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Direct Optofluidic Measurement of the Lipid Permeability of Fluoroquinolones

View Article: PubMed Central - PubMed

ABSTRACT

Quantifying drug permeability across lipid membranes is crucial for drug development. In addition, reduced membrane permeability is a leading cause of antibiotic resistance in bacteria, and hence there is a need for new technologies that can quantify antibiotic transport across biological membranes. We recently developed an optofluidic assay that directly determines the permeability coefficient of autofluorescent drug molecules across lipid membranes. Using ultraviolet fluorescence microscopy, we directly track drug accumulation in giant lipid vesicles as they traverse a microfluidic device while exposed to the drug. Importantly, our measurement does not require the knowledge of the octanol partition coefficient of the drug – we directly determine the permeability coefficient for the specific drug-lipid system. In this work, we report measurements on a range of fluoroquinolone antibiotics and find that their pH dependent lipid permeability can span over two orders of magnitude. We describe various technical improvements for our assay, and provide a new graphical user interface for data analysis to make the technology easier to use for the wider community.

No MeSH data available.


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Schematic of the optofluidic assay.(a) Vesicles and antibiotic molecules are mixed using a T junction microfluidic chip. Under UV illumination, the antibiotic molecules are autofluorescent. The channel width is 40 μm. Vesicles can be detected at two time points in the same field of view. As a vesicle traverses the channel, the drug permeates into the vesicle and hence the drug autofluorescence intensity within the vesicle increases. This increase in drug autofluorescence inside the vesicle (Iin) is used to determine the permeability coefficient of the drug across the vesicle lipid membrane on the basis of a simple diffusion model4. (b) Typical experimental data, depicting the permeation of pefloxacin into DPhPC lipid vesicles at pH 6. Each point in the scatter plot represents an individual vesicle. The dark grey squares refer to vesicles detected at the initial (t = 0) time point whereas the blue triangles refer to vesicles detected at the later detection point (on average 8.5 s further downstream for this particular measurement). The downward shift in ΔI with time is a result of the increase in autofluorescence within the vesicles (Iin) and is thus a direct measurement of pefloxacin permeation into the vesicles. Similar scatter plots for the other drug/pH conditions measured in this paper are presented in the Supplementary Information. (c) Chemical structures of the fluoroquinolone antibiotics investigated in this paper arranged in order of decreasing octanol partition coefficients (Table 1).
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f1: Schematic of the optofluidic assay.(a) Vesicles and antibiotic molecules are mixed using a T junction microfluidic chip. Under UV illumination, the antibiotic molecules are autofluorescent. The channel width is 40 μm. Vesicles can be detected at two time points in the same field of view. As a vesicle traverses the channel, the drug permeates into the vesicle and hence the drug autofluorescence intensity within the vesicle increases. This increase in drug autofluorescence inside the vesicle (Iin) is used to determine the permeability coefficient of the drug across the vesicle lipid membrane on the basis of a simple diffusion model4. (b) Typical experimental data, depicting the permeation of pefloxacin into DPhPC lipid vesicles at pH 6. Each point in the scatter plot represents an individual vesicle. The dark grey squares refer to vesicles detected at the initial (t = 0) time point whereas the blue triangles refer to vesicles detected at the later detection point (on average 8.5 s further downstream for this particular measurement). The downward shift in ΔI with time is a result of the increase in autofluorescence within the vesicles (Iin) and is thus a direct measurement of pefloxacin permeation into the vesicles. Similar scatter plots for the other drug/pH conditions measured in this paper are presented in the Supplementary Information. (c) Chemical structures of the fluoroquinolone antibiotics investigated in this paper arranged in order of decreasing octanol partition coefficients (Table 1).

Mentions: The T junction microfluidic chips used in the experiments (shown schematically in Fig. 1a) were constructed using standard photo- and soft lithography techniques41824. The chips are made of Sylgard 184 polydimethylsiloxane (PDMS, Dow Corning) using an elastomer : curing agent ratio of 9:1. The chips are designed such that two time points may be observed in the same field of view (60× magnification). We have constructed multiple designs with different lengths of the mixing channel, which enable us to observe the vesicles over different timescales. For fast permeation experiments (on the order of a few seconds), we used a chip design where the channel length from the T junction to the outlet reservoir was 28 mm18. For measurements where the permeation was found to be slower (minutes), we used a chip with a channel length of approximately 380 mm from the T junction to the outlet reservoir4. In our experiments, vesicles typically flow through the channel at speeds of around 1 mm/s. AutoCAD drawings of the different designs are available on request.


Direct Optofluidic Measurement of the Lipid Permeability of Fluoroquinolones
Schematic of the optofluidic assay.(a) Vesicles and antibiotic molecules are mixed using a T junction microfluidic chip. Under UV illumination, the antibiotic molecules are autofluorescent. The channel width is 40 μm. Vesicles can be detected at two time points in the same field of view. As a vesicle traverses the channel, the drug permeates into the vesicle and hence the drug autofluorescence intensity within the vesicle increases. This increase in drug autofluorescence inside the vesicle (Iin) is used to determine the permeability coefficient of the drug across the vesicle lipid membrane on the basis of a simple diffusion model4. (b) Typical experimental data, depicting the permeation of pefloxacin into DPhPC lipid vesicles at pH 6. Each point in the scatter plot represents an individual vesicle. The dark grey squares refer to vesicles detected at the initial (t = 0) time point whereas the blue triangles refer to vesicles detected at the later detection point (on average 8.5 s further downstream for this particular measurement). The downward shift in ΔI with time is a result of the increase in autofluorescence within the vesicles (Iin) and is thus a direct measurement of pefloxacin permeation into the vesicles. Similar scatter plots for the other drug/pH conditions measured in this paper are presented in the Supplementary Information. (c) Chemical structures of the fluoroquinolone antibiotics investigated in this paper arranged in order of decreasing octanol partition coefficients (Table 1).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5015079&req=5

f1: Schematic of the optofluidic assay.(a) Vesicles and antibiotic molecules are mixed using a T junction microfluidic chip. Under UV illumination, the antibiotic molecules are autofluorescent. The channel width is 40 μm. Vesicles can be detected at two time points in the same field of view. As a vesicle traverses the channel, the drug permeates into the vesicle and hence the drug autofluorescence intensity within the vesicle increases. This increase in drug autofluorescence inside the vesicle (Iin) is used to determine the permeability coefficient of the drug across the vesicle lipid membrane on the basis of a simple diffusion model4. (b) Typical experimental data, depicting the permeation of pefloxacin into DPhPC lipid vesicles at pH 6. Each point in the scatter plot represents an individual vesicle. The dark grey squares refer to vesicles detected at the initial (t = 0) time point whereas the blue triangles refer to vesicles detected at the later detection point (on average 8.5 s further downstream for this particular measurement). The downward shift in ΔI with time is a result of the increase in autofluorescence within the vesicles (Iin) and is thus a direct measurement of pefloxacin permeation into the vesicles. Similar scatter plots for the other drug/pH conditions measured in this paper are presented in the Supplementary Information. (c) Chemical structures of the fluoroquinolone antibiotics investigated in this paper arranged in order of decreasing octanol partition coefficients (Table 1).
Mentions: The T junction microfluidic chips used in the experiments (shown schematically in Fig. 1a) were constructed using standard photo- and soft lithography techniques41824. The chips are made of Sylgard 184 polydimethylsiloxane (PDMS, Dow Corning) using an elastomer : curing agent ratio of 9:1. The chips are designed such that two time points may be observed in the same field of view (60× magnification). We have constructed multiple designs with different lengths of the mixing channel, which enable us to observe the vesicles over different timescales. For fast permeation experiments (on the order of a few seconds), we used a chip design where the channel length from the T junction to the outlet reservoir was 28 mm18. For measurements where the permeation was found to be slower (minutes), we used a chip with a channel length of approximately 380 mm from the T junction to the outlet reservoir4. In our experiments, vesicles typically flow through the channel at speeds of around 1 mm/s. AutoCAD drawings of the different designs are available on request.

View Article: PubMed Central - PubMed

ABSTRACT

Quantifying drug permeability across lipid membranes is crucial for drug development. In addition, reduced membrane permeability is a leading cause of antibiotic resistance in bacteria, and hence there is a need for new technologies that can quantify antibiotic transport across biological membranes. We recently developed an optofluidic assay that directly determines the permeability coefficient of autofluorescent drug molecules across lipid membranes. Using ultraviolet fluorescence microscopy, we directly track drug accumulation in giant lipid vesicles as they traverse a microfluidic device while exposed to the drug. Importantly, our measurement does not require the knowledge of the octanol partition coefficient of the drug – we directly determine the permeability coefficient for the specific drug-lipid system. In this work, we report measurements on a range of fluoroquinolone antibiotics and find that their pH dependent lipid permeability can span over two orders of magnitude. We describe various technical improvements for our assay, and provide a new graphical user interface for data analysis to make the technology easier to use for the wider community.

No MeSH data available.


Related in: MedlinePlus