Multiparameter Quantification of Liposomal Nanomedicines at the

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Multiparameter Quantification of Liposomal Nanomedicines at the Single-particle Level by High Sensitivity Flow Cytometry Chaoxiang Chen, Shaobin Zhu, Shuo Wang, Wenqiang Zhang, Yu Cheng, and Xiaomei Yan ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b01867 • Publication Date (Web): 04 Apr 2017 Downloaded from http://pubs.acs.org on April 9, 2017

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ACS Applied Materials & Interfaces

Multiparameter Quantification of Liposomal Nanomedicines at the Single-particle Level by High Sensitivity Flow Cytometry

Chaoxiang Chen, Shaobin Zhu, Shuo Wang, Wenqiang Zhang, Yu Cheng, and Xiaomei Yan*

The MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, The Key Laboratory for Chemical Biology of Fujian Province, Collaborative Innovation Center of Chemistry for Energy Materials, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, Fujian 361005, P. R. China

Corresponding Author * To whom correspondence should be addressed. Phone: 86-592-2184519. E-mail: [email protected].

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ABSTRACT Drug-encapsulated liposomes have been considered the most clinically acceptable drug delivery systems. However, current methods fall short in the quantitative characterization of individual nanoliposomes due to their small sizes and large heterogeneity. Here we report a high-throughput method for the absolute quantification of particle size, drug content, fraction of drug encapsulation, and particle concentration of liposomal nanomedicines at the single-particle level. A laboratory-built high sensitivity flow cytometer (HSFCM) was used to simultaneously detect the side scattered and fluorescence signals generated by individual nanomedicine particles at a speed up to 10000 nanoparticles per minute. To cope with the size dependence of refractive index of liposomal nanomedicines, different sizes of doxorubicin-loaded liposomes (DELs) were fabricated and characterized to serve as the calibration standards for both the particle size and drug content measurement. This method provides a highly practical platform for the characterization of liposomal nanomedicines, and broad applications can be envisioned.

KEYWORDS: doxorubicin-encapsulated liposomes, nanomedicine, single-particle detection, nanoparticle characterization, flow cytometry

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INTRODUCTION Liposomal nanoparticles, which are characterized by high biological compatibility and the ability to incorporate large quantities of various drugs, are considered the most successful drug delivery systems known to date.1-5 Pegylated liposomal doxorubicin (Doxil) was the first FDA-approved nano-drug (1995) and has been successfully applied to treat several types of cancer.6 Due to the complexity and the large intrinsic heterogeneity of liposomal nanomedicines in particle size and composition, rigorous characterization of their physicochemical properties at the single-particle level is of fundamental importance for successful clinical translation.7-9 Cryogenic transmission electron microscopy (cryo-TEM) and freeze-fracture TEM (FF-TEM) are the central techniques used to provide precise measurements of the size, morphology, and structure of drug-encapsulating liposomes.10-13 Although cryo-electron tomography has been applied to measure the volume of the doxorubicin core in Doxil,14 the actual number of doxorubicin (Dox) molecules in each liposome remains unknown. Moreover, the routine use of cryo-TEM for the characterization of drug-encapsulating liposomes is impractical due to the low accessibility, high cost, and tedious sample preparation and analysis procedures. Thus, current analyses of liposome-based nanomedicines have been mainly relied on bulk measurements, including dynamic light scattering (DLS), size-exclusion chromatography, and field-flow fractionation (FFF).15-18 However, these methods fail to reveal size distribution in high resolution and can hardly provide biochemical information such as how many drug molecules are enclosed or how many ligand molecules are immobilized on the surface of each individual liposome particle. The lack of state-of-the-art methodologies for precise nanoparticle characterization has 3 / 25



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been recognized as the biggest challenge to the field of nanomedicine.19, 20 Recently, many newly developed single-particle techniques have been applied to the characterization of liposomal nanoparticles, such as nanoparticle tracking analysis (NTA), tunable-resistive pulse sensing (TRPS), high-resolution flow cytometry (FCM), chip-based flow cytometer, and electrochemical methods,21-27 along with many microscopic techniques, such as fluorescence microscopy, confocal laser scanning microscopy (CLSM), atomic force microscopy (AFM), and environmental scanning electron microscopy (ESEM).29-32 However, high-throughput methods facilitating multiparameter quantification of liposomal nanomedicines at the single-particle level and down to 50 nm particle size remain to be developed. Integrating light scattering with strategies for single-molecule fluorescence detection in a sheathed flow, we have developed high sensitivity flow cytometry (HSFCM) that enables multiparameter analysis of silica nanoparticles (SiNPs) down to 24 nm.33-35 Recently, analysis of single viruses with a resolution comparable to that of electron microscopy has been demonstrated.36 With respect to the characterization of liposome-based nanomedicines, HSFCM is sufficiently sensitive to detect both the scattered light and the intrinsic fluorescence of the doxorubicin emitted from each individual liposome of Doxoves (~60 nm, a research-grade product of PEGylated liposomal doxorubicin) and to detect both the light scattering and the fluorescence of SYTO dye-labeled siRNA of siRNA-loaded lipid nanoparticles (54 ± 13 nm).35 Here, we report an HSFCM-based method for the absolute quantification of particle size, drug content, fraction of drug encapsulation (the ratio of drug-loaded liposomes to all liposomes), and

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particle concentration of liposomal nanomedicines at the single-particle level with an analysis rate up to 10000 particles per minute.

EXPERIMENTAL SECTION Reagents and chemicals. Hydrogenated soy L-α-phosphatidylcholine (HSPC), cholesterol and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000] (ammonium salt) (DSPE-PEG2000) were purchased from Avanti Polar Lipids, Inc. (Alabaster, Alabama, USA). Doxorubicin hydrochloride was purchased from J&K Scientific Co., Ltd. (Beijing, China). Doxove was purchased from FormuMax Scientific Inc. (Palo Alto, California, USA). The generic version of Doxil was obtained in China. PD-10 Desalting Columns and Polycarbonate Membrane Filters with pore diameter of 30 nm, 50 nm, 80 nm, 100 nm, 200 nm, and 400 nm were purchased from GE Healthcare Life Sciences (Piscataway, New Jersey, USA). Fluorescent silica nanoparticles of 80 nm diameter were synthesized in the laboratory and the particle concentration was measured by calibration against 100 nm Orange FluoSpheres of known concentration (Molecular Probes, Rugen, USA) via single particle enumeration using HSFCM.34 Distilled, deionized water supplied by a Milli-Q RG unit (Millipore, Bedford, Massachusetts, USA) was filtered through a 0.22-µm filter and used for buffer preparation and served as the sheath fluid of the HSFCM. All the buffers were filtered through a 0.22-µm filter and used within three weeks. Laboratory-built HSFCM system. The laboratory-built HSFCM system was described before.35, 36 In brief, a 20-mW, 532-nm continuous-wave solid-state Nd:YAG laser was used as 5 / 25



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the excitation light source. A half-wave plate and a polarizing beam splitter provided polarization control and continuously variable attenuation of the laser light. The laser excitation power of 16 mW was used in the present study. The light emitted by each nanoparticle was collected by an infinity-corrected microscopic objective and was then directed by a dichroic beam splitter into two distinct light paths for SS and FL detection. The reflected light was spectrally filtered by a bandpass filter (FF01-524/24, Semrock, Inc.) to reduce the interference of fluorescence and was then focused by an aspheric lens onto a single-photon counting avalanche photodiode detector (APD) for side scatter detection. The small active area (∼180 µm in diameter) of the APD detector works as a limiting aperture to exclude the background scattering from the cuvette windows and the sheath fluid. The transmitted light was spectrally filtered by a Raman edge filter (LP03-532RS, Semrock, Inc.) and a bandpass filter (FF01-582/75, Semrock, Inc.), and was then focused onto the second APD by another aspheric lens for FL detection. The output signals were simultaneously counted and processed by a National Instruments DAQ card and a custom program written in LabVIEW 2012. HSFCM fluidics system was the same as that described before.33-36 In brief, ultrapure water filtered through 0.22-µm filters was used as the sheath fluid via a gravity feed, and the flow rate was controlled by adjusting the relative height between the sheath supply bottle and the waste container. Normally, the sheath-flow rate was approximately 40 µL/min and the measured sample volumetric flow rate was approximately 2 nL/min. Based on the overlap of the focused laser spot (approximately 16 µm) and the sample stream (approximately 1.4 µm in diameter), the calculated detection volumes (defined as the product of the size of the laser-beam spot and the sample-stream area) were approximately 25 fL. According 6 / 25


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to Poisson statistics, for a nanoparticle concentration of approximately 5 × 109/mL, the probability that two nanoparticles will pass through the probe volume (25 fL) simultaneously is 0.7%. Preparation

of

doxorubicin-encapsulated

liposomes

as

reference

standards.

Doxorubicin-encapsulating liposomes (DELs) of different sizes were prepared by film hydration and extrusion as described in the literature.37 Briefly, HSPC, cholesterol, and DSPE-PEG2000 were mixed and dissolved in chloroform at a molar ratio of 56.3 : 38.4 : 5.3 with a total lipid concentration of 40 mM. Once the lipids were thoroughly mixed, the solvent was dried on a rotary evaporator into a lipid film and kept in vacuo overnight to remove the residual chloroform. The lipid film was hydrated in 250 mM ammonium sulfate solution at 65 °C for 1 h and then subjected to 10 freeze-thaw cycles between liquid nitrogen and a 65 °C water bath to form a multilamellar liposome suspension. Then, the size of the liposomes was decreased by extruding them through polycarbonate filters with pore size of 400 nm and 200 nm, followed by 100 nm, 80 nm, 50 nm or 30 nm, respectively to obtain liposomes with four different sizes. After 20 cycles of extrusion, the transmembrane ammonium gradient of the suspension was established by dialysis against the stocking solution (10% sucrose, 10 mM histidine, pH 6.5). Then the lipid concentrations of the four liposomal standards were measured by the Stewart assay. Doxorubicin was added to each liposome suspension and diluted to a final lipid and drug concentration of 20 mM and 2 mg/mL, respectively. The bulk encapsulation efficiency of doxorubicin was measured by detecting the fluorescence intensity before and after purification through a PD-10 column (diluted to the same lipid concentration with stocking solution). The drug encapsulation 7 / 25



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efficiencies of the liposomal standards extruded through 30-nm, 50-nm, 80-nm and 100-nm filters were found to be 99.4%, 98.5%, 97.6% and 93.9%, respectively. The liposomes were sterilized by filtration through a 0.22-µm membrane to be used as reference standards and stored at 4 °C and protected from light for long-term stability. All the liposomal samples for HSFCM and cryo-TEM analysis were diluted in the stocking solution. Quantification of the doxorubicin content for DEL reference standards. The doxorubicin content of the as synthesized DEL reference standards was quantified in units of molecules of Dox per particle. A Shimadzu RF-5301 spectrofluorometer was used for the fluorescence measurement. The calibration curve of the concentration of doxorubicin standard solutions ranging from 0.25 µM to 5.00 µM, and fluorescence intensity at 556 nm was established. Then, the DEL standards were lysed in a 0.5% Triton X-100 solution and diluted 100-fold prior to the fluorescence measurement under the same instrument conditions to obtain the corresponding doxorubicin concentration via the calibration curve. Meanwhile, the particle concentrations of the DEL standards were measured via single-particle enumeration by the HSFCM. The DEL standards were diluted 2 × 104-fold and mixed with a diluted suspension of 80 nm diameter fluorescent silica nanoparticles of known particle concentration. By comparing the number of events measured in 1 min between the DEL standards and that of the internal standard (fluorescent silica nanoparticles), an accurate particle concentration of DELs standards can be acquired.

RESULTS AND DISCUSSION 8 / 25


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A laboratory-built HSFCM equipped with a 20-mW, 532-nm continuous-wave solid-state Nd:YAG laser was used as the excitation source.35, 36 Ultrapure water (distilled, deionized water filtered through a 0.22-µm filter) served as the sheath fluid of the HSFCM. In HSFCM, the focused laser spot (~ 16 µm in diameter) is much larger than the hydrodynamically focused sample stream (~ 1.4 µm in diameter). Therefore on the light path of side scattering detection (perpendicular to the incident laser beam), part of the sheath fluid that is illuminated by the laser beam can also be sensed by the detector though not at the image plane of the detector. Due to the much larger sheath flow rate than that of the sample fluid, the number of background particles detected by a flow-cytometric system is mainly contributed by the impurity particles in the sheath fluid. Thus, the much slower sheath-flow velocity (approximately 20 mm/s) of HSFCM than that of the conventional FCM (several m/s) results in a significant reduction in the detected event rate of impurity particles in the sheath fluid by three to four orders of magnitude. When the ultrapure water was injected as the sample, the average count rate of impurity particles was around 120 per minute (data not shown). Assessment of Encapsulation Efficiency. To assess the performance of the instrument in analyzing liposomal nanomedicine, doxorubicin-encapsulating liposomes (DELs) were used as the model system and a DEL sample with large heterogeneity in particle size and Dox content was specially fabricated. The nanomedicine particles were passed sequentially through the tightly focused laser beam of the HSFCM, and the side scatter (SS) and fluorescence (FL) signals emitted from each individual particle were detected simultaneously. Figure. 1a shows the representative burst traces of side scatter (SS) and fluorescence signals. We can see that there 9 / 25



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existed some liposomal particles from which no fluorescence was detected concurrently with the side-scattered light and can be inferred to be empty liposomes. Among 6126 events detected in 1 minute of data acquisition, two distinct populations can be identified in the bivariate dot-plot of fluorescence burst area versus side scatter burst area (Figure. 1b), and approximately 30% of the population exhibits negligible fluorescence signal. This fraction of liposomes may have lost their transmembrane ammonium gradient prior to the drug loading, which resulted in the unsuccessful encapsulation of doxorubicin. Therefore, by enabling simultaneous side scatter and fluorescence detection of single Dox-loaded liposomes, HSFCM provides the unique advantage in revealing empty vesicles and assessing the fraction of drug encapsulation which otherwise have been masked by bulk measurements.

Figure 1. HSFCM analysis of a DEL sample with large heterogeneity. a) Representative side scatter and fluorescence burst traces. b) Bivariate dot-plot of the FL versus SS burst area. The dot-plot was derived from data collected over 1 min. Laser excitation power: 16 mW; focused laser spot: 16 µm diameter. 10 / 25


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Establishment of Size Quantification Method. Elastic light scattering represents the simplest and most direct method for single-particle detection, yet particle size is usually measured in a relative manner. To enable absolute particle sizing via intensity measurement of the scattered light by individual nanoparticles, a calibration curve built upon size reference standards of a closely matched refractive index is a prerequisite. However, this is rather difficult for drug-loaded liposomes. At a certain wavelength, the average refractive index of a liposome of spherical shape is determined by the refractive indices of the lipid and the aqueous compartment and by their volume fractions in the vesicle, which can be described as below:38, 39

=

+ 1 −

1,

where nLiposome is the refractive index of the liposome; nL and nw are the refractive indices of the lipid bilayer and the inner water phase, respectively; and f is the volume fraction of lipid in the vesicle. Figure. 2a shows the calculated dependence of the refractive index of empty liposomes on the particle size by using 1.49 and 1.33 for nL and nw, respectively.38 Because of the volume fractions of the lipid and aqueous compartment scale with the square and cube of the linear dimension, respectively, the much lower refractive index of the aqueous phase results in a continual decrease in the refractive index of empty liposomes with the increase of particle size. Using these refractive indices, the normalized side scattering intensities of empty liposomes were calculated based on the Rayleigh scattering theory, of which the scattering cross section (σscatt) is given by: σ =

2

! & "#$% 3(&

* − 1 ) ) 2, * +2


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where d is the particle diameter, λ is the wavelength of the incident light, nmed is the refractive index of the medium surrounding the particle (1.33 for water), and m is the ratio of the refractive indices of the particle and the medium. For SiNPs and PNPs, the refractive indices at 532 nm are 1.46 and 1.59, respectively. The normalized side scattering intensities of empty liposomes were compared with those of SiNPs and polystyrene nanoparticles (PNPs), which have been widely used as the reference standards in light scattering-based size measurement. Figure. 2b shows that, in the size range of 40 to 140 nm, the large difference in refractive index that separates PNPs and SiNPs from empty liposomes results in a significant discrepancy in the scattered light intensity, particularly for larger size particles. Therefore, for an accurate size measurement of liposomes or vesicles of which the refractive index varies with particle size, it is necessary to have a size reference standard of closely matched size-dependent refractive index for the calibration of the scattered light intensity of nanoparticles. For Dox-encapsulating liposomes, their ellipsoid shape and the crystallization of doxorubicin inside liposomes lead to a more complicated situation of the refractive index.

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Figure 2. Theoretical calculations of a) the size dependence of the refractive index of empty liposomes and b) the normalized side scattering intensity of polystyrene nanoparticles (PNPs), SiNPs, and empty liposomes.

To perfectly match the refractive index of Doxil, we synthesized DELs of different sizes in strict accordance with the lipid composition and drug-to-lipid ratio of Doxil. These DELs were meant to be used as a dual-purpose standard for both the particle size and drug content calibration of Doxil. Cryo-TEM was used to measure the absolute particle size of the DEL standards extruded from polycarbonate membranes with each pore size (Figure. 3a). Note that when doxorubicin molecules are loaded into the liposome via a transmembrane ammonium sulfate gradient, they are accumulated in the intra-liposome aqueous phase and most of them are in the form of aggregated (crystallized) [doxorubicin]2SO4 salt with a rod-like shape.6,14,41 For DELs extruded from polycarbonate membranes with pore sizes of 30, 50, 80, and 100 nm, the measured particle sizes were 59 ± 10 nm, 72 ± 10 nm, 95 ± 13 nm and 115 ± 15 nm, respectively (Figure. 3b). Liposome extrusion is normally carried out at a temperature above the gel to liquid-crystalline phase transition temperature of the lipid bilayer. In this liquid phase, the liposomes are very flexible. During the extrusion, liposomes will be compressed into flat-ellipse shaped form and then reformed to spherical shape after passing through the pores. Therefore, the actual size of liposomes will be larger than the pore size.

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Figure 3. Fabrication of DEL reference standards for the size calibration of Doxil. a) Representative cryo-TEM images of the DEL standards extruded from polycarbonate membranes of different sizes. b) Particle size distribution histograms for DEL standards of different sizes. For each reference standard, 250 individual liposomes in the cryo-TEM micrographs were examined. The particle size of oval-shaped liposome was measured as the average of the long and short axes. Instrument: FEI Tecnai F20 transmission electron microscope operating at 200 kV.

Then, these four DEL standards were analyzed on the HSFCM. Figure. 4a shows the representative side scatter and fluorescence burst traces of DELs prepared by 50-nm polycarbonate membrane extrusion. Meanwhile, the intrinsic fluorescence of doxorubicin is dramatically quenched compared with that of their free form in solution. However, thanks to the superior sensitivity of the HSFCM, the extremely weak intrinsic fluorescence of encapsulated doxorubicin from individual liposomes can be well detected above the background. The SS burst area distribution histograms of all four sizes of DEL standards were plotted (Figure. 4b) upon 14 / 25


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interrogating thousands of particles individually for each size of the DEL standard. By plotting the median SS burst area against the particle size measured by cryo-TEM, a calibration curve was obtained, exhibiting approximately three orders of magnitude of dependence of the SS intensity on liposome size (Figure. 4c). To verify the importance of matching the refractive index in the design of appropriate size reference standards, four sizes of monodisperse SiNPs were synthesized with comparable sizes to those of the DEL standards and analyzed on the HSFCM at the same instrument settings. The resulting curve exhibited an approximately sixth order dependence of intensity on particle size (Figure. 4c). The two calibration curves of DELs and SiNPs intersect around a particle size of 56 nm and deviate largely from each other with increasing particle size. These data highlight the necessity and importance of using size standards with comparable refractive indices to ensure accurate particle size measurement based on the intensity of scattered light. Establishment of Drug Content Quantification Method. In addition to the particle size measurement, quantification of loaded doxorubicin content in each liposome is indispensable for ensuring the biological function of nanomedicines. In our earlier report, we showed that HSFCM is sensitive enough to detect the intrinsic doxorubicin fluorescence emitted from each individual liposome particles of Doxove.35 Here, we move forward to the absolute quantification of loaded doxorubicin content in units of molecules of Dox per particle. First, the doxorubicin concentration of the DEL standard was obtained by using a spectrofluorometer to measure the fluorescence emission spectra of doxorubicin standard solutions along with DEL standards upon solubilization (lysed in a 0.5% Triton X-100 solution, Figure. 4d). In the HSFCM setup, the 15 / 25



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hydrodynamically focused sample stream (ca. 1.4 µm) was completely illuminated within the central region of the focused laser beam (~ 16 µm), and all the particles going through the flow cell could be detected.33-36 Therefore, the particle concentrations of the DEL standard can be accurately analyzed on the HSFCM via single-particle enumeration by using 80 nm diameter fluorescent SiNPs of known particle concentration as the internal standard (Figure. 4e).35 By dividing the doxorubicin concentration of a solubilized DEL standard by its particle concentration, the average number of doxorubicin molecules per liposome particle can be derived. The average molecules of Dox per particle were measured to be 9.0 × 103, 1.1 × 104, 1.7 × 104, and 2.3 × 104 for these four DEL standards. Figure. 4f shows a linear calibration curve of molecules of Dox per particle with the median FL burst area of these four DEL standards. This strict linear relationship facilitates the drug content quantification of Dox-loaded liposomes via fluorescence detection. Thus, with the calibration curves of scattered light intensity against particle size and fluorescence intensity against molecules of Dox per particle, the side scatter and fluorescence signals of each individual drug-encapsulating liposome can be converted to a particle size in nanometers and a Dox content in molecules of Dox. With an analysis rate of up to 10 000 particles per minute, statistically robust distributions of the particle size and drug content of a DEL sample can be acquired in minutes.

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Figure 4. Particle size and Dox content characterization of the DEL reference standards. a) Representative side scatter and fluorescence burst traces for a DEL standard prepared using 50-nm PC membrane extrusion. b) Side scatter burst area distribution histograms for all four sizes of DEL standards. c) Dependence of scattered light intensity on the particle size for DEL standards and monodisperse SiNPs. d) Spectrofluorometric measurement of doxorubicin standard solutions and the solubilized DEL standard. Inset: The calibration curve between the doxorubicin concentration and the integrated fluorescence intensity. e) Particle concentration measurement of DEL using 80 nm diameter fluorescent SiNPs of known concentration as an internal standard on the HSFCM. f) Linear relationship between the number of doxorubicin molecules per particle and the FL burst area for four sizes of DEL standards.

Applications in the Characterization of Commercial DEL Samples. The DEL standards were used for the quantitative characterization of both the particle size and doxorubicin content of commercially available samples. In addition to Doxove, a generic version of Doxil (gDoxil, 17 / 25



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manufactured in China) was also analyzed. First, the four DEL standards were analyzed separately on the HSFCM to construct the calibration curves of SS intensity versus particle size and FL intensity versus molecules of Dox per particle. Then, gDoxil and Doxove were measured using the same instrument settings. Based on the calibration curves, the brightness of the side scatter and fluorescence for each liposome can be converted to the particle size and doxorubicin content. As shown in Figure. 5, the large inherent polydispersity can be disclosed in minutes by the HSFCM. For the sample of Doxove, the measured particle size was 63 ± 12 nm, which is in good agreement with the cryo-TEM measurement of 62 ± 11 nm.35 Moreover, the doxorubicin content and its distribution are reported for the first time as (9.2 ± 4.8) × 103 molecules per nanoliposomes. Our method uniquely enables the absolute measurement of drug content in single liposomes, which can hardly be achieved currently by other methods. For the sample of gDoxil, the particle size and doxorubicin content were measured to be 71 ± 14 nm and (1.7 ± 0.7) × 104 molecules per particle, respectively. A positive correlation of particle size and drug content was identified for both samples and the doxorubicin encapsulation efficiency was measured to be approximately 100% at the single-particle level for both Doxove and gDoxil. Moreover, through the single-particle enumeration, the liposome concentrations of Doxove and gDoxil were determined to be 2.7 × 1014 and 1.5 × 1014 particles/mL. It is worth mentioning that the DEL standards are stable for at least twelve months when stored at the 4 °C and protected from light.

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Figure 5. Multiparameter quantification of Dox-loaded liposome samples. a) and b) Bivariate dot-plots of the doxorubicin content versus the particle size for Doxove (a, 40,000-fold dilution in stocking solution) and gDoxil (b, 20,000-fold dilution in stocking solution).

CONCLUSIONS To summarize, using doxorubicin-encapsulating liposomes as a model, we demonstrate the application of a laboratory-built HSFCM for the absolute quantification of particle size, drug content, fraction of drug encapsulation, and particle concentration of liposomal nanomedicines at the single-particle level for the first time. Superior instrumentation sensitivity facilitating simultaneous detection of the scattered light and fluorescence signals of each individual nanoliposomes as small as 30 nm in size is the key point. Therefore, the fluorescence of the drug encapsulated in a nanocarrier, either by intrinsic fluorescence or via fluorescent labeling, can be correlated with the side scatter signal at the single-particle level to quantitatively determine the fraction of drug encapsulation. Understanding the fraction of drug encapsulation and the drug 19 / 25



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content in each individual liposomal particle opens up new routes in the effort to synthesize more uniform nanomedicines for drug delivery. For the accurate particle size measurement of liposomal nanomedicines, both the theoretical calculations and experimental results highlight the significant importance of matching the refractive indices of size reference standards with liposomal samples. Doxorubicin-loaded liposomes of different sizes were fabricated and characterized to serve as the calibration standards for the measurement of both the particle size and drug content. Distributions of particle size and drug content of a specimen can be obtained in minutes by converting the distributions of side scatter and fluorescence intensity via the calibration curves, respectively. The HSFCM method provides a versatile and highly practical platform for the characterization of liposomal nanomedicine.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] ORCID Xiaomei Yan: 0000-0002-7482-6863 Notes The authors declare no competing financial interests.

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ACKNOWLEDGMENTS We acknowledge support from the National Natural Science Foundation of China (21475112, 21225523, 21027010, and 21521004), the National Key Basic Research Program of China (2013CB933703), the National Fund for Fostering Talents of Basic Science (J1310024), and the Program for Changjiang Scholars and Innovative Research Team in University (IRT13036). The authors thank Prof. Gaolin Liang (University of Science and Technology of China) for assistance with cryo-TEM analysis.

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Multiparameter Quantification of Liposomal Nanomedicines at the

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