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Influence of Cubosome Surface Architecture on its Cellular Uptake Mechanism Sonal Deshpande, and Neetu Singh Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b04423 • Publication Date (Web): 21 Mar 2017 Downloaded from http://pubs.acs.org on March 27, 2017

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Influence of Cubosome Surface Architecture on its Cellular Uptake Mechanism Sonal Deshpande1 and Neetu Singh*1,2 1

Centre for Biomedical Engineering, Indian Institute of Technology-Delhi, Hauz Khas, New

Delhi-110016, India. 2

Biomedical Engineering Unit, All India Institute of Medical Sciences, Ansari Nagar, New

Delhi-110029, India Keywords: cell membrane, cholesterol, cubosomes, surface architecture, uptake mechanisms

Abstract: Interaction of nanoparticles with biological systems is a key factor influencing their efficacy as a drug delivery vehicle. The inconsistency in defining the optimal design parameters across different nanoparticle types, suggests that information gained from one model system need not apply to other systems. Therefore, selection of a versatile model system is critical for such studies. Cubosomes are one of the potential drug delivery vehicles due to their biocompatibility, stability, ability to carry hydrophobic, hydrophilic and amphiphilic drugs and ease of surface modification. Here we report, the importance of surface architecture of cubosomes by comparing their cellular uptake mechanism with poly-ε-lysine (PεL) coated cubosomes. Uncoated cubosomes entered cells by energy-independent, cholesterol dependent mechanism, whereas PεL coated cubosomes relied on energy-dependent mechanisms and enter

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the endosomes. As endosomal entrapment was evaded by uncoated cubosomes, they can be preferably used for cytosolic delivery of therapeutic agents.

INTRODUCTION Nanoparticles have found wide applications in the field of medicine owing to the advantages they provide over conventional therapeutic agents. However, their application as drug delivery vehicles and imaging agents is restricted because of their toxicity,1–3 instability,4 undesired interaction with proteins5,6 and limitation to the amount of cargo they can carry. As a result, efforts are constantly being made to either improve the properties of the existing nanoparticles or to synthesize altogether a new nanoparticle. Both these approaches can lead to the development of multifunctional nanoparticles. However for designing an efficacious drug delivery system, insight into the interaction of nanoparticles with biological system is very critical.7,8 Depending upon their applications, nanoparticles are desired to enter specific cell organelles or the cytosol. Nanoparticles typically enter cells via different uptake mechanisms like phagocytosis,

clathrin-mediated

endocytosis,

caveolae-mediated

endocytosis

and

macropinocytosis.9–12 These mechanisms result in entrapment of the nanoparticles into endosomes and eventually exposing them to the acidic environment of lysosomes, which may degrade and affect the activity of the therapeutic agent.13,14 Endosomal entrapment is also one of the bottlenecks in RNAi, as the cargo needs to escape out of the endosome and enter the cytosol to interact with the cellular machinery for further processing.15 To circumvent this issue, strategies like endosomal escape of nanoparticles have been investigated. As the toxicity associated with nanoparticles has also been attributed to the immune system activation by their interaction with endosomal toll-like receptors (TLRs)16, exploring strategies involving alternative


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uptake mechanisms might be helpful. Other energy independent uptake mechanisms, where entrapment in endosomal vesicle may not occur, like clathrin and caveolae-independent uptake and translocation across the cell membrane via lipid interaction needs to be exploited.17–19 As the intracellular fate of nanoparticles depends upon the mechanism by which they are internalized by the cells, extensive investigations have been performed by varying various design aspects of the nanoparticle that can influence the cellular interaction, such as size, shape, charge,20–22 crosslinking density (for polymeric nanoparticles)23 and surface lipophilicity24,25. Interestingly, the observations from such nanoparticle-cell interaction studies are mostly restricted to the model system under investigation and the system might have limitations that can affect its applications. For example, even though a number of studies have shown the effect of size, shape and surface charge of gold nanoparticles on its cellular uptake, as the type and amount of drugs that can be loaded onto them is limited, it cannot be applied to obtain a versatile delivery system. On the other hand, although loading of the nanoparticles can be easily achieved in polymeric systems, it is rather difficult to modulate the surface architecture, thereby compromising the flexibility in their applications. Therefore, for a potential translation of the information derived from such nanomaterial-cell interaction studies, it is essential to develop a system where such studies can be performed and utilized to tune the uptake mechanism easily. This will thus provide a platform system that can be modified based on the application. Cubosomes are self-assembled lyotropic liquid crystalline nanoparticles with an intrinsic property of patterned surface and are a potential drug delivery vehicle with ability to carry different types of drugs and imaging agents.26–28 Though initial studies in the field were limited to the material characterization, recent focus has shifted to their biological evaluation.29–33 Studies on a unit cell of cubic phase indicated the presence of water channel openings on the


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surface surrounded by lipid bilayer resulting into a specific hydrophilic-hydrophobic pattern,34,35 a structure difficult to obtain at nanoscale. Recently, our group has taken advantage of this feature and reported the use of cubosomes as a theranostic vehicle.36 We demonstrated that it can be easily functionalized to modify the surface, is biocompatible, stable in biological milieu, easily internalized by the cells and have the ability to carry hydrophilic, hydrophobic as well as amphiphilic molecules. Thorough understanding of their interaction with biological systems will aid in obtaining information required to modulate their application based properties thereby taking cubosomes one step closer into clinics. In this work, we have studied cellular uptake of two different cubosomes, one without any surface modification and other with a polymer coating of the surface. While in the uncoated cubosomes, the patterned surface is maintained, a simple coating with the cationic polymer, poly-ε-lysine, masks the pattern, thereby making the surface hydrophilic. Since, surface architecture is known to alter the cellular interactions we hypothesize that there should be a difference in the uptake of these nanoparticles. Here, we report a systematic study probing the difference in the uptake for a hydrophobic-hydrophilic patterned and hydrophilic surface cubosomes. EXPERIMENTAL SECTION Materials Rylo MG 20 Pharma (henceforth called Rylo), a commercial grade monoolein-based emulsifier, was received as generous gift from Danisco Corporation (India) and was used as received. Pluoronic F127 was obtained from BASF and Poly-ε-lysine (Mw=20000) from CMS Chemicals Ltd. (UK). Cholesterol was obtained from SRL chemicals. Methyl-β-cyclodextrin was obtained from TCI chemicals. HeLa, MDA-MB-231and NIH-3T3 cells were obtained from


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National Centre for Cell Sciences, Pune, India. Dulbecco's Modified Eagle Medium (DMEM, high glucose), Dulbecco’s Phosphate buffered saline (DPBS), calcein, Lysotracker DND-22, trypsin, fetal bovine serum (FBS) and 4′,6-diamidino-2-phenylindole (DAPI) were procured from ThermoFisher Scientific. Nile Red, lovastatin, Tween 20, chlorpromazine and paraformaldehyde were obtained from Sigma Aldrich. Cubosome preparation Cubosomes were prepared by a previously reported method.36 Briefly, Rylo/Nile Red mixture, with final concentration of 0.5 mg/g of Nile Red, was prepared and melted at 80°C. The bulk phase was then prepared by adding deionized water (DI) to the molten Rylo/Nile Red mixture, such that their ratio was 40:60 respectively. The bulk phase was allowed to form a crystal clear homogeneous phase and characterized by SAXS. To prepare cubosomes, 0.2 g of bulk phase was homogenized in 2 mL of aqueous Pluronic F127 solution such that Rylo:Pluronic F127 was in the ratio of 100:5 (w/w). Homogenization was carried out using IKA Ultraturrax T18 at 12000 rpm for 30 min. The synthesized cubosomes were characterized by cryo-TEM and dynamic light scattering (DLS). The average size of cubosomes (designated as 𝑅𝐹 𝑁𝑅 ) was obtained by DLS using Malvern Nano ZS90TM particle size analyzer. From the average size and the amount of Rylo added, number density of cubosomes was estimated. This gives the surface area of cubosomes in the solution, which was used for calculating the amount of functional polymer required for surface functionalization. Calculated amount of poly-ε-lysine solution was then added dropwise, in order to avoid phase separation, to get polymer coating density of 0.5 mg/m 2 𝑁𝑅 in the solution. Coated cubosomes were designated as 𝑅𝐹𝑃𝜀𝐿 . Coating of cubosomes was

confirmed by analyzing its zeta potential by Malvern Nano ZS90TM particle size analyzer. For cell studies, cubosomes were sterilized by UV exposure for 1 h.


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Time dependent stability of cubosomes under biologically relevant conditions was analyzed by monitoring the hydrodynamic diameter by DLS. Samples were prepared by incubating cubosomes in phosphate buffered saline containing 10% fetal bovine serum. Interaction of cubosomes with cholesterol Cholesterol (1 mg) was suspended in 1 mL DPBS by vigorous shaking. Cubosomes were added to the suspension at the final concentration of 4 mg/mL and mixed well by vortexing. The suspension was incubated for 14 h at room temperature, and an aliquot was viewed under Olympus IX73 inverted fluorescence microscope using TRITC filter for observing the fluorescence from Nile Red loaded in the cubosomes. Cholesterol crystals were removed by a brief spin of 2 min using Remi RM-02 minicentrifuge and change in cubosome size and phase was monitored by Malvern Nano ZS90TM particle size analyzer and Cryo-TEM, respectively. Hemolysis assay Hemolysis assay was performed by adding cubosomes at different concentrations to 50 µL of erythrocytes, to a final volume of 250 µL in PBS. After incubation at 37°C for 30 min, cells were centrifuged at 1000 g for 5 min and absorbance of the supernatant was measured at 415 nm to quantify cell lysis. Cells untreated with cubosomes were used as negative control while cells incubated with water as positive control. Blocking energy dependent mechanisms Circular coverslips (12 mm) were sterilized overnight in absolute ethanol, placed in 24 well plate and washed with DPBS. HeLa cells were then seeded in DMEM+10% FBS at a density of 5×104 cells/well and incubated overnight at 37°C under 5% CO2. Media was removed and the cells were then washed with DPBS and pre-incubated at 4°C in serum free DMEM for 1 h.


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Media was then replaced with cold serum free DMEM containing cubosomes at a final concentration of 0.05 mg/mL and incubated for another 1 h and 3 h at 4°C. Cells incubated at 37°C for 15min, 30min, 1h, 3h and 4h with cubosomes were used as control. Cells without cubosomes treatment were used as blank to eliminate cellular auto-fluorescence. After incubation with cubosomes, cells were first washed three times with chilled DPBS on ice. They were then briefly washed with ice cold 0.05% Tween 20 in DPBS, followed by three washes of ice cold DPBS. Cells were then fixed at room temperature with 4% (w/v) paraformaldehyde in DPBS for 15 min, followed by three washes of DPBS. After fixing, the cells were counterstained for nuclei using 300 nM DAPI for 30 s, washed with DPBS and observed under Carl Zeiss fluorescence microscope using TRITC and DAPI filters. The images were processed using the NIH ImageJ software. The uptake was also quantified by flow cytometry. For preparation of samples for flow cytometry, instead of fixing, the cells were trypsinized, passed through 40 µm BD strainer and analyzed by excitation with 488 nm laser and emission under PE channel of BD AccuriC6 flow cytometer. All measurements were done in triplicate and the standard deviations were plotted as error bars. Statistical analysis was done using Student’s t-test. Uptake of cubosomes by MDAMB-231 and NIH 3T3 cells at 4°C and 37°C for 1 h was also analyzed by flow cytometry using same protocol. Energy dependence of coated cubosomes was further investigated by specifically inhibiting the clathrin-coated pit formation, by chlorpromazine. Briefly, HeLa cells were seeded on sterile coverslips in a 24 well plate, at a density of 5×104 cells/well and incubated overnight. Cell were then pre-incubated with 10 µg/mL chlorpromazine in serum-free DMEM, for 20 min at 37°C. Media was then replaced with serum-free DMEM containing cubosomes at a final concentration of 0.05 mg/mL, along with the inhibitor. Cells were then incubated for 30 min at 37°C, washed


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three times with DPBS and processed for flow cytometry, according to the previous protocol. Statistical analysis was done using Student’s t-test. Endosomal uptake of cubosomes by colocalization studies HeLa cells were seeded in DMEM+10% FBS at density of 5×104 cells/well in a 24 well plate on sterilized coverslips and incubated overnight. They were then washed with DPBS and preincubated at 4°C for 1 h in serum-free DMEM. Calcein is a membrane-impermeable fluorescent dye that gets entrapped in the endocytic vesicles, therefore is used to study endosomal uptake. Calcein and cubosomes at final concentration of 0.1 mg/mL and 0.05 mg/mL, respectively, were added to the medium and further incubated for 1 h at 4°C. Cells were then incubated at 37°C for 2 h. Cells without cubosomes and calcein treatment were used as blank to eliminate cellular autofluorescence. They were then washed with DPBS, followed by brief wash with Tween 20 and three more washes with DPBS. For fixing, cells were incubated in 4% (w/v) paraformaldehyde for 15 min, washed with DPBS and observed under Olympus confocal microscope using TRITC and FITC filters. The images were processed and colocalization was quantified using Coloc2 plugin of NIH ImageJ (Fiji) software. The endosomal fate of cubosomes was then investigated by labelling lysosomes using Lysotracker Blue DND-22. HeLa cells were pre-incubated at 4°C for 1 h in serum-free DMEM. Cubosomes at the final concentration of 0.05 mg/mL were added to the medium and further incubated for 1 h at 4°C. Cells were then incubated at 37°C for 2 h, followed by 3 washes of DPBS. They were then stained using Lysotracker Blue DND-22 (75 nM) for 30 min at 37°C and washed 3 times with DPBS, before imaging by Nikon confocal microsope using DAPI and TRITC filters. The images were processed and pseudo coloured using the NIH ImageJ software. Colocalization was quantified using Coloc2 plugin of NIH ImageJ (Fiji) software.


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Lipid vesicle formation after cubosome treatment HeLa cells were seeded in DMEM+10% FBS at density of 5×104 cells/well in 24 well plate on sterilized coverslips and incubated overnight. They were then washed with DPBS and incubated for 4 h at 37°C in presence of 0.05 mg/mL cubosomes in serum-free media. Cells were then washed 3 times with DPBS, stained with 300 nM Nile Red for 30 min at 37°C, followed by 3 more washes of DPBS. Cells were then imaged using Olympus confocal and IX73 inverted fluorescence microscope using TRITC and FITC filters. Colocalization was quantified using Coloc2 plugin of NIH ImageJ (Fiji) software. Cellular cholesterol depletion HeLa cells at density of 5×104 cells/well were seeded on coverslips in a 24 well plate. After overnight incubation, they were washed with DPBS and pre-incubated for 1 h at 37°C in serum free DMEM containing the inhibitors, methyl-β-cyclodextrin and lovastatin, at the final concentration of 2.5 mM and 1 µg/mL, respectively. Cubosomes (0.05 mg/mL) were then added to the inhibitor containing media and incubation was continued for another 1 h at 37°C. Cells incubated in serum free media without inhibitors were used as control. Cells without inhibitor as well as cubosomes were used as blank to eliminate any cellular auto-fluorescence. Cells were then washed three times with DPBS, followed by a brief wash with 0.05% Tween 20 and three more washes of DBPS. Cells were then imaged under Olympus IX73 inverted fluorescence microscope using red channel. The images were processed using the NIH ImageJ software. For flow cytometry, cells were trypsinized, passed through 40 µm BD strainer and analyzed by excitation with 488 nm laser and emission under PE channel of BD FACS Aria flow cytometer. All measurements were done in triplicate and the standard deviations were plotted as error bars.


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For blocking energy dependent as well as cholesterol dependent mechanisms, same protocol was followed, only the pre-incubation, incubation with cubosomes and washing was done at 4°C. Statistical analysis was done using Students’s T-test. RESULTS AND DISCUSSION Characterization of cubosomes Cubosomes were prepared using our previously reported method.36 The ratio of Rylo:deionized water used for the preparation of cubosomes was 60:40, which gave a crystal clear, Pn3m phase at room temperature. Nile Red, a hydrophobic fluorescent dye, loaded in the cubosomes was used for their intracellular tracking by fluorescence microscopy. On the basis of our previous study, 0.05% Nile Red was loaded into bulk phase, as at this concentration, the Pn3m phase is maintained. The cubic phase was verified by small angle X-ray scattering analysis, which showed typical peaks for cubic phase (Figure S1a).37 Homogenization of the bulk phase yielded a turbid suspension of cubosomes. Cubosomes thus formed were analyzed by DLS and were found to be of 152±19 nm in diameter with a zeta potential of -20±4 mV (Figure S1b and S1c). Poly-ε-lysine (PεL), a cationic polymer, coats the negatively charged surface of cubosomes via electrostatic interaction. Coated cubosomes were 169±28 nm in size (Figure S1b) and had a near neutral surface charge of +1±0.5 mV (Figure S1c). Cubosomes of sub 200 nm, along with few vesicles, were also observed by cryo-TEM (Figure S1d and S1e). Further, incubation of cubosomes with phosphate buffered saline containing 10% FBS, did not affect their hydrodynamic diameter (Figure S1f). A recent study by Azmi et. al.38 showed change in phase of phytantriol based cubosomes from cubic to a neat hexagonal phase when incubated with human plasma. Interestingly, when phytantriol was replaced with monoolein, the cubosomes were found to be more stable over a prolonged time.


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Hereafter, we denote the Nile Red loaded uncoated cubosomes as 𝑅𝐹 𝑁𝑅 and coated cubosomes 𝑁𝑅 as 𝑅𝐹𝑃𝜀𝐿 .

Cryo-TEM (Figure S1d and S1e) as well as theoretical models of cubosomes have suggested presence of water nanochannel openings on the surface, surrounded by lipid bilayers resulting into a patterned surface of the uncoated cubosomes.34,35 The nanoparticles are further stabilized commonly by using a tri block copolymer, Pluronic F127. The exact interaction of pluronic with the surface of cubosomes and its ability to stabilize the surface is still not well understood and is being investigated by many groups.39–41 Depending upon the orientation of pluronic chains, which will further depend on its concentration etc. it could partially mask the surface with weak interactions. We anticipate that any other hydrophobic interactions42 should be able to easily destabilize the pluronic-hydrophobic region interaction on the cubosome surface, hence, exposing the lipid region on the surface. On the other hand, poly-ε-lysine coating can mask this hydrophilic-hydrophobic surface, yielding a slightly positively charged hydrophilic surface. Thus, providing cubosomes with two different surface structures, one with patterned hydrophilic𝑁𝑅 hydrophobic surface (𝑅𝐹 𝑁𝑅 ) and the other with only hydrophilic surface (𝑅𝐹𝑃𝜀𝐿 ). The cell

membrane is composed of a lipid bilayer embedded with different types of proteins and show presence of discrete membrane microdomains rich in cholesterol and sphingolipids, termed as lipid rafts.43 Thus, the cell membrane also exhibits a mosaic of regions with varying degree of hydrophobicity, which can be correlated to the patterned surface of cubosomes. Our study is based on a simple hypothesis that the hydrophobic-hydrophilic pattern on the uncoated cubosomes can interact with the hydrophobic regions on the cell surface and might enter the cells via mechanisms other than those known for charged hydrophilic nanoparticles. Before carrying out cellular uptake investigations, we studied the interaction of the cubosomes


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with cholesterol in-vitro and analyzed the stability of cubosomes in maintaining its cubic phase and size. We observed that the cubosomes with patterned surface, 𝑅𝐹 𝑁𝑅 , interacted with the hydrophobic cholesterol crystals and formed large vesicles, which is indicated by shift in the position of histogram peaks (Figure S2a) when measured by DLS. The large vesicles were also observed under fluorescence microscope (Figure S2b). On the other hand, the coated cubosomes showed no aggregation as indicated by no change in the size (Figure S2a). Visualization of the coated cubosomes after incubation with cholesterol under fluorescence microscope also suggested absence of aggregation (Figure S2b). To get a further insight on cholesterol interaction and internal structure of cubosomes, cryo-TEM was performed. As can be observed in Figure S2c, internal structure of uncoated cubosomes was altered, while it was maintained in case of coated cubosomes. The results suggest that as PεL is a hydrophilic polymer, coating cubosomes with PεL makes its surface more hydrophilic, thereby preventing its interaction with the hydrophobic cholesterol crystals. We have earlier reported the biocompatibility of cubosomes using HeLa cell lines.36 For further investigation, we performed hemolysis assay and observed that cubosomes did not show profound effect on erythorcytes stability upto 150 µg/mL (Figure S3). 44 Time dependent uptake We began our studies by first observing the cellular uptake of the two cubosomes, 𝑅𝐹 𝑁𝑅 and 𝑁𝑅 𝑅𝐹𝑃𝜀𝐿 , at 37°C on 4 h incubation time. Interestingly, we did not observe any significant

difference in the uptake of the two cubosomes (Figure S4a and S4b). This could be because all the uptake mechanisms are active at 37°C resulting into saturation of uptake after a longer incubation time.45,46 However, since cellular uptake first requires the binding of nanoparticles to


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the cell surface, followed by the internalization of nanoparticles by the specific endocytosis mechanisms, a preferred cell surface binding will be able to transport more nanoparticles inside at early time points. We therefore analyzed the kinetics of cubosome uptake by incubating cells with cubosomes for 15 min, 30 min, 1 h and 3 h at 37°C. After 15 min and 30 min incubation, we were surprised to observe higher uptake (52±3% and 65±2%, respectively) of 𝑅𝐹 𝑁𝑅 as 𝑁𝑅 compared to that of 𝑅𝐹𝑃𝜀𝐿 (38±5% and 51±3%, respectively) (Figure S4a). This difference was

not significant after 1 h incubation time, as almost similar cellular uptake was observed for both the nanoparticles (Figure S4a and S4b).These observations thus suggest that as hypothesized, indeed the kinetics of the cellular uptake of the two cubosomes is different. The results were even more surprising as 𝑅𝐹 𝑁𝑅 , with a negative surface charge (Figure S1c), was expected to show lesser cellular uptake. On the contrary, the higher uptake observed, indicates exploitation of some factors other than surface charge, by the cubosomes to enter the cells. Energy dependent uptake mechanism Intrigued by the slightly higher uptake of RF NR at 0.5) in comparison with 𝑅𝐹 𝑁𝑅 . Therefore, we confirmed that the 𝑁𝑅 uptake of 𝑅𝐹𝑃𝜀𝐿 was indeed by energy dependent mechanisms, which entraps them into the

endosomes, whereas, the 𝑅𝐹 𝑁𝑅 translocate across the membrane avoiding the endosomal entrapment. Interestingly, very recently Hinton et.al.,55 observed that cubosomes (without any coating) were present in punctate vesicles intracellularly. They speculated the punctate vesicles to be endosomes/lysosomes. Following this, another report by Falchi et. al.56 observed a time dependent increase in intracellular lipid droplets (observed as punctate spots) after incubating cells with the cubosomes. They further stained the punctate spots and demonstrated them to be lipid droplets. 56 Taking advantage of the Nile Red, a dye whose fluorescence is dependent on its environment hydrophobicity, loaded into cubosomes, we further examined the intracellular localization of cubosomes. After the cubosome uptake by the cells, we stained them with more free Nile Red in order to stain any lipid vesicles formed. Here, the hypothesis was that the Nile Red in cubosomes or similar hydrophobic environment will be red and in lipid droplets will be green. Interestingly, while the positive control, where cells were treated with free oleic acid to create lipid droplets, showed punctate green spots, the cells treated with uncoated cubosomes showed significantly higher green punctate fluorescence compared to the cells treated with coated cubosomes (Figure S8). Further investigation by confocal imaging (Figure S9) to observe if the green fluorescence from the lipid droplets and the red fluorescence from the cubosomes colocalize, confirmed that the cubosomes were observed to be localized with the lipid droplets (Pearson’s coefficient > 0.5). This study suggested that while lipid droplets were formed in the cells treated with uncoated cubosomes, cells treated with coated cubosomes had very few punctate green spots and mostly diffused green fluorescence suggesting slightly lower lipid droplet formation ability (Figure S8).


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Cholesterol depletion

Figure 3. a) Fluorescence microscopy images of HeLa cells treated with cholesterol inhibitors, methyl-β-cyclodextrin (2.5 mM) and lovastatin (1 µg/mL), at 37°C, showing significant inhibition of Nile Red loaded uncoated cubosomes (𝑅𝐹 𝑁𝑅 ) as compared to poly-ε-lysine coated 𝑁𝑅 cubosomes (𝑅𝐹𝑃𝜀𝐿 ). b) and c) Flow cytometry analysis of cubosome uptake by HeLa cells after

cholesterol inhibition at 37°C. Percent relative uptake indicates intensity of Nile Red (cubosomes) in cells, with respect to cells without cubosomes (Blank). Error bar indicates standard deviation between triplicates of two independent experiments. *** = P

Influence of Cubosome Surface Architecture on Its ... - ACS Publications

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