Fluorescence lifetime microscopy unveils the supramolecular organization of liposomal Doxorubicin

Tentori, Paolo; Signore, Giovanni; Camposeo, Andrea; Carretta, Annalisa; Ferri, Gianmarco; Pingue, Pasqualantonio; Luin, Stefano; Pozzi, Daniela; Gratton, Enrico; Beltram, Fabio; Caracciolo, Giulio; Cardarelli, Francesco

doi:10.1039/d2nr00311b

Cited by 19 publications

(43 citation statements)

References 21 publications

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“…Interestingly, these FLIM-based estimates are in keeping with previous ones obtained by others using NAD(P)H as test molecule 11 . In addition, it should be kept in mind that what is measured by FLIM are the so-called “fractional intensities” of the two NAD(P)H forms, which are related to their actual molar fractions by means of the quantum yields (QYs) of the pure bound and free species 33 . Taking into account that the average QY for the bound NAD(P)H form is ~8.5 times larger than that for the free NAD(P)H form (estimated by the difference in lifetimes 20 , 34 ), the measured fractional intensities can be corrected and used to extract the molar fractions of free and bound NAD(P)H from FLIM data (Eq.…”

Section: Resultsmentioning

confidence: 99%

Single-cell imaging of α and β cell metabolic response to glucose in living human Langerhans islets

et al. 2022

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Here we use a combination of two-photon Fluorescence Lifetime Imaging Microscopy (FLIM) of NAD(P)H free/bound ratio in living HIs with post-fixation, immunofluorescence-based, cell-type identification. FLIM allowed to measure variations in the NAD(P)H free/bound ratio induced by glucose; immunofluorescence data allowed to identify single α and β cells; finally, matching of the two datasets allowed to assign metabolic shifts to cell identity. 312 α and 654 β cells from a cohort of 4 healthy donors, 15 total islets, were measured. Both α and β cells display a wide spectrum of responses, towards either an increase or a decrease in NAD(P)H free/bound ratio. Yet, if single-cell data are averaged according to the respective donor and correlated to donor insulin secretion power, a non-random distribution of metabolic shifts emerges: robust average responses of both α and β cells towards an increase of enzyme-bound NAD(P)H belong to the donor with the lowest insulin-secretion power; by contrast, discordant responses, with α cells shifting towards an increase of free NAD(P)H and β cells towards an increase of enzyme-bound NAD(P)H, correspond to the donor with the highest insulin-secretion power. Overall, data reveal neat anti-correlation of tissue metabolic responses with respect to tissue insulin secretion power.

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Section: Resultsmentioning

confidence: 99%

Single-cell imaging of α and β cell metabolic response to glucose in living human Langerhans islets

et al. 2022

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“…The excitation wavelength was set to 470 nm in order to selectively excite the intrinsic fluorescence of DOX. Based on our previous study [7], we know that three pure species concur to generate the measured lifetime of Doxoves®, each with its own mono-exponential characteristic lifetime and relative abundance: crystallized DOX (DOXc), free DOX (DOXf), and DOX associated with the liposomal membrane (DOXb) (Figure 1B). As a result, the FLIM signature of Doxoves® falls within the triangle having the lifetimes of the three pure species as vertices, as expected from the well-known algebraic rules of phasor composition [6].…”

Section: Molar Composition Of Doxoves®mentioning

confidence: 83%

“…Then, the exact molar fractions of the three species are determined by combining phasor-FLIM with quantitative absorption/fluorescence spectroscopy on DOXc, DOXf, and DOXb pure standards. The final picture of the Doxoves® formulation comprises most of the drug in the crystallized form (>98%), with the remaining minor fractions divided between free (~1%) and membrane-bound drug (<1%) [7].…”

Section: Introductionmentioning

confidence: 99%

Monitoring drug stability by label-free fluorescence lifetime imaging: a case study on liposomal doxorubicin

Carretta,

Cardarelli

2023

J. Phys.: Conf. Ser.

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In a previous report, we demonstrated that Doxorubicin (DOX) intrinsic fluorescence can be exploited in combination with the phasor approach to fluorescence lifetime imaging microscopy (FLIM) and quantitative absorption/fluorescence spectroscopy to resolve the supramolecular organization of the drug within its FDA-approved nanoformulation, Doxil®. The resulting ‘synthetic identity’ comprises three co-existing physical states of the drug within Doxil®: a dominating fraction of crystallized DOX (DOXc >98%), and two minor fractions of free DOX (DOXf∼1%), and DOX associated with the liposomal membrane (DOXb <1%). This result serves as a benchmark here to address the time evolution of Doxil® synthetic identity. We probe the effect of temperature for a total duration of 6 months in a non-invasive way by FLIM. We confirm Doxil® stability if stored at 4°C, while we detect marked changes in its synthetic identity at 37°C: crystallized DOX gets progressively disassembled in time, in favor of the other two physical states, free and membrane-associated DOX. Our phasor-FLIM-based approach paves the way to time-resolved biochemical assays on the supramolecular organization of encapsulated fluorescent drugs potentially all the way from the production phase to their state within living matter.

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“…The study demonstrated the power of FLIM as a microscopic technique and the fascinating data it can deliver while investigating therapeutically relevant molecules. Out of its many strengths as an imaging tool, what emerged to be particularly relevant from the perspective of this study was the insensitivity of the fluorescence lifetime of a fluorophore toward its concentration, emission intensity, and bleaching effect. − As a dye, FITC was a stable pH-sensitive marker for microscopy. The fluorescence lifetime of FITCor other fluorescein derivatives per se, such as C-SNARF-4 or Oregon Green 488decreases with pH. − For example, the lifetime of FITC is 1.6 ns at pH 3, 4.5 ns at pH 5.8, and 7.8 ns at pH 9.…”

Section: Discussionmentioning

confidence: 99%

An Investigation into the Acidity-Induced Insulin Agglomeration: Implications for Drug Delivery and Translation

Fagihi,

Premathilaka,

O’Neill

et al. 2023

ACS Omega

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Insulin undergoes agglomeration with (subtle) changes in its biochemical environment, including acidity, application of heat, ionic imbalance, and exposure to hydrophobic surfaces. The therapeutic impact of such unwarranted insulin agglomeration is unclear and needs further evaluation. A systematic investigation was conducted on recombinant human insulinwith or without labeling with fluorescein isothiocyanatewhile preparing insulin suspensions (0.125, 0.25, and 0.5 mg/mL) at pH 3. The suspensions were incubated (37 °C) and analyzed at different time points (t = 2, 4, 24, 48, and 72 h). Transmission electron microscopy and nanoparticle tracking analysis identified colloidally stable (zeta potential 15 ± 5 mV) spherical agglomerates of unlabeled insulin (100–500 nm). Circular dichroism established the preservation of insulin’s secondary structure rich in α-helices despite exposure to an acidic environment (pH 3) for 72 h. Furthermore, fluorescence lifetime imaging microscopy illustrated an acidic core inside these spherical agglomerates, while the acidity gradually lessened toward the periphery. Some of these smaller agglomerates fused to form larger chunks with discrete zones of acidity. The data indicated a primary nucleation-driven mechanism of acid-induced insulin agglomeration under physiologically relevant conditions.

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Fluorescence lifetime microscopy unveils the supramolecular organization of liposomal Doxorubicin

Abstract: The supramolecular organization of Doxorubicin (DOX) within the standard Doxoves® liposomal formulation (DOX®) is investigated using visible light and phasor approach to fluorescence lifetime imaging (phasor-FLIM).

Cited by 19 publications

References 21 publications

Single-cell imaging of α and β cell metabolic response to glucose in living human Langerhans islets

Single-cell imaging of α and β cell metabolic response to glucose in living human Langerhans islets

Monitoring drug stability by label-free fluorescence lifetime imaging: a case study on liposomal doxorubicin

An Investigation into the Acidity-Induced Insulin Agglomeration: Implications for Drug Delivery and Translation

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