Branched PEI (Mn 10,000), PLGA (50:50, Mw 40,000–75,000), poly(vinyl alcohol) (PVA, 87–89% hydrolyzed, Mw 13,000–23,000), rhodamine B isothiocyanate (RITC, mixed isomers), dichloromethane (DCM), pyridine, p-nitrophenyl chloroformate (p-NPC), triethyleamine (TEA), diethyl ether, and cholesterol were all obtained from Sigma-Aldrich (St. Louis, MO). Amine-terminated methoxy PEG (mPEG-NH2) (Mw 5,000) was obtained from Nektar (Huntsville, AL). 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-mPEG-2000 (DSPE-PEG 2000), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) sodium salt (DOPG) were purchased from Avanti Polar Lipids Inc. (Alabaster, AL). All other chemicals used in this study were purchased from Sigma-Aldrich unless specified otherwise.
Preparation and characterization of PEI-RITC conjugates
PEI was fluorescently labeled by conjugation with RITC using a similar method as described earlier9
RITC (5.4 mg, 1.0×10−5
mol) dissolved in 1 mL deionized distilled water (ddH2
O) was added to PEI (20.0 mg, 2.0×10−6
mol) dissolved in 4 mL ddH2
O. The pH of the mixture was adjusted to 9.0 using 1.0 N hydrochloric acid (HCl), followed by vigorous mixing at room temperature (RT) for 24 h. Unreacted RITC was removed using membrane dialysis (Spectra/Por dialysis membrane, MWCO 3,500, Spectrum Laboratories Inc., Rancho Dominguez, CA) in 3 L of phosphate buffered saline (PBS) for 2 days, changing the buffer every 12 h, followed by dialysis in ddH2
O for 2 days, changing the water every 12 h. The purified PEI-RITC conjugates were lyophilized over 2 days using a Labconco FreeZone 4.5 system (Kansas City, MO) and stored at −20 °C.
A series of RITC solutions in ddH2O (6.3, 12.5, 25.0, 37.5, and 50.0 µg/mL) were prepared and used as standards to calculate the RITC content of the conjugates in subsequent measurements. PEI-RITC conjugates were dissolved in ddH2O at a concentration of 100 µg/mL. UV spectra were recorded against ddH2O using a DU800 UV/Vis Spectrophotometer (Beckman Coulter, CA). A standard curve of RITC absorbance versus concentration was constructed, and the concentration of RITC in the PEI-RITC solution was calculated based on Beer’s Law. The number of RITC molecules per PEI chain was determined based on the amount of RITC in the PEI-RITC solution.
Synthesis and characterization of PEG-PLGA copolymer
PEG-PLGA block copolymer used in this study was synthesized from mPEG-NH2
and PLGA using a similar method as described earlier.19
Five hundred milligrams of PLGA (1.3×10−5
mol) were dissolved in 8 mL of DCM, to which 5.1 µL (6.3×10−5
mol) of pyridine were added. p-NPC (12.6 mg, 6.3×10−5
mol) was dissolved in 1 mL of DCM, and then added dropwise to the PLGA and pyridine solution under vigorous stirring, and the reaction was carried out at RT for 24 h. The reaction product (p-NP-PLGA) was then precipitated using ice-cold diethyl ether and vacuum filtered. Next, p-NP-PLGA (400 mg, 1.0×10−5
mol) was dissolved in 8 mL of DCM. mPEG-NH2
(93.8 mg, 1.9×10−5
mol) was dissolved in 3 mL of DCM, to which 7 µL (5.0×10−5
mol) of TEA were added. The mPEG-NH2
and TEA solution was then added dropwise to the p-NP-PLGA solution under vigorous stirring, and the reaction was carried out at RT for 24 h. The final product (PEG-PLGA) was precipitated using ice-cold diethyl ether and vacuum filtered. PEG-PLGA was characterized using 1
H NMR in CDCl3
using a 400 MHz Bruker DPX-400 spectrometer (Bruker BioSpin Corp., Billerica, MA).
Encapsulation of PEI-RITC conjugates into polymeric NPs
PLGA and PEG-PLGA NPs were prepared using a double emulsion method as described previously.20
Briefly, 20 mg of either PLGA or PEG-PLGA were dissolved in 1 mL of DCM. PEI-RITC was dissolved in ddH2
O at a concentration of 1 mg/mL, and 100 µL of the solution were added to either PLGA or PEG-PLGA solution in DCM. The mixture was sonicated for 1 min using a Misonix XL Ultrasonic Processor (100% duty cycle, 475 W, 1/8” tip, QSonica, LLC, Newtown, CT). Two milliliters of 3% PVA solution in ddH2
O was then added to the mixture, followed by sonication for 1 min at 100% duty cycle. The double emulsion was then poured into 20 mL of 0.3% PVA in ddH2
O, and vigorously stirred at RT for 24 h to evaporate DCM. The resulting aqueous solution was transferred to Nalgene high-speed centrifuge tubes (Fisher Scientific, Pittsburg, PA). PVA and unencapsulated PEI-RITC were removed by ultracentrifugation at 20,000 rpm for 30 min using a Beckman Avanti J25 Centrifuge (Beckman Coulter, Brea, CA). After washing the NPs five times with ddH2
O, the pellet was resuspended in ddH2
O, lyophilized over 2 days, and stored at −20 °C.
Characterization of the PEI-RITC-encapsulated polymeric NPs
Particle size (diameter, nm) and surface charge (zeta potential, mV) of the NP-based nanohybrids were obtained from three repeat measurements by quasi-elastic laser light scattering using a Nicomp 380 Zeta Potential/Particle Sizer (Particle Sizing Systems, Santa Barbara, CA). The nanohybrid particles were suspended in ddH2O at a concentration of 100 µg/mL, filtered through a 0.45 µm syringe filter, and briefly vortexed prior to each measurement. Loading was defined as the PEI-RITC content of the NP-based nanohybrids. Five milligrams of NP-based nanohybrids were completely dissolved in 1 mL of 0.5 M NaOH, followed by filtration through a 0.45 µm syringe filter. The fluorescence intensity from the filtrates containing PEI-RITC was then measured using a SpectraMAX GeminiXS microplate spectrofluorometer (Molecular Devices, Sunnyvale, CA). The amount of PEI-RITC released was determined from a standard curve of PEI-RITC fluorescence versus concentration in 0.5 M NaOH. Loading was expressed as µg PEI-RITC/mg PLGA or PEG-PLGA. Loading efficiency was defined as the ratio of the actual loading obtained to the theoretical loading (amount of PEI-RITC added divided by the mass of PLGA or PEG-PLGA used in each formulation).
Scanning Electron Microscopy (SEM)
The surface morphology of PLGA and PEG-PLGA NPs was examined using a JEOL-JSM 6320F field emission microscope (JEOL USA, Peabody, MA). Freeze dried NP samples were placed onto a carbon adhesive strip mounted on an aluminum stub. Samples were sputter-coated with Pt/Pd at a coating thickness of 6 nm (Polaron E5100 sputter coater system, Polaron, UK) and then visualized at an accelerating voltage of 5.0 mV and 8.0 mm working distance.
Encapsulation of PEI-RITC conjugates into liposomes
Unilamellar liposomes were prepared using a film hydration method followed by extrusion as described previously.16
Briefly, DOPG (5.0 mg, 6.3×10−6
mol), DSPC (4.9 mg, 6.2×10−6
mol), Cholesterol (2.4 mg, 6.2×10−6
mol, and DSPE-PEG 2000 (1.8 mg, 6.3×10−7
mol) were dissolved in 5 mL of DCM in a round-bottom flask. The flask was connected to a rotary evaporator (Rotavapor RII, Buchi, Switzerland) at 50 °C for 1 h to evaporate DCM until completely dried. The dried lipid film was hydrated in 1 mL of 0.1 mg/mL PEI-RITC solution in ddH2
O, followed by vortexing for 15 min to form multilamellar liposomes. Multilamellar liposomes were sonicated in a bath sonicator for 30 min, and then extruded 20 times through a polycarbonate membrane of 100 nm pore size using a Lipofast Pneumatic extruder (Avestin Inc., Ottawa, Canada). The resulting unilamellar liposome suspension was centrifuged at 20,000 rpm for 1 h to remove residual PEI-RITC. The pellet was resuspended in 1 mL of 5% sucrose, lyophilized over 2 days, and stored at −20 °C.
Characterization of the PEI-RITC-encapsulated liposomes
Particle size (diameter, nm) and surface charge (zeta potential, mV) were measured using the same method described for polymeric NPs. Loading was determined by dissolving 10 mg of lyophilized liposomes in 1 ml of 0.1% Triton X-100, followed by filtration through a 0.45 µm syringe filter and measuring the fluorescence of the filtrate. The amount of PEI-RITC released was determined from a standard curve of PEI-RITC fluorescence versus concentration in 0.1% Triton X-100. Loading was expressed as µg PEI-RITC/mg lipids. Loading efficiency was calculated from the ratio of the actual measured loading to the theoretical loading (amount of PEI-RITC added divided by the mass of lipids and sucrose used in the formulation).
Transmission Electron Microscopy (TEM)
Size and shape of liposomal nanohybrids was examined using TEM. Liposomes were dissolved in ddH2O at a concentration of 1 mg/mL. One drop of the solution was then placed on a 300-mesh copper grid and left to dry overnight, followed by negative staining with 2% phosphotungstic acid (PTA). TEM images were acquired using a JEOL JEM 1220 (JEOL USA) at an accelerating voltage of 80 kV.
Release kinetics study of PEI-RITC-encapsulated nanohybrids
Five milligrams of each nanohybrid in microcentrifuge tubes were dispersed in 1 mL PBS (pH 7.4) or acetate buffer (pH 4.0) in triplicates, and the solutions were placed in a shaking water bath (37 °C, 100 rpm). At designated time points (30 min, 1 h, 2, 4, 6, 8, 10, 12 and 24 h; every 2 days thereafter), solutions were centrifuged at 20,000 rpm for 5 min and the supernatants were collected. The nanohybrid systems were then redispersed in fresh PBS or acetate buffer and placed back in the water bath. The fluorescence of the supernatants was measured and the cumulative amount of PEI-RITC released over time was determined from a standard curve of PEI-RITC fluorescence versus concentration in either PBS or acetate buffer.
Cytotoxicity of PEI-RITC-encapsulated nanohybrids
MCF-7 cell line was obtained from ATCC (Manassas, VA) and grown continuously as a monolayer in GIBCO Dulbecco’s modified Eagle medium (DMEM, Invitrogen Corporation, Carlsbad, CA) in a humidified incubator at 37 °C and 5% CO2. DMEM was supplemented with penicillin (100 units/mL), streptomycin (100 mg/mL), and 10% heat-inactivated fetal bovine serum (FBS) (Invitrogen Corporation, Carlsbad, CA) before use. For the assay, MCF-7 cells were seeded in 96-well plates at a density of 2×104 cells/well and grown in DMEM for 24 h. Cells (n=4) were then treated with PEI-RITC or nanohybrid systems (PEI-RITC encapsulated liposomes, PEG-PLGA NPs, and PLGA NPs) at 4 PEI-RITC concentrations (1, 5, 10, and 30 µg/mL) for 1, 4, 24, and 48 h. After each incubation time, cells were washed and incubated for an additional 24 h in a normal culture condition. Cell viability was assessed using a CellTiter 96 AQueous One Solution (MTS) Assay (Promega, Madison, WI) according to the manufacturer’s protocol. The UV absorbance was measured at 490 nm using a Labsystems Multiskan Plus microplate reader (Labsystems, Finland). Mean cell viabilities were determined relative to a negative control (untreated cells). Statistical analysis was performed using OriginPro 8.1 (OriginLab, Northampton, MA). Mean cell viabilities were compared using 1-way ANOVA followed by Tukey’s post hoc test at p < 0.05.
Cellular uptake of PEI-RITC-encapsulated nanohybrids: Confocal microscopy observation
MCF-7 cells were seeded in 4-well chamber slides (Millicell EZ Slide, Millipore, Billerica, MA) at a density of 2.0×105 cells/well and incubated in DMEM for 24 h. PEI-RITC (0.5 µg), liposomes (67 µg), PEG-PLGA NPs (242 µg), and PLGA NPs (106 µg) were each dispersed in 1 mL of DMEM to make the concentration of PEI-RITC constant at 0.5 µg/mL throughout all nanohybrids. Cells were treated with the three nanohybrids and unencapsulated PEI-RITC for 1, 4, 24, and 48 h. Following the treatment, cells were washed with PBS three times, and then 50 µL of Wheat Germ Agglutinin Alexa Fluor® 488 conjugate (WGA-AF488, 5 µg/mL, Invitrogen Corporation, Carlsbad, CA) was added to each dish and incubated for 10 min at RT to stain the cell membrane. Cells were washed again with PBS, followed by fixation in 500 µL of 4% paraformaldehyde for 10 min at RT. After washing excess paraformaldehyde, cells were mounted with antiphotobleaching mounting media with DAPI (Vector Laboratory Inc., Burlingame, CA), and covered with glass cover slips. Cellular uptake was visualized using a Zeiss LSM 510 confocal laser scanning microscope (CLSM, Carl Zeiss, Germany). The 488 nm line of a 30 mW tunable argon laser was used for excitation of AF488, a 1 mW HeNe at 543 nm for RITC, and a 25 mW diode UV 405 nm laser for DAPI. Emission was filtered at 505–530 nm, 565–595 nm, and 420 nm for AF488, RITC, and DAPI, respectively.
Cellular uptake of PEI-RITC-encapsulated nanohybrids: Flow cytometry measurements
MCF-7 cells were seeded in 12-well plates at a density of 1×106 cells/well and incubated in DMEM for 24 h. Cells were then treated with unencapsulated PEI-RITC and the three nanohybrids under that same condition described in the cellular uptake experiment. After each incubation period, cells were washed with PBS and then suspended with trypsin/EDTA. Cell suspensions were centrifuged at 3500 rpm for 5 min, resuspended in 500 µL of 1% paraformaldehyde, and transferred to flow cytometry sample tubes. Fluorescence signal intensities from the samples were measured using a MoFlo cell sorter (BD, Franklin Lakes, NJ) and data analysis was performed using Summit v4.3 software (Dako Colorado, Fort Collins, CO).