Image Cytometry: Versatile Tool for Probing Cellular Uptake Kinetics of Biodegradable Nanoparticles

Research Article

Austin J Radiol. 2015;2(5): 1031.

Image Cytometry: Versatile Tool for Probing Cellular Uptake Kinetics of Biodegradable Nanoparticles

Roohi Gupta¹,²*, Mishra P¹ and Mittal A²

¹Department of Biochemical Engineering & Biotechnology, Indian Institute of Technology, India

²Kusuma School of Biological Sciences, Indian Institute of Technology, India

³Department of Radiation Oncology, Fox Chase Cancer Center, USA

*Corresponding author: Roohi Gupta, Department of Radiation Oncology, Fox Chase Cancer Center, P0103, 333 Cottman Avenue, Philadelphia, PA 19111, USA

Received: February 09, 2015; Accepted: August 18, 2015; Published: August 20, 2015


Biodegradable nanoparticles have been extensively investigated to facilitate intracellular delivery of therapeutics. Uptake kinetic studies and subsequent quantitative analysis are important in order to develop efficient drug delivery vehicle. In the present work, image cytometry is used for studying intra cellular uptake kinetics of biodegradable nanoparticles with two model systems- HeLa and Caco-2 cells. Nanoparticles (NPs) were formulated using biodegradable Poly D, L-lactic-co-Glycolic Acid (PLGA) polymer containing 6-coumarin as a fluorescent marker and characterized for size, surface morphology, surface charge, loading efficiency and encapsulation efficiency. Cells were incubated with coumarin loaded NPs for defined time intervals (0 to 2 hours) and at different temperature conditions (4°C, 25°C and 37°C). Intracellular amount of nanoparticles (in terms of its fluorescence intensity) was quantified by analyzing the captured fluorescent images of cells in MATLAB (using selfwritten algorithms). Both types of cells took up PLGA NPs quite readily with similar uptake profile and saturation was observed after one hour incubation period. The internalization of NPs was found to be more in Caco-2 cells when compared with HeLa, but the initial rate of uptake was found to be higher in HeLa cells. Also, no statistical difference in uptake kinetics was observed between synchronized and non-synchronized cells. The internalization of NPs was confirmed by confocal laser scanning microscopy. We report for the first time rigorous image analysis using MATLAB for studying uptake kinetics of NPs thus providing a new quantitative tool in studying drug delivery. Also, the results described herein will enhance our basic understanding of the cellular uptake of biodegradable NPs with cells in vitro which in turn could help in developing efficient nanocarrier systems for intracellular delivery of therapeutics.

Keywords: Image cytometry; Nanoparticles; Cell synchronization; Drugdelivery


Drug delivery vehicles (e.g. Liposomes, Polymeric nanoparticles, Dendrimers) are promising carriers for delivering therapeutic molecules (e.g. drugs, proteins) to physiological systems [1-6]. Polymeric biodegradable nanoparticles are of particular interest due to their biocompatibility, biodegradability and ability to maintain therapeutic drug levels for sustained periods of time [7-11]. PLGA has been the most extensively investigated polymer used in the formulation of biodegradable NPs [8-10]. PLGA has US Food and Drug Administration approval and due to polyester in nature, it undergoes hydrolysis after entering into the body, forming biologically compatible and metabolizable moieties (lactic acid and glycolic acid) that are eventually removed from the body by the citric acid cycle [10,12].

Pharmaceutical applications of NPs depend upon delivery of these nanoparticles (encapsulating therapeutic molecules) to target cells followed by proper intracellular uptake. Uptake kinetic studies and subsequent quantitative analysis are crucial in order to develop efficient drug delivery vehicle [13]. Previous studies indicated that much emphasis is laid on studying nanosystems of different compositions and its methods of formulations, but little information is available on understanding their interactions with the biological environment (target cells) and their intracellular uptake. Present work attempts to provide insight in to these relatively unexplored areas by studying the uptake kinetics of biodegradable PLGA NPs. The mammalian cancer cell lines, HeLa (the best characterized cell line in terms of its cell biology) and CaCo-2 (widely used and established in vitro cell line to evaluate the intestinal permeability and metabolism of drugs) were used as model systems for present investigations [14,15]. NPs were loaded with a hydrophobic fluorescent probe (model hydrophobic drug) as fluorescence labeling makes cellular uptake of nanoparticles readily detectable by fluorescence microscopy. 6-coumarin was used as a fluorescent marker because the marker shows high fluorescence activity even at low concentration and it does not leach away from nanoparticles in acidic pH present in endolysosomes ensuring accurate intracellular nanoparticles distribution pattern [16]. 6-coumarin loaded PLGA NPs were incubated with cells for defined time intervals and at different temperature conditions. Incubated cells were observed using fluorescence microscopy and images were captured. Quantification of uptake of fluorophore loaded nanoparticles have been estimated using flow cytometry [6,17] or using HPLC analysis [18,19] of fluorophore extracted from cell lysates in previous reports. Fluorescent intensities of internalized nanoparticles have also been quantified using analysis of captured fluorescent images by various commercially available software’s like Image J software [20]. But we report rigorous image analysis using MATLAB for studying uptake kinetics of NPs in contrast to expensive (flow cytometry) or cumbersome procedure (HPLC analysis) as published earlier. Own scripts were written in MATLAB for performing specific image analysis tasks. Uptake studies were also performed with synchronized cells. Confocal laser scanning microscopy was used to confirm internalization of nanoparticles. These studies will be useful to establish the efficacy of biodegradable nanoparticles for intracellular drug delivery and emphasizing the applicability of image cytometry in pharmaceutical research.

Materials and Methods


Poly (D, L-lactide- co- glycolide) (PLGA, MW 40,000-75,000, copolymer ratio 50:50), Polyvinyl alcohol (PVA, MW 30,000- 70,000), 6- Coumarin (MW 350.44), Thymidine and Nocodazole were purchased from Sigma Chemical (St. Louis, MO, USA). Dichloromethane (HPLC grade) and Formaldehyde were from Qualigens Fine Chemicals (Mumbai, Maharashtra, India). New Born Calf Serum (NBCS), Foetal Bovine Serum (FBS), Dulbecco’s Phosphate Buffer Solution (DPBS) and Dulbecco’s Modified Eagles Medium (DMEM) were purchased from Gibco BRL (Grand Island, NY, USA). Kanamycin acid sulphate, Trypsin-EDTA solution and Propidium Iodide were purchased from Himedia Laboratories Pvt. Ltd (Mumbai, Maharashtra, India). Tissue culture plates (35 mm diameter) and tissue culture flasks (T-25) used were from Corning Life Sciences (Corning, NY, USA).

Formulation of coumarin loaded nanoparticles

Nanoparticles containing a lipophilic fluorescent dye, 6-coumarin [Ex (λ) 450nm/ Em (λ) 490 nm] were formulated by using single emulsion- solvent evaporation technique as described previously [18,21]. In a typical procedure, 90 mg PLGA was dissolved in 3 ml of dichloromethane. A 2% solution of PVA was prepared in cold distilled water, and centrifuged at 1000 rpm for 5 min and then filtered through a 0.22 μm hydrophilic polysulfonic membrane syringe filter (25 mm Millipore filter unit, Millipore, Bedford, MA, USA) to remove any undisclosed PVA. 50μg of 6-coumarin (stock solution 0.5 mg/ml in dichloromethane) was added to the PLGA solution followed by vortexing. It was then placed on an ice bath for 5 min and emulsified using a micro tip probe sonicator (Soniprep 150, MSE Scientific Instruments, Crawley, UK) for 30s, to obtain a primary emulsion. The primary emulsion was then added in two portions to 12 ml of the PVA solution with intermittent vortexing to obtain oil in water emulsion. The emulsion was placed on an ice bath for 5 min and then sonicated for 2 min. The o/w emulsion was stirred overnight on a magnetic stirring plate (Spinit, New Delhi, India) to allow the evaporation of dichloromethane and formation of the nanoparticles. The suspension of nanoparticles was stirred in a vacuum desiccators placed on the magnetic stirring plate for an additional hour to ensure complete removal of the organic solvent. The suspension was transferred into centrifuge tubes and centrifuged at 27000 rpm (110,000×g) for 20 min at 4 °C in an ultracentrifuge (Beckman L8- 60M Ultracentrifuge, Fullerton, CA, USA). The pellet obtained was resuspended in double distilled water and sonicated for 30s on an ice bath to disperse any aggregates. Again ultracentrifugation of the sonicated suspension was carried out under same conditions as mentioned above. The pellet obtained again was resuspended in double distilled water, sonicated and ultra centrifuged was carried out. These centrifugation steps were meant to remove PVA from the formulation. After the last centrifugation, the coumarin loaded PLGA nanoparticles were resuspended in 7 ml of double distilled water and sonicated for 30s on an ice bath. The nanoparticles were then centrifuged at 1000 rpm for 10 min at 4 °C to remove any large aggregates. The supernatant was collected and frozen at -70 °C for 45 min and subsequently lyophilized for 2 days (Labconco Freeze Dry system/ Free zone 4.5, Kansas City, MO, USA). The lyophilized (powdered form) coumarin loaded PLGA nanoparticles were then stored at 4 °C.

Nanoparticle characterization

Size of the nanoparticles was determined by Photon Correlation Spectroscopy (PCS) using quasi elastic light scattering equipment (Brookhaven Instruments Corp., Holtsville, NY, USA) and 90 Plus Particle Sizing Software (Version 3.42). To measure particle size, a dilute suspension (200μg/ml) of nanoparticles was prepared in double distilled water and sonicated on an ice bath for 30s to break the aggregates [18]. The sample was filled in a cuvette and subjected to particle size analysis. Similarly, Zeta potential of the nanoparticles was measured using Malvern Zetasizer Nano ZS (Worcestershire, United Kingdom). The surface morphology of the formulated nanoparticles was visualized by Scanning Electron Microscope (Zeiss EVO 50). Fluorescence techniques were used to evaluate the actual amount of 6-coumarin dye encapsulated in the particles. Because 6-coumarin has inherent fluorescent properties (excitation/ emission= 450 nm/490nm), spectroscopy (PerkinElmer Life And Analytical Sciences, Inc., Waltham, MA, USA) was used to generate a calibration curve of fluorescence values at known concentrations of the dye that allowed quantification of the percent loading of the dye in the NPs. To determine the amount of coumarin encapsulated in the particles, a known quantity of 6-coumarin NPs was dissolved in 1 ml of dichloromethane and kept overnight. The sample was centrifuged at 10,000 rpm for 5 min followed by collection and spectroscopic analysis of the supernatant [6]. The encapsulation efficiency was calculated by indirect method using the following formula [18]. Encapsulation efficiency (%) = [Total dye added (μg) – free dye (μg) / Total dye added (μg)] x 100. The supernatant and the washings (obtained during formulation of NPs) were stored to determine the amount of coumarin that was not entrapped in the NPs (free dye).

Cell culture

Human cervical adenocarcinoma (HeLa cells) and Human epithelial colorectal adenocarcinoma (Caco-2 cells) were used for studying the cellular uptake of coumarin loaded nanoparticles. Both cells were procured from National Centre for Cell Sciences (NCCS), Pune, and Maharashtra, India. HeLa cells were cultured regularly in T-25 tissue culture flasks in DMEM (Dulbecco’s Modified Eagle’s Medium) supplemented with 10% New Born Calf Serum at 37°C and 5% CO2 in a CO2 incubator (Shellab CO2 water jacketed incubator, Cornelius, OR, USA). Caco-2 cells were also cultured regularly in DMEM supplemented with 20% Foetal Bovine Serum at 37°C in 5% CO2. The cells were passaged in a split ratio of 1:2 or 1:3. All the experiments were performed with cells between passages 12 to 14.

Cellular nanoparticle uptake studies: Both HeLa and Caco- 2 cells (in separate sets of experiments) were seeded in 35 mm diameter tissue culture petridishes and were allowed to grow till 80% confluency. A suspension of nanoparticles (2 mg/ml) in DMEM was prepared as stock [19]. The medium in the petri dishes was replaced with the suspension of the nanoparticles (100μg/ml) and incubated for different time intervals 0 min, 15 min, 30 min, 60 min, 90 min and 120 min. After specific time durations the cells were washed three times with DPBS (with Ca/Mg) to remove any non- cell associated nanoparticles [10,19]. Then the cells were fixed with 4% formaldehyde [22] and were observed using an inverted fluorescence microscope IX51 Olympus (Olympus Inc., Tokyo, Japan). Green channel data for Coumarin (Co) visualization was acquired by using the mirror unit U-MWB2 (Olympus Inc., Tokyo, Japan) with excitation filter of 460- 490 nm, emission filter of 520 nm and a dichromatic mirror at 500 nm. The images were acquired using a cooled CCD camera (DP70, Olympus Inc., Tokyo, Japan). The uptake studies were performed at temperatures 4°C, 25°C and 37°C independently for both cell lines. For each case the exposure time for image acquisition (using DP70) was kept same (fixed manually) to allow signal comparisons.

The studies were also conducted with live cells for both cell lines. In live cell imaging, the cells were visualized immediately (after washing step) without fixing and observed using IX51 Olympus and images were captured using Olympus DP70 camera. All the experiments were conducted in triplicates and each result is presented as mean ± SD with n=3.

Cell synchronization: HeLa and Caco-2 cells were synchronized using thymidine and nocodazole [23]. To synchronize cells at the G1 / S boundary, cells were treated at 50% confluence with 2 mM thymidine. After 16 h, cells were washed and fed with fresh medium for 6 h before addition of nocodazole (600 ng/ml from a stock of 5 mg/ml prepared in DMSO) for 1h. The cells were then fixed [22], stained with propidium iodide (0.03mg/ml prepared in DPBS) [nucleic acid binding fluorescent dye, Ex (λ) 530nm/Em (λ) 615 nm], observed using IX51 Olympus and images were captured using Olympus DP70 camera. The synchronized cells (for both cell lines) were also incubated with suspension of the nanoparticles (100 μg/ ml) at 37°C for different time intervals 0 min, 15 min, 30 min, 60 min, 90 min and 120 min, fixed, stained with Propidium Iodide (PI), observed using IX51 Olympus and images were captured using Olympus DP70 camera. Red channel data for PI visualization was acquired by using the mirror unit U-MWG2 (Olympus Inc., Tokyo, Japan) with excitation filter of 510-550 nm, emission filter of 590 nm and a dichromatic mirror at 570 nm. All Images were acquired at same manual camera settings to allow image data comparison. The experiments were conducted in triplicates and each result is presented as mean ± SD with n=3.

Quantification of images in MATLAB

All the images observed (of random fields) using IX51 Olympus microscope (using specific filters) and captured using DP70 camera were analyzed in MATLAB (The Math Works, Inc.). Images were acquired at same manual camera settings to allow image data comparison. For quantification of fluorescence intensity associated with cells, own algorithms were written in MATLAB (for performing specific image analysis tasks) and the total pixels associated with cell in X-Y coordinate system were quantified in terms of population assay and single cell assay. In case of population assay, total fluorescence in a given field of view as well as average fluorescence per cell (obtained by analyzing total fluorescence intensity per field divided by total number of cells in the given field) was analyzed. In case of single cell assay, 25 randomly selected cells were analyzed individually per field of view. The fluorescence intensities at different times were normalized (for all sets of data) with that of 0 min incubation time to compensate for variations in excitation intensities and/or camera output.

Confocal laser scanning microscopy (CLSM)

Intracellular localization was confirmed by confocal laser scanning microscopy (Olympus Fluoview FV 1000 equipped with lasers 405, 488, 515, 543 and 633, Olympus Inc., Tokyo, Japan). HeLa and Caco-2 cells incubated with 6-coumarin labeled PLGA NP’s were excited with 488 nm and detected in channel 1 (dichroic mirror 570 nm, filter 530 nm) and acquisitions were saved.

Results and Discussion

Characterization of nanoparticles

Nanoparticles (NPs) were prepared from PLGA polymer containing free carboxylic end groups, loaded with 6-coumarin molecules using single emulsion solvent evaporation method. The nanoparticles size (obtained from quasi-light scattering) ranged from 132 to 246 nm with polydispersity index ranging from 0.02-0.154, suggesting uniformity in the particle size distribution. Previous reports have shown that particle size is an important property that affects the intracellular uptake of nanoparticles. Though different cell types have different property (specific cell size for uptake) but in general particles < 500nm in size can be internalized by the cells by endocytotic processes [15,16]. Therefore, our formulations of nanoparticles were suited for the cellular uptake studies. The zeta potential of NPs ranged from -8.4 mV to –12.37mV, indicating location of few free carboxylic end groups of the PLGA polymer on the surface of nanoparticles. The negative charge on surface of NPs prevented aggregation of NPs. Loading efficiency of coumarin particles was found out to be 60% w/w with encapsulation efficiency 62%.

The SEM image of the coumarin-loaded PLGA nanoparticles revealed their regular spherical shape (Figure 1A). Their surface morphology was smooth, without any visible pinholes or cracks within the conventional SEM resolution.