeneities. CONCLUSIONS: This extensive multi-laboratory exercise provides a detailed assessment of near-infrared Diffuse optical instruments and can be used for reference grading. The dataset-available soon in an open data repository-can be evaluated in multiple ways, for instance, to compare different analysis tools or study the impact of hardware implementations.


Assuntos
Laboratórios , Óptica e Fotônica , Imagens de Fantasmas , Reprodutibilidade dos Testes , Análise Espectral
3.
J Biomed Opt ; 27(3)2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35332743

RESUMO

SIGNIFICANCE: Diffuse optical tomography is an ill-posed problem. Combination with ultrasound can improve the results of diffuse optical tomography applied to the diagnosis of breast cancer and allow for classification of lesions. AIM: To provide a simulation pipeline for the assessment of reconstruction and classification methods for diffuse optical tomography with concurrent ultrasound information. APPROACH: A set of breast digital phantoms with benign and malignant lesions was simulated building on the software VICTRE. Acoustic and optical properties were assigned to the phantoms for the generation of B-mode images and optical data. A reconstruction algorithm based on a two-region nonlinear fitting and incorporating the ultrasound information was tested. Machine learning classification methods were applied to the reconstructed values to discriminate lesions into benign and malignant after reconstruction. RESULTS: The approach allowed us to generate realistic US and optical data and to test a two-region reconstruction method for a large number of realistic simulations. When information is extracted from ultrasound images, at least 75% of lesions are correctly classified. With ideal two-region separation, the accuracy is higher than 80%. CONCLUSIONS: A pipeline for the generation of realistic ultrasound and diffuse optics data was implemented. Machine learning methods applied to a optical reconstruction with a nonlinear optical model and morphological information permit to discriminate malignant lesions from benign ones.


Assuntos
Neoplasias da Mama , Tomografia Óptica , Mama/diagnóstico por imagem , Mama/patologia , Neoplasias da Mama/patologia , Feminino , Humanos , Imagens de Fantasmas , Tomografia Óptica/métodos , Ultrassonografia
4.
Biomed Opt Express ; 12(2): 1105-1122, 2021 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-33680561

RESUMO

A recent upgrade of the time domain multi-wavelength optical mammograph developed by Politecnico di Milano achieved good performance in laboratory tests [Biomed. Opt. Express9, 755 (2018).10.1364/BOE.9.000755]. However, it proved unsatisfactory when in vivo measurements were finally performed. That led to a further upgrade, including the replacement of the time-to-digital converter with a new model, and the related set-up changes. The new instrument version offers improved laboratory performance (as assessed through established protocols: BIP and MEDPHOT) and good in vivo performance (extension of the scanned breast area, repeatability, consistency of estimated tissue composition with physiology). Besides introducing the new set-up and detailing its laboratory and in vivo performance, we highlight the importance of systematic in vivo testing before entering clinical trials.

5.
Biomed Opt Express ; 11(5): 2779-2793, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32499960

RESUMO

The human abdominal region is very heterogeneous and stratified with subcutaneous adipose tissue (SAT) being one of the primary layers. Monitoring this tissue is crucial for diagnostic purposes and to estimate the effects of interventions like caloric restriction or bariatric surgery. However, the layered nature of the abdomen poses a major problem in monitoring the SAT in a non-invasive way by diffuse optics. In this work, we examine the possibility of using multi-distance broadband time domain diffuse optical spectroscopy to assess the human abdomen non-invasively. Broadband absorption and reduced scattering spectra from 600 to 1100 nm were acquired at 1, 2 and 3 cm source-detector distances on ten healthy adult male volunteers, and then analyzed using a homogeneous model as an initial step to understand the origin of the detected signal and how tissue should be modeled to derive quantitative information. The results exhibit a clear influence of the layered nature on the estimated optical properties. Clearly, the underlying muscle makes a relevant contribution in the spectra measured at the largest source-detector distance for thinner subjects related to blood and water absorption. More unexpectedly, also the thin superficial skin layer yields a direct contamination, leading to higher water content and steeper reduced scattering spectra at the shortest distance, as confirmed also by simulations. In conclusion, provided that data analysis properly accounts for the complex tissue structure, diffuse optics may offer great potential for the continuous non-invasive monitoring of abdominal fat.

6.
Analyst ; 144(23): 6871-6880, 2019 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-31686068

RESUMO

Surface functionalization is a key step in biosensing since it is the basis of an effective analyte recognition. Among all the bioreceptors, antibodies (Abs) play a key role thanks to their superior specificity, although the available immobilization strategies suffer from several drawbacks. When gold is the interacting surface, the recently introduced Photochemical Immobilization Technique (PIT) has been shown to be a quick, easy-to-use and very effective method to tether Abs oriented upright by means of thiols produced via tryptophan mediated disulphide bridge reduction. Although the molecular mechanism of this process is quite well identified, the detailed morphology of the immobilized antibodies is still elusive due to inherent difficulties related to the microscopy imaging of Abs. The combination of Mass Spectrometry, Surface-Enhanced Raman Spectroscopy and Ellman's assay demonstrates that Abs irradiated under the conditions in which PIT is realized show only two effective disulphide bridges available for binding. They are located in the constant region of the immunoglobulin light chain so that the most likely position Ab assumes is side-on, i.e. with one Fab (i.e. the antigen binding portion of the antibody) exposed to the solution. This is not a limitation of the recognition efficiency in view of the intrinsic flexibility of the Ab structure, which makes the free Fab able to sway in the solution, a feature of great importance in many biosensing applications.


Assuntos
Anticorpos Imobilizados/química , Anticorpos Monoclonais Murinos/química , Regiões Constantes de Imunoglobulina/química , Sequência de Aminoácidos , Anticorpos Imobilizados/efeitos da radiação , Anticorpos Monoclonais Murinos/efeitos da radiação , Técnicas Biossensoriais/instrumentação , Dissulfetos/efeitos da radiação , Regiões Constantes de Imunoglobulina/efeitos da radiação , Nanopartículas Metálicas/química , Conformação Proteica , Prata/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Análise Espectral Raman , Propriedades de Superfície , Raios Ultravioleta
7.
Biomed Opt Express ; 10(8): 3899-3915, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31452983

RESUMO

Recently, multimodal imaging has gained an increasing interest in medical applications thanks to the inherent combination of strengths of the different techniques. For example, diffuse optics is used to probe both the composition and the microstructure of highly diffusive media down to a depth of few centimeters, but its spatial resolution is intrinsically low. On the other hand, ultrasound imaging exhibits the higher spatial resolution of morphological imaging, but without providing solid constitutional information. Thus, the combination of diffuse optical imaging and ultrasound may improve the effectiveness of medical examinations, e.g. for screening or diagnosis of tumors. However, the presence of an ultrasound coupling gel between probe and tissue can impair diffuse optical measurements like diffuse optical spectroscopy and diffuse correlation spectroscopy, since it may provide a direct path for photons between source and detector. A systematic study on the effect of different ultrasound coupling fluids was performed on tissue-mimicking phantoms, confirming that a water-clear gel can produce detrimental effects on optical measurements when recovering absorption/reduced scattering coefficients from time-domain spectroscopy acquisitions as well as particle Brownian diffusion coefficient from diffuse correlation spectroscopy ones. On the other hand, we show the suitability for optical measurements of other types of diffusive fluids, also compatible with ultrasound imaging.

8.
Analyst ; 144(6): 1876-1880, 2019 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-30810548

RESUMO

The first detailed analysis of FLIM applications for Mg cell imaging is presented. We employed the Mg-sensitive fluorescent dye named DCHQ5, a derivative of diaza-18-crown-6 ethers appended with two 8-hydroxyquinoline groups, to perform fluorescence lifetime imaging in control and Mg deprived SaOS-2 live cells, which contain different concentrations of magnesium. We found that the lifetime maps are almost uniform all over the cells and, most relevantly, we showed that the ratio of the amplitude terms is related to the magnesium intracellular concentration.


Assuntos
Neoplasias Ósseas/metabolismo , Magnésio/metabolismo , Imagem Óptica/métodos , Osteossarcoma/metabolismo , Espectrometria de Fluorescência/métodos , Humanos , Magnésio/análise , Células Tumorais Cultivadas
9.
Sci Rep ; 8(1): 10015, 2018 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-29968735

RESUMO

Thyroid plays an important role in the endocrine system of the human body. Its characterization by diffuse optics can open new path ways in the non-invasive diagnosis of thyroid pathologies. Yet, the absorption spectra of tyrosine and thyroglobulin-key tissue constituents specific to the thyroid organ-in the visible to near infrared range are not fully available. Here, we present the optical characterization of tyrosine (powder), thyroglobulin (granular form) and iodine (aqueous solution) using a time domain broadband diffuse optical spectrometer in the 550-1350 nm range. Various systematic errors caused by physics of photo migration and sample inherent properties were effectively suppressed by means of advanced time domain diffuse optical methods. A brief comparison with various other known tissue constituents is presented, which reveals key spectral regions for the quantification of the thyroid absorbers in an in vivo scenario.


Assuntos
Iodo/análise , Análise Espectral/métodos , Tireoglobulina/análise , Glândula Tireoide/diagnóstico por imagem , Nódulo da Glândula Tireoide/patologia , Tirosina/análise , Humanos , Iodo/química , Fenômenos Ópticos , Tireoglobulina/química , Tirosina/química
10.
Biomed Opt Express ; 9(5): 2068-2080, 2018 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-29760970

RESUMO

We present the recipe and characterization for preparing liquid phantoms that are suitable for both near-infrared spectroscopy and diffuse correlation spectroscopy. The phantoms have well-defined and tunable optical and dynamic properties, and consist of a solution of water and glycerol with fat emulsion as the scattering element. The recipe takes into account the effect of bulk refractive index changes due to the addition of glycerol, which is commonly used to alter the sample viscosity.

11.
Biomed Opt Express ; 9(2): 755-770, 2018 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-29552410

RESUMO

A novel detection chain, based on 8 Silicon Photomultipliers (forming a wide-area custom-made detection probe) and on a time-to-digital converter, was developed to improve the signal level in multi-wavelength (635-1060 nm) time domain optical mammography. The performances of individual components and of the overall chain were assessed using established protocols (BIP and MEDPHOT). The photon detection efficiency was improved by up to 3 orders of magnitude, and the maximum count rate level was increased by a factor of 10 when compared to the previous system, based on photomultiplier tubes and conventional time-correlated single-photon counting boards. In the estimate of optical parameters, the novel detection chain provides performances comparable to the previous system, widely validated in clinics, but with higher signal level, higher robustness, and at a lower price per channel, thus targeting important requirements for clinical applications.

12.
Biophys Chem ; 229: 130-134, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28733103

RESUMO

Elastin is a key structural protein of dynamic connective tissues widely found in the extracellular matrix of skin, arteries, lungs and ligaments. It is responsible for a range of diseases related to aging of biological tissues. The optical characterization of elastin can open new opportunities for its investigation in biomedical studies. In this work, we present the absorption spectra of elastin using a broadband (550-1350nm) diffuse optical spectrometer. Distortions caused by fluorescence and finite bandwidth of the laser source on estimated absorption were effectively accounted for in measurements and data analysis and compensated. A comprehensive summary and comparison between collagen and elastin is presented, highlighting distinct features for its accurate quantification in biological applications.


Assuntos
Elastina/química , Animais , Bovinos , Elastina/metabolismo , Matriz Extracelular/metabolismo , Espectrofotometria Infravermelho
13.
Opt Express ; 25(5): 4585-4597, 2017 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-28380730

RESUMO

We present a new technique, frequency offset Raman spectroscopy (FORS), to probe Raman spectra of diffusive media in depth. The proposed methodology obtains depth sensitivity exploiting changes in optical properties (absorption and scattering) with excitation wavelengths. The approach was demonstrated experimentally on a two-layer tissue phantom and compared with the already consolidated spatially offset Raman spectroscopy (SORS) technique. FORS attains a similar enhancement of signal from deep layers as SORS, namely 2.81 against 2.62, while the combined hybrid FORS-SORS approach leads to a markedly higher 6.0 enhancement. Differences and analogies between FORS and SORS are discussed, suggesting FORS as an additional or complementary approach for probing heterogeneous media such as biological tissues in depth.

14.
J Biomed Opt ; 22(1): 15006, 2017 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-28138693

RESUMO

Reduction in scattering, high absorption, and spectral features of tissue constituents above 1000 nm could help in gaining higher spatial resolution, penetration depth, and specificity for in vivo studies, opening possibilities of near-infrared diffuse optics in tissue diagnosis. We present the characterization of collagen absorption over a broadband range (500 to 1700 nm) and compare it with spectra presented in the literature. Measurements were performed using a time-domain diffuse optical technique. The spectrum was extracted by carefully accounting for various spectral distortion effects, due to sample and system properties. The contribution of several tissue constituents (water, lipid, collagen, oxy, and deoxy-hemoglobin) to the absorption properties of a collagen-rich in vivo bone location, such as radius distal in the 500- to 1700-nm wavelength region, is also discussed, suggesting bone diagnostics as a potential area of interest.


Assuntos
Colágeno/farmacocinética , Absorção Ocular , Hemoglobinas/metabolismo , Metabolismo dos Lipídeos , Óptica e Fotônica , Espalhamento de Radiação , Sensibilidade e Especificidade , Espectroscopia de Luz Próxima ao Infravermelho
15.
Sci Rep ; 7: 40683, 2017 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-28091596

RESUMO

Several techniques are being investigated as a complement to screening mammography, to reduce its false-positive rate, but results are still insufficient to draw conclusions. This initial study explores time domain diffuse optical imaging as an adjunct method to classify non-invasively malignant vs benign breast lesions. We estimated differences in tissue composition (oxy- and deoxyhemoglobin, lipid, water, collagen) and absorption properties between lesion and average healthy tissue in the same breast applying a perturbative approach to optical images collected at 7 red-near infrared wavelengths (635-1060 nm) from subjects bearing breast lesions. The Discrete AdaBoost procedure, a machine-learning algorithm, was then exploited to classify lesions based on optically derived information (either tissue composition or absorption) and risk factors obtained from patient's anamnesis (age, body mass index, familiarity, parity, use of oral contraceptives, and use of Tamoxifen). Collagen content, in particular, turned out to be the most important parameter for discrimination. Based on the initial results of this study the proposed method deserves further investigation.


Assuntos
Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/patologia , Mama/diagnóstico por imagem , Mama/patologia , Mamografia/métodos , Área Sob a Curva , Composição Corporal , Diagnóstico Diferencial , Feminino , Humanos , Processamento de Imagem Assistida por Computador , Análise Espectral
16.
PLoS One ; 11(12): e0168426, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27997565

RESUMO

Non-invasive in vivo diffuse optical characterization of human bone opens a new possibility of diagnosing bone related pathologies. We present an in vivo characterization performed on seventeen healthy subjects at six different superficial bone locations: radius distal, radius proximal, ulna distal, ulna proximal, trochanter and calcaneus. A tailored diffuse optical protocol for high penetration depth combined with the rather superficial nature of considered tissues ensured the effective probing of the bone tissue. Measurements were performed using a broadband system for Time-Resolved Diffuse Optical Spectroscopy (TRS) to assess mean absorption and reduced scattering spectra in the 600-1200 nm range and Diffuse Correlation Spectroscopy (DCS) to monitor microvascular blood flow. Significant variations among tissue constituents were found between different locations; with radius distal rich of collagen, suggesting it as a prominent location for bone related measurements, and calcaneus bone having highest blood flow among the body locations being considered. By using TRS and DCS together, we are able to probe the perfusion and oxygen consumption of the tissue without any contrast agents. Therefore, we predict that these methods will be able to evaluate the impairment of the oxygen metabolism of the bone at the point-of-care.


Assuntos
Consumo de Oxigênio , Sistemas Automatizados de Assistência Junto ao Leito , Rádio (Anatomia) , Tomografia de Coerência Óptica , Ulna , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Rádio (Anatomia)/diagnóstico por imagem , Rádio (Anatomia)/metabolismo , Espectrofotometria/instrumentação , Espectrofotometria/métodos , Tomografia de Coerência Óptica/instrumentação , Tomografia de Coerência Óptica/métodos , Ulna/diagnóstico por imagem , Ulna/metabolismo
17.
J Biomed Opt ; 21(9): 091311, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27403837

RESUMO

Diffuse optical imaging and spectroscopy of the female breast is an area of active research. We review the present status of this field and discuss the broad range of methodologies and applications. Starting with a brief overview on breast physiology, the remodeling of vasculature and extracellular matrix caused by solid tumors is highlighted that is relevant for contrast in optical imaging. Then, the various instrumental techniques and the related methods of data analysis and image generation are described and compared including multimodality instrumentation, fluorescence mammography, broadband spectroscopy, and diffuse correlation spectroscopy. We review the clinical results on functional properties of malignant and benign breast lesions compared to host tissue and discuss the various methods to improve contrast between healthy and diseased tissue, such as enhanced spectroscopic information, dynamic variations of functional properties, pharmacokinetics of extrinsic contrast agents, including the enhanced permeability and retention effect. We discuss research on monitoring neoadjuvant chemotherapy and on breast cancer risk assessment as potential clinical applications of optical breast imaging and spectroscopy. Moreover, we consider new experimental approaches, such as photoacoustic imaging and long-wavelength tissue spectroscopy.


Assuntos
Neoplasias da Mama/diagnóstico por imagem , Mama/diagnóstico por imagem , Mamografia , Imagem Óptica , Feminino , Humanos , Espectroscopia de Luz Próxima ao Infravermelho , Tomografia Óptica
18.
PLoS One ; 10(6): e0128941, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26029912

RESUMO

BACKGROUND: Breast tissue composition is recognized as a strong and independent risk factor for breast cancer. It is a heritable feature, but is also significantly affected by several other elements (e.g., age, menopause). Nowadays it is quantified by mammographic density, thus requiring the use of ionizing radiation. Optical techniques are absolutely non-invasive and have already proved effective in the investigation of biological tissues, as they are sensitive to tissue composition and structure. METHODS: Time domain diffuse optical spectroscopy was performed at 7 wavelengths (635-1060 nm) on 200 subjects to derive their breast tissue composition (in terms of water, lipid and collagen content), blood parameters (total hemoglobin content and oxygen saturation level), and information on the microscopic structure (scattering amplitude and power). The dependence of all optically-derived parameters on age, menopausal status, body mass index, and use of oral contraceptives, and the correlation with mammographic density were investigated. RESULTS: Younger age, premenopausal status, lower body mass index values, and use of oral contraceptives all correspond to significantly higher water, collagen and total hemoglobin content, and lower lipid content (always p < 0.05 and often p < 10-4), while oxygen saturation level and scattering parameters show significant dependence only on some conditions. Even when age-adjusted groups of subjects are compared, several optically derived parameters (and in particular always collagen and total hemoglobin content) remain significantly different. CONCLUSIONS: Time domain diffuse optical spectroscopy can probe non-invasively breast tissue composition and physiologic blood parameters, and provide information on tissue structure. The measurement is suitable for in vivo studies and monitoring of changes in breast tissue (e.g., with age, lifestyle, chemotherapy, etc.) and to gain insight into related processes, like the origin of cancer risk associated with breast density.


Assuntos
Neoplasias da Mama/patologia , Mama/patologia , Adulto , Idoso , Índice de Massa Corporal , Densidade da Mama , Demografia/métodos , Feminino , Humanos , Glândulas Mamárias Humanas/anormalidades , Glândulas Mamárias Humanas/patologia , Menopausa/fisiologia , Pessoa de Meia-Idade , Óptica e Fotônica/métodos , Pré-Menopausa/fisiologia , Fatores de Risco , Tomografia Óptica/métodos
19.
Biomed Opt Express ; 5(10): 3684-98, 2014 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-25360382

RESUMO

The optical characterization of malignant and benign breast lesions is presented. Time-resolved transmittance measurements were performed in the 630-1060 nm range by means of a 7-wavelength optical mammograph, providing both imaging and spectroscopy information. A total of 62 lesions were analyzed, including 33 malignant and 29 benign lesions. The characterization of breast lesions was performed applying a perturbation model based on the high-order calculation of the pathlength of photons inside the lesion, which led to the assessment of oxy- and deoxy-hemoglobin, lipids, water and collagen concentrations. Significant variations between tumor and healthy tissue were observed in terms of both absorption properties and constituents concentration. In particular, benign lesions and tumors show a statistically significant discrimination in terms of absorption at several wavelengths and also in terms of oxy-hemoglobin and collagen content.

20.
Photochem Photobiol Sci ; 13(12): 1680-9, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25308511

RESUMO

Polyplexes are nanoparticles formed by the self-assembly of DNA/RNA and cationic polymers specifically designed to deliver exogenous genetic material to cells by a process called transfection. There is a general consensus that a subtle balance between sufficient extracellular protection and intracellular release of nucleic acids is a key factor for successful gene delivery. Therefore, there is a strong need to develop suitable tools and techniques for enabling the monitoring of the stability of polyplexes in the biological environment they face during transfection. In this work we propose time-resolved fluorescence spectroscopy in combination with SYBR Green I-DNA dye as a reliable tool for the in-depth characterization of the DNA/vector complexation state. As a proof of concept, we provide essential information on the assembly and disassembly of complexes formed between DNA and each of three cationic polymers, namely a novel promising chitosan-graft-branched polyethylenimine copolymer (Chi-g-bPEI), one of its building block 2 kDa bPEI and the gold standard transfectant 25 kDa bPEI. Our results highlight the higher information content provided by the time-resolved studies of SYBR Green I/DNA, as compared to conventional steady state measurements of ethidium bromide/DNA that enabled us to draw relationships among fluorescence lifetime, polyplex structural changes and transfection efficiency.


Assuntos
Cátions/química , DNA/química , Nanopartículas/química , Compostos Orgânicos/química , Polímeros/química , Benzotiazóis , Sobrevivência Celular , Quitosana/análogos & derivados , Quitosana/química , Diaminas , Células HeLa , Heparina/química , Humanos , Polietilenoimina/análogos & derivados , Polietilenoimina/química , Quinolinas , Espectrometria de Fluorescência , Transfecção
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