Chemicals
PLGA ester-terminated (lactide to glycolide ratio 50:50, molecular weight 45,000–55,000), Nile Red dye, acetonitrile HPLC grade 99.93%, and curcumin were obtained from Sigma-Aldrich (St Louis, MO). 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (ammonium salt) (DSPE-PEG (2000) Amine (2790.486) (average MW due to polydispersity of PEG)) was purchased from Avanti (Alabaster, Alabama). Roswell Park Memorial Institute (RPMI) 1640 medium, fetal bovine serum, and penicillin/streptomycin were purchased from Hyclone. MilliQ water (Millipore) was used for all experiments.
Microfluidic production of nanolipomer nanoparticles
The basis of the microfluidic synthesis strategy reported in this manuscript utilizes addition of reagents at two inlet ports. One port with the aqueous phase which contains a lipid/stabilizing component. The second port with the organic phase contains polymer and drug. When these channels combine, a nanoprecipitation reaction occurs and rapid mixing in milliseconds [11] allows for coating the lipid/stabilizing agent onto the polymeric nanoparticle core. Overall, this process allows for high flow rates.
All optimization experiments were conducted using the NanoAssemblr Benchtop instrument (manufactured by Precision Nanosystems). PLGA nanoparticles were synthesized by dissolving PLGA ± curcumin in acetonitrile at varying concentrations, this organic solvent mixture was injected in one inlet port in the system. Simultaneously, the DSPE-PEG (at varying concentrations) in 4% ethanol mixture was injected in the other inlet port of the system. Immediately prior to inlet port injection of the DSPE-PEG solution, it was heated in a water bath at 60 °C for 30 s (Fig. 1).
Instrument parameters optimization
This was achieved by manipulating control of total flow rate (TFR) 2–12 ml/min at PLGA concentrations 5–20 mg/ml and DSPE-PEG concentration of 40% w/w. Next, flow rate ratios of aqueous:solvent (1:1) through (9:1) with 10 mg/ml PLGA in acetonitrile and 40% DSPE-PEG in 4% ethanol while TFR was held at 12 ml/min. NP product was gathered in 15 ml falcon tube while separately disposing the initial volume of 0.25 ml and the final 0.05 ml of NP solution. After the nanolipomer (NLPs) were manufactured, they were processed to remove the aceteonitrile with a solvent exchange method to water in which NLPs were diluted and centrifuged thrice at 1600g for 30 min runs in Amicon Ultra Centrifugal Filters 10,000 NMWL. The NLP size and distribution were tested in distilled water and PBS with the Malvern Zetasizer ZS instrument.
Optimization of NLP formulation parameters
PLGA and DSPE-PEG parameters were optimized with polymer concentration 5, 10, and 20 mg/ml and lipid concentrations of 0, 5, and 10% w/w. This was followed by solvent exchange and determination of size and polydispersity as described above.
After determination of optimized formulation parameters to this point, the encapsulation of curcumin was performed. Curcumin was dissolved in 10 mg/ml of PLGA in acetonitrile so initial concentration of curcumin was varied between 0 and 7.5% curcumin w/w polymer. 10% DSPE-PEG w/w of lipid to polymer ratio was used with a TFR of 12 ml/min and a flow rate ratio (FRR) of 1:1 (aqueous channel input to organic channel input). Solvent exchange to remove non-encapsulated curcumin was performed and the amount of encapsulated curcumin was quantified by UV–Vis plate reader against a standard curve of curcumin in acetonitrile using absorbance at wavelength of 450 nm.
Drug loading and encapsulation efficiency were determined for the different initial concentrations of curcumin. Encapsulation efficiency (EE) was calculated using the following equation: EE = (actual amount of drug encapsulated in nanoparticles)/(starting amount of drug used in nanoparticles) × 100%. Drug loading (DL) was calculated with the equation: DL = (weight of drug in nanoparticles)/(weight of nanoparticles) × 100%.
NLP stability was assessed by incubating 100 μl of 10 mg/ml NLP formulation into 1 ml of molecular biology reagent water (Sigma-Aldrich). Nanoparticles were stored at 4 °C then size and PDI were measured daily for a period of 7 days.
Fluorescent characterization studies
Time-resolved measurements
Fluorescence lifetime and anisotropy decay were measured using FluoTime200 (PicoQuant, GmbH, Berlin, Germany) time domain fluorometer. This instrument, equipped with microchannel plate detector (Hamamatsu, Japan) and a 470 nm pulsed picosecond laser diode provided resolution of 4 ps/channel. The fluorescence lifetime of curcumin-loaded NP was measured at magic angle conditions and data were analyzed with a FluoFit version 5.0 software (PicoQuant, GmbH, Berlin, Germany). The lifetime data were fitted to the multi-exponential deconvolution model:
$$I\left( t \right) = \int_{ - \infty }^{t} {IRF\left( {t^{\prime}} \right)} \mathop \sum \limits_{i} \alpha_{i} e^{{\frac{{ - t - t^{\prime}}}{{\tau_{i} }}}}$$
where IRF(t′) represents the instrument response function at time \(t^{\prime}\), τ
i
is the lifetime of the ith component, and α
i
is the amplitude of decay of the ith component at time t. The average values were calculated as:
$$\bar{\tau } = \sum\limits_{i} {f_{i} \tau_{i} } \quad f_{i} = \frac{{\alpha_{i} \tau_{i} }}{{\mathop \sum \nolimits_{i} \alpha_{i} \tau_{i} }}$$
The anisotropy decays were measured using VV and VH polarizer orientation on the emission side with a 470 nm laser diode. Anisotropy decays were analyzed with multi-exponential fitting model in FluoFit3 program from PicoQuant, Inc (Germany) using following equation.
$$r\left( t \right) = \sum\limits_{i} {r_{i} e^{{ - t/\Phi _{i} }} }$$
Steady-state measurements
All measurements were performed in a 1 cm × 1 cm quartz cuvette at room temperature (20 °C). On account of poor water solubility, Curcumin dissolved in 100% ethanol was used as a reference. The nanoparticles were easily dissolved in water. Absorption spectra were collected on a Cary 50 Bio UV–visible spectrophotometer (Varian Inc., Australia). The absorption was scanned from 300 to 500 nm and water was used as a baseline reference. Emission spectra were measured using Cary Eclipse spectrofluorometer (Varian Inc., Australia). The samples were excited at 375 nm, and the emission scanned from 450 to 700 nm in a square geometry set-up.
Cell viability assay
Prostate cancer cell line C4-2B-luciferase was used to assess cell viability after treatment with NLP or free curcumin. C4-2B-luciferase cells were generously provided by Dr. Even Keller (University of Michigan). Cells were grown in RPMI-Media supplemented with 10% FBS and 1% antibiotic–antimycotic. Upon reaching 70% confluency, cells were subcultured and plated on 96 well flat bottom plates at a seeding density of 2000 cells per well in quadruplicate with the same culture conditions as described above. Cells were allowed to attach for 24 h, media was replaced with fresh media and then cells were treated with NLPs or free drug for a period of either 48, or 72 h with concentration range 0–20 μM curcumin or equivalent dose of NLP. At respective time points, 10 μl Tiazolyl Blue Tetrazolium Bromide (MTT) suspended in PBS at a concentration of 10.5 mg/ml was added to the 200 μl media in each 96 well plate. Cells were incubated with MTT reagent for 3 h, media was then removed and 150 μl of DMSO was added to all wells and mixed. Absorbance was read on BioTek Synergy two Multi-Mode Plate Reader at 570 nm.
Cellular uptake studies
C4-2B-luciferase cells were plated on glass cover slips in 6 well plates at a density of 0.5 × 106 cells per well and attachment allowed for 24 h. During NLP synthesis step, Nile Red dye was incorporated into PLGA core and excess dye washed out of the formulation. NLP was added to cell culture media at a concentration of 200 μg/ml for 0–6 h time points and at appropriate time washed three times with PBS to remove NLP that was not taken up by cells. Cells were fixed with 4% paraformaldehyde for 10 min, washed, then mounted on slides with Prolong Gold antifade reagent with DAPI. Imaging was performed with the Zeiss LSM 510 confocal microscope.
NLP retention study
To determine in vivo retention time of NLP, Nile red was incorporated into the NLP formulation as described above. Male nude mice aged 5 weeks were injected via tail vein with 2 mg of final NLP formulation. Fluorescent signal after injection was monitored between 25 min and 24 h using the IVIS animal imaging system (Perkin Elmer, USA). All institutional IACUC animal protocols were followed.