Targeted Plasma Membrane Delivery of a Hydrophobic Cargo Encapsulated in a Liquid Crystal Nanoparticle Carrier

Podsumowanie

A liquid crystal nanoparticle (LCNP) nanocarrier is exploited as a vehicle for the controlled delivery of a hydrophobic cargo to the plasma membrane of living cells.

Streszczenie

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. Recently, nanoparticles (NPs) have shown significant promise in the development of such delivery systems. Here, a liquid crystal NP (LCNP)-based delivery system has been employed for the controlled delivery of a water-insoluble dye, 3,3′-dioctadecyloxacarbocyanine perchlorate (DiO), from within the NP core to the hydrophobic region of a plasma membrane bilayer. During the synthesis of the NPs, the dye was efficiently incorporated into the hydrophobic LCNP core, as confirmed by multiple spectroscopic analyses. Conjugation of a PEGylated cholesterol derivative to the NP surface (DiO-LCNP-PEG-Chol) enabled the binding of the dye-loaded NPs to the plasma membrane in HEK 293T/17 cells. Time-resolved laser scanning confocal microscopy and Förster resonance energy transfer (FRET) imaging confirmed the passive efflux of DiO from the LCNP core and its insertion into the plasma membrane bilayer. Finally, the delivery of DiO as a LCNP-PEG-Chol attenuated the cytotoxicity of DiO; the NP form of DiO exhibited ~30-40% less toxicity compared to DiO_free delivered from bulk solution. This approach demonstrates the utility of the LCNP platform as an efficient modality for the membrane-specific delivery and modulation of hydrophobic molecular cargos.

Wprowadzenie

Since the advent of interfacing nanomaterials (materials ≤100 nm in at least one dimension) with living cells, a continuing goal has been to take advantage of the unique size-dependent properties of nanoparticles (NPs) for various applications. These applications include cell and tissue labeling/imaging (both in vitro and in vivo), real-time sensing, and the controlled delivery of drugs and other cargos¹. Examples of such relevant NP properties include the size-dependent emission of semiconductor nanocrystals (quantum dots, QDs); the photothermal properties of gold nanoparticles; the large loading capacity of the aqueous core of liposomes; and the ballistic conductivity of carbon allotropes, such as single-wall carbon nanotubes and graphene.

More recently, significant interest has arisen in the use of NPs for the controlled modulation of drugs and other cargos, such as contrast/imaging agents. Here, the rationale is to significantly enhance/optimize the overall solubility, delivered dose, circulation time, and eventual clearance of the drug cargo by delivering it as an NP formulation. This has come to be known as NP-mediated drug delivery (NMDD), and there are currently seven FDA-approved NP drug formulations for use in the clinic to treat various cancers and hundreds more in various stages of clinical trials. In essence, the goal is to "achieve more with less;" that is, to use the NP as a scaffold to deliver more drug with fewer dosing administrations by taking advantage of the large surface area:volume (e.g., hard particles, such as QDs and metal oxides) of NPs or their large interior volume for loading large cargo payloads (e.g., liposomes or micelles). The purpose here is to reduce the necessity for multiple systemically delivered dosing regimens while at the same time promoting aqueous stability and enhanced circulation, particularly for challenging hydrophobic drug cargos that, while highly effective, are sparingly soluble in aqueous media.

Thus, the goal of the work described herein was to determine the viability of using a novel NP scaffold for the specific and controlled delivery of hydrophobic cargos to the lipophilic plasma membrane bilayer. The motivation for the work was the inherent limited solubility and difficulty in the delivery of hydrophobic molecules to cells from aqueous media. Typically, the delivery of such hydrophobic molecules requires the use of organic solvents (e.g., DMSO) or amphiphilic surfactants (e.g., Poloxamers), which can be toxic and compromise cell and tissue viability², or micelle carriers, which can have limited internal loading capacities. The NP carrier chosen here was a novel liquid crystal NP (LCNP) formulation developed previously³ and that had been shown previously to achieve a ~40-fold improvement in the efficacy of the anticancer drug doxorubicin in cultured cells⁴.

In the work described herein, the representative cargo selected was the potentiometric membrane dye, 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO). DiO is a water-insoluble dye that has been used for anterograde and retrograde tracing in living and fixed neurons, membrane potential measurements, and for general membrane labeling^5,6,7,8,9. Due to its hydrophobic nature, DiO is typically added directly to cell monolayers or tissues in a crystalline form¹⁰, or it is incubated at very high concentrations (~1-20 µM) after dilution from a concentration stock solution^11,12.

Here, the approach was use to the LCNP platform, a multifunctional NP whose inner core is completely hydrophobic and whose surface is simultaneously hydrophilic and amenable to bioconjugation, as a delivery vehicle for DiO. DiO is incorporated into the LCNP core during synthesis, and the NP surface is then functionalized with a PEGylated cholesterol moiety to promote the membrane binding of the DiO-LCNP ensemble to the plasma membrane. This approach resulted in a delivery system that partitioned the DiO into the plasma membrane with greater fidelity and membrane residence time than the free form of DiO delivered from bulk solution (DiO_free). Further, this method showed that the LCNP-mediated delivery of DiO substantially modulates and drives the rate of specific partitioning of the dye into the lipophilic plasma membrane bilayer. This is achieved while concomitantly reducing the cytotoxicity of the free drug by ~40% by delivering it as an LCNP formulation.

It is anticipated that the methodology described herein will be a powerful enabling technique for researchers whose work involves or requires the cellular delivery of highly hydrophobic cargos that are sparingly soluble or completely insoluble in aqueous solution.

Protokół

1. Preparation of DiO-LCNP and DiO-LCNP-PEG-Chol

1. Dissolve liquid crystalline diacrylate cross-linking agent (DACTP11, 45 mg), 3,3′-dioctadecyloxacarbocyanine perchlorate (DiO, 2 mg), and a free radical initiator (azobisisobutyronitrile, 1 mg) for polymerization in 2 ml of chloroform. Add this to an aqueous solution of acrylate-functionalized surfactant (AC10COONa, 13 mg in 7 ml).
2. Stir the mixture for 1 hr and sonicate at 80% amplitude for 5 min to produce a miniemulsion consisting of small droplets of the organic material surrounded by polymerizable surfactant in water.
3. Heat the mixture to 64 °C in an oil bath to initiate the polymerization of both the cross-linking agent and surfactant as the chloroform slowly evaporates, leaving a DiO-containing NP suspension that is stabilized by the surfactant.
4. Filter the NP suspension (3 times) through a 0.2 µm syringe filter to remove any aggregation. Store the filtered NP solution at 4 °C until further use.
5. Conjugation of PEG-Chol to DiO-LCNP via EDC coupling.
   1. Dissolve Chol-PEG-NH₂·HCl (PEG-Chol, 0.9 mM) in 25 mM HEPES buffer (pH 7.0).
   2. Prepare a working solution containing N-hydroxy-sulfosuccinimide sodium salt (NHSS, 40 mM) and 1-ethyl-3-(3-(dimethylamino)-propyl) carbodiimide hydrochloride (EDC, 400 mM) in HEPES buffer from concentrated stock solutions.
   3. Immediately add 20 µl of the freshly prepared working solution of NHSS/EDC to 1.0 ml of DiO-LCNP in HEPES buffer and stir for 5 min.
   4. Add 20 µl of stock solution of Chol-PEG-NH₂·HCl to this mixture and stir for 2 hr.
   5. Briefly centrifuge the reaction mixture at maximum speed (~2,000 x g) for 30 sec using a tabletop mini centrifuge and pass the supernatant through a PD-10 size exclusion chromatography column¹³ equilibrated with Dulbecco's phosphate-buffered saline (DPBS, 0.1x).

2.  Characterization of DiO-LCNP and DiO-LCNP-PEG-Chol

1. Confirm the successful covalent conjugation of PEG-Chol to the DiO-LCNP surface by gel electrophoresis.
   1. Dissolve 0.5 g of agarose in 50 ml (1x) of tris-borate electrophoresis (TBE; 89 mM Tris (pH 7.6), 89 mM boric acid, and 2 mM EDTA) buffer. Heat the solution in a microwave oven to dissolve the agarose. Allow the solution to cool slightly and pour the contents into a gel plate in the electrophoresis box. Insert a gel comb to create sample wells.
   2. Once the gel has solidified, add the required amount (enough to submerge the gel in the chamber) of TBE running buffer to the chamber.
   3. Add 35 µl of DiO-LCNP sample (amended with glycerol, 5% v/v) to the wells of the gel and run for 20 min at a voltage of 110 V.
   4. Image the gel using a gel imaging system with excitation and emission filters at 488 nm and 500-550 nm, respectively.
2. Assess particle size and distribution by dynamic light scattering (DLS) by diluting the DiO-LCNP solution (~200-fold dilution) in PBS (pH ~8, 0.1x) and measuring on a DLS instrument¹⁴. Measure the zeta-potential of the DiO-LCNPs using an appropriate zeta potential measurement instrument.

3. Preparation of Cell Culture Dishes for Delivery Experiments and Imaging

NOTE: DiO-LCNP labeling is performed on HEK 293T/17 human embryonic kidney cells (between passages 5 and 15) that are cultured as described previously⁴. Perform the delivery experiments and the subsequent cell imaging as described below.

1. Prepare 35 mm diameter tissue culture dishes (fitted with 14 mm No. 1 coverglass inserts) by coating them with bovine fibronectin (~100 µl at a concentration of 20 µg/ml) in DPBS for 2 hr at 37 °C.
2. Remove the fibronectin coating solution from the culture dish. Harvest HEK 293 T1/7 cells from the T-25 flask by first washing the cell monolayer with 3 ml of DPBS and then by adding 2 ml of trypsin-EDTA (0.5% trypsin-0.25% EDTA).
3. Incubate the flask at 37 °C for ~3 min. Remove the trypsin-EDTA and return the flask to the incubator. Once cells are detached from the flask, neutralize the trypsin by adding 4 ml of complete medium (Dulbecco's Modified Eagle Medium; DMEM; amended to contain 10% fetal bovine serum, 5% sodium pyruvate, and 5% antibiotic/antimycotic) to the flask. Determine the cell concentration in the suspension by counting them in a cell counter.
4. Adjust the cell concentration to ~8 x 10⁴ cells/ml with growth medium. Add 3 ml of the cell suspension to the dish and culture in the incubator overnight; the next day, the cells should be at ~70% confluency and should be ready for use. Adequate cell density is critical for robust and efficient labeling of a high percentage of cells.

4. Cellular Delivery of DiO and DiO-LCNPs and Imaging of Fixed Cells

1. Prepare 1 ml solutions of DiO, DiO-LCNP, and DiO-LCNP-PEG-Chol in delivery medium (HEPES-DMEM; DMEM containing 25 mM HEPES) by diluting stock solutions of DiO_free and DiO-LCNPs; appropriate DiO concentrations for incubation on cells will be ~1-10 µM (expressed as either the concentration of DiO_free or DiO in the form of DiO-LCNP).
2. Remove the growth medium from the culture dishes using a serological pipette and wash the cell monolayers (see step 3) two times with HEPES-DMEM (2 ml each wash). Perform the washes by gently adding and removing HEPES-DMEM using a pipette to/from the edge of the dishes.
3. Add 0.2 ml of the prepared DiO_free or DiO-LCNP delivery solutions to the center of the culture dishes and return the dishes to the incubator for an appropriate period of time (typically 15 or 30 min, depending on the needs of the experiment). Longer incubation times add to more nonspecific cellular labeling of non-membranous areas (e.g., cytosol).
4. After the incubation period, remove the delivery solutions using a serological pipette. Wash the cell monolayers two times with DPBS (2 ml each wash). Perform the washes by gently adding and removing DPBS to/from the edge of the dish.
5. Fix the cell monolayers using 4% paraformaldehyde (prepared in DPBS) for 15 min at room temperature.
   CAUTION: Paraformaldehyde is flammable, a respiratory irritant, and a suspected carcinogen.
6. Remove the paraformaldehyde solution using a pipette and gently wash the cells 1 time with DPBS (2 ml) by adding and removing DPBS using pipette to/from the edge of the dishes.
7. The fixed cells are ready to be imaged for the presence of a membranous fluorescence signal using confocal laser scanning microscopy (CLSM). Perform the imaging immediately or, alternatively, replace the medium with DPBS containing 0.05% NaN₃and store the dishes at 4 °C.
   CAUTION: NaN₃is toxic; exercise extreme caution when using NaN₃.
   NOTE: Fixed samples stored in this manner should be imaged within 48 hr for optimum results.

5. Cellular Delivery of DiO and DiO-LCNPs and FRET Imaging in Live Cells

NOTE: In this method, cells are colabeled with 6 µM each DiO-LCNP-PEG-Chol (where DiO is a FRET donor) and 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI_free, where DiI is a FRET acceptor). The release of DiO from DiO-LCNP-PEG-Chol and its incorporation into the plasma membrane is confirmed by an observed increase in energy transfer from the DiO donor to the DiI acceptor.

1. Label cells sequentially with DiO-LCNP-PEG-Chol and DiI_free using the method described in step 4.
2. After staining, wash the cells 1 time with DPBS (2 ml) using a serological pipette and replace the wash buffer with 2 ml of live cell imaging solution (LCIS).
3. On a microscope stage equipped with a heated incubation chamber, image the live cell sample using a confocal microscope (60X objective) with a FRET imaging configuration at 30 min intervals over a 4 hr period by exciting the DiO donor at 488 nm and collecting full emission spectra of both the DiO donor and the DiI acceptor from 490-700 nm with a 32-channel spectral detector.
4. NOTE: For more information on FRET imaging, please see reference¹⁵.
5. Determine the time-resolved emission intensity of both the DiO donor and the DiI acceptor from cells stained with DiO-LCNP-PEG-Chol and DiI. Calculate the time-resolved acceptor/donor FRET ratio (DiI_emi/DiO_emi) for the images, which will steadily increase and eventually plateau once the amount of DiO donor partitioned into the membrane has reached a maximum¹⁶.

6. Cytotoxicity Assay of DiO and DiO-LCNPs to the HEK 293T/17 Cells

NOTE: The cytotoxicity of the DiO-LCNP materials is assessed using a tetrazolium dye-based proliferation assay¹⁷. Cells are cultured in a multiwell plate in the presence of varying concentrations of the materials under conditions that emulate delivery/labeling. The cells are then cultured for 72 hr to allow for proliferation. A dye (MTS, (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) is then added to the wells, and metabolically active cells convert the dye into a blue formazan product. The amount of color formation is directly proportional to the number of viable cells.

1. Harvest HEK 293 T1/7 cells from the T-25 flask by following the procedure described in step 3.2.Seed HEK 293T/17 cells (5,000 cells/100 µl/well) to the wells of a 96-well tissue culture-treated plate and culture for 24 hr.
2. Completely remove the culture media from the wells using a micropipette and add 50 µl of HEPES-DMEM containing DiO_free, DiO-LCNPs, or DiO-LCNP-PEG-Chol at increasing concentrations to replicate wells. Incubate on the cells at 37 °C for 30 min.
   NOTE: Typically, replicates done in triplicate or quadruplicate are sufficient to yield statistically reliable data.
   1. After incubation, remove the delivery medium containing the materials using a micropipette and replace it with 100 µl of growth medium. Culture the cells for 72 hr.
3. Add 20 µl of the tetrazolium substrate to each well, return the plate to the incubator, and allow color formation to proceed at 37 °C for 4 hr. Read the absorbance (abs) of the formazan product at 570 nm (absorption minima for the DiO-LCNPs used in this study) and 650 nm (for subtraction of nonspecific background absorbance) using a microtiter plate reader.
4. Plot the differential absorbance value (abs₅₇₀ - abs₆₅₀) versus material concentration and report the results as percent of control cell proliferation (degree of proliferation of cells in cell culture medium only).

7. Data Analysis

1. Statistically analyze the data with a univariate analysis of variance (ANOVA). For multiple comparisons, apply the Bonferroni's post hoc test. Provide all average values as ± standard error of mean (SEM) unless otherwise mentioned.
   NOTE: The acceptable probability for significance was p < 0.05.

Wyniki

LCNPs were prepared in which the hydrophobic core of the NP was loaded with a representative membrane-labeling probe to demonstrate the utility of the LCNP as an efficient delivery vehicle for hydrophobic cargos. For this purpose, the cargo chosen was the highly water-insoluble potentiometric membrane-labeling dye, DiO. DiO-loaded LCNPs (DiO-LCNPs) were synthesized using a two-phase mini-emulsion technique with the chemical components DACTP11, AC10COONa, and DiO, as shown in Figure 1

Dyskusje

A continuing goal of NMDD is the controlled targeting and delivery of drug formulations to cells and tissues, combined with simultaneous improved drug efficacy. One specific class of drug molecules for which this has posed a significant challenge is hydrophobic drugs/imaging agents that have sparingly to no solubility in aqueous media. This problem has plagued the transition of potent drugs from in vitro cell culture systems to the clinical setting and has resulted in a number of promising drug molecules being &...

Materiały

Name	Company	Catalog Number	Comments
1-ethyl-3-(3-(dimethylamino)-propyl)carbodiimide hydrochloride (EDCA)	ThermoFisher	E2247
3,3′-dioctadecyloxacarbocyanine perchlorate (DiO)	Sigma Aldrich	D4292-20MG	Hazardous; make stock solution in DMSO
Cholesterol poly(ethylene glycol) amine hydrochloride	Nanocs, Inc.	PG2-AMCS-2k
Countess automated cell counter	ThermoFisher	C10227
Dioctadecyl-3,3,3′⁠,3′-tetramethylindocarbocyanine perchlorate (DiI)	Sigma Aldrich	468495-100MG	Hazardous; make stock solution in DMSO
Dulbecco's Modified Eagle's Medium (DMEM)	ThermoFisher	21063045	Warm in 37 °C before use
Dulbecco's Phosphate Buffered Saline (DPBS)	ThermoFisher	14040182	Warm in 37 °C before use
Dynamic light scattering instrument	ZetaSizer NanoSeries (Malvern Instruments Ltd., Worcestershire, UK)
Fibronectin Bovine Protein, Plasma	ThermoFisher	33010018	Make stock solution 1 mg/ml using DPBS. Use 20-30 µg/ml for coating MetTek dish, 2 hr at 37 °C
Formaldehyde (16%, W/V)	ThermoFisher	28906	Hazardous; dilute to 4% using DPBS
Human embryonic kidney cells (HEK 293T/17)	American Type Culture Collection	ATCC® CRL-11268™
Live cell imaging solution (LCIS)	ThermoFisher	A14291DJ	Warm in 37 °C before use
MatTek 14 mm # 1.0 coverglass insert cell culture dish	MatTek corporation	P35G-1.0-14-C
Modified Eagle Medium (DMEM) containing 25 mM HEPES	ThermoFisher	21063045	Warm in 37 °C before use
N-hydroxysulfosuccinimide sodium salt (NHSS)	ThermoFisher	24510
Nikon A1si spectral confocal microscope	Nikon Instruments
Trypan Blue Stain (0.4%)	ThermoFisher	T10282	mix as a 50% to the cell suspension before counting the cells
Zeta potential instrument	ZetaSizer NanoSeries (Malvern Instruments Ltd., Worcestershire, UK)
Ultrasonic Processor	Sonics and Materials Inc	GEX 600-5
Mini Cetntrifuge	Benchmark	Mini-fuge-04477
PD-10 Sephadex™ G-25 Medium	GE Healthcare	17-0851-01
Bio-Rad ChemiDoc XRS Imaging System	Bio-RAD	76S/07434
Trypsin-EDTA (0.25%), phenol red	ThermoFisher	25200056