Hyaluronic Acid-decorated PLGA-PEG Nanoparticles for Targeted Delivery of SN-38 to Ovarian Cancer

Materials and Methods

Chemicals. PLGA (average M_w 33 kDa) was purchased from Lakeshore biomaterials, Birmingham, AL, USA. HA (average M_w 16.9 kDa) was obtained from Lifecore Biomedical (Chaska, MN, USA) and used after de-salting by dialysis against deionized water. SN-38 was purchased from AK Scientific, Union City, CA, USA. N,N’-Dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were purchased from Thermo Scientific, Rockford, IL, USA. Polyethylene glycol bis amine (PEG bis amine, M_w 2000 Da), poly vinyl alcohol (M_w 31,000-50,000 g/mol), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT), fluorescein isothiocyanate (FITC), hexadecyl-trimmethylammonium bromide (CTAB) and colchicine were purchased from Sigma Aldrich, St. Louis, MO, USA.

Cell culture. SKOV-3 (human ovarian carcinoma) and Chinese hamster ovary (CHO) cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Dulbeco's modified Eagle's medium (DMEM) and F12 medium, respectively. OVCAR-8 (human ovarian carcinoma) cells were obtained from the National Cancer Institute (NCI), Bethesda, MD, USA and cultured in RPMI-1640 medium. All the media were supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin-streptomycin solution. Cells were incubated in a CO₂ incubator at 37°C and 95% relative humidity. All cell lines used were of passage number 20-40.

Preparation of HA decorated Nanoparticles. HA-decorated PLGA nanoparticles were prepared in two steps. Firstly, PLGA was conjugated with PEG bis amine. Secondly, HA was conjugated onto PLGA-PEG-NH₂ nanoparticles by EDC coupling.

Synthesis and characterization of PLGA-PEG-NH₂. PLGA-PEG-NH₂ was synthesized as reported elsewhere (24).

Activation of COOH group on PLGA. PLGA dissolved in anhydrous methylene chloride was reacted with DCC and NHS for 24 h at room temperature (RT) under nitrogen atmosphere at a stoichiometric molar ratio of PLGA:DDC:NHS was 1:10:10. By product N,N’-dicyclohexylurea was removed by filtering through a 0.45-μm Teflon syringe filter. The filtrate was precipitated and washed with ice cold diethyl ether. The precipitate was vacuum dried to obtain activated PLGA.

Conjugation of PEG bis amine with activated PLGA. Activated PLGA and PEG bis amine were dissolved in anhydrous methylene chloride (1:1 molar ratio) and reacted overnight at RT under nitrogen. The reaction mixture was precipitated and washed with cold methanol. The precipitate was then vacuum dried to obtain PLGA-PEG-NH₂. The conjugate was dissolved in CDCl₃ to a 5 mg/ml solution and was analyzed by nuclear magnetic spectrophotometer (NMR) (500 MHz Bruker, Billerica, MA, USA).

Nanoparticle preparation. PLGA-PEG-NH₂ nanoparticles were prepared by single-emulsion (O/W) solvent evaporation method. Briefly, 20-60 mg of polymer PLGA-PEG-NH₂ was dissolved in 4 ml of methylene chloride. This organic phase was added to 10 ml of 1% polyvinyl alcohol (PVA) aqueous solution and sonicated at 20 W using a probe sonicator (Barson sonifier 250; Barson Ultrasonics, Danbury, CT, USA) for 4 min at 10 s pulse in an ice bath. Organic solvent was evaporated by stirring at RT overnight. The nanosuspension was then centrifuged at 20,000 ×g on a Sorvall RC 6+ centrifuge (Thermo Scientific) for 20 min to separate unloaded material and nanoparticles were washed with deionized water to remove excess of PVA.

FITC- or SN-38-loaded nanoparticles were also prepared by same method: 500 μl of 3 mg/ml FITC solution in acetone was added to the organic phase, whereas in the case of SN-38, solution in dimethyl sulfoxide (DMSO) was added to the organic phase. For loading SN-38 into nanoparticles, the pH of the 1% PVA solution was adjusted to 3.1 with 0.1 N HCl. Unencapsulated FITC or SN-38 was removed by centrifugation followed by passing the nanoparticles through sephadex™ G-25 column (PD10; GE Healthcare Bio-Sciences Corp, Piscataway, NJ, USA). Nanoparticles were lyophilized with 1% sucrose as cryoprotectant.

Preparation of HA-conjugated nanoparticles. HA was conjugated onto the surface of nanoparticles as reported earlier (25). Briefly, HA was presoaked in pH 4.7 2-(N-morpholino)ethanesulfonic acid (MES) buffer overnight for complete solubilization and swelling. −COOH groups of HA were activated by treating with EDC and NHS for 2 h in the same buffer. PLGA-PEG-NH₂ nanoparticles were added to the activated HA and stirred overnight to obtain nanoparticles decorated with HA. Excess of HA was removed by three cycles of centrifugation at 15000 × g for 20 min.

CTAB turbidimetric assay. HA conjugation onto nanoparticles was confirmed and quantified by a reported indirect method using a CTAB turbidimetric assay (25). The amount of unconjugated HA in the supernatant after centrifugation was quantified and the amount of conjugated HA was calculated by subtracting the amount in the supernatant fraction from the total amount used in the reaction. Briefly, 50 μl of HA standard solution (0.2-1 mg/ml) or supernatant sample was added in triplicate to a 96-well plate. All the samples were incubated with 50 μl of 0.2 M sodium acetate buffer, pH 5.5 at 37°C for 10 min. Then 100 μl of 10 mM CTAB solution was added to the wells and the absorbance of the precipitated complex was read within 10 minutes at 570 nm using Novostar microplate reader (BMG Lab Technologies, Cary, NC, USA).

Size and zeta potential. Nanoparticles were measured for their size, polydispersity and zeta potential by dynamic light scattering using ZetaPALS zeta potential analyzer (Brookhaven Instruments Corporation, Holtsville, NY, USA).

Toxicity of PLGA-PEG-HA nanoparticles. Toxicity of PLGA-PEG-HA nanoparticles was evaluated by MTT assay on CHO cells. CHO cells were plated at a density of 10,000 cells per well in a 96 well plate. When cells were 80% confluent, they were treated with PLGA-PEG-HA nanoparticles at a concentration of 1-100 μg/ml and incubated for 8 h at 37°C. Following the incubation period, all the media were aspirated and 50 μl of 0.5 mg/ml of MTT solution in serum-free media was added. After 4 h of incubation, 150 μl of DMSO was added to each well to dissolve the formazan crystals and plates were incubated for a further 1 h. After thorough mixing of each well, the optical density was measured at 570 nm using a microplate reader. Viability of the cells exposed to nanoparticles was expressed as a percentage of the viability of cells grown in the absence of nanoparticles.

CD44 receptor expression analysis by flow cytometry. BD Biosciences (San Jose, CA, USA) support protocol was followed for the CD44 expression by flow cytometer. Briefly, 0.5×10⁶ cells/sample were suspended in 50 μl of cold stock buffer [0.2% bovine serum albumin (BSA) in 1× phosphate buffer saline (PBS)]. Then cell suspension was treated with 10 μl of FITC-labeled CD44 antibody (BD Biosciences) in amber-colored microcentrifuge tube as per supplier's instructions, mixed properly and incubated for 30 min at 4°C in the dark. Cells were washed three times with cold stock buffer, resuspended in stock buffer and analyzed by flow cytometry (BD Accuri C6 Flow Cytometer; BD Biosciences, San Jose, CA, USA) and receptor expression was measured as percentage of cells exhibiting fluorescence.

Specificity of HA conjugated PLGA nanoparticles. For specificity experiments, FITC was loaded into nanoparticles as described earlier and unencapsulated FITC was removed by size-exclusion chromatography. The FITC-loaded nanoparticles were immediately used for cellular uptake studies. Uptake of the nanoparticles was estimated and compared in CD44⁺ (SKOV-3, OVCAR-8) and CD44⁻ (CHO) cell lines. Briefly, 1×10⁵ cells/well were plated in a 12-well plate, and after reaching 70-80% confluence, cells were treated with 5 μg/ml and 25 μg/ml (from a stock of 5 mg/ml nanosuspension) of FITC loaded PLGA-PEG and PLGA-PEG-HA nanoparticle suspension in serum-free media separately. After 30 min of incubation, cells were washed three times with cold PBS, trypsinized, washed and suspended in stock buffer and analyzed for the percentage of cells with fluorescence using BD Accuri C6 flow cytometer (BD Biosciences).

Mechanism of cellular internalization. To investigate the internalization mechanism of HA-conjugated nanoparticles, cells were pre- and co-incubated with excess of HA (1 mg/ml) or 20 μM of colchicine before the addition of FITC-loaded HA nanoparticles. The concentration of excess HA was selected based on a previous report (25). To differentiate the physical surface adsorption of the HA-conjugated nanoparticles from internalized nanoparticles, cellular uptake was tested at 4°C and 37°C.

SN-38 encapsulation efficiency. Encapsulation efficiency was determined by dissolving a known amount of nanoparticles in DMSO and analyzing SN-38 content on a UV-visible spectrophotometer at 380 nm. The minimum detection limit was 1 μg/ml, with R² value of 0.9996, and no interference was observed with PLGA and PEG at 380 nm. The percentage encapsulation efficiency (%EE) was calculated using the following formula (26):

Assessment of SN-38-loaded nanoparticle stability. Stability of SN-38 in nanoparticles was assessed in PBS pH 7.4 and in FBS. Briefly, freez-dried nanoparticle samples (equivalent to 150 μg/ml of SN-38) were dispersed in 3 ml of PBS or FBS, sonicated to disperse any possible lumps and incubated at RT for three days in dark. Each day after thorough mixing, 1 ml of sample was withdrawn and centrifuged at 20000 ×g. Supernatant (0.5 ml) was taken and mixed with an equal quantity of acetonitrile acidified with 0.1 N HCl, mixed well and kept aside for 15-20 minutes before analysis by high performance liquid chromatography (HPLC). Particle size and poly dispersity index of the nanoparticles were tested before centrifugation.

HPLC analysis. All stability samples were analyzed on a reverse-phase HPLC system (Waters, Milford, MA, USA) with a binary pump and dual λ absorbance UV-visible detector. We used an HPLC method that can analyze lactone and carboxylate forms of SN-38 simultaneously, as reported earlier with little modifications (27). The chromatographic separations were achieved using an Atlantis® 5 μM 4.6×250 mm C18 column at RT. An isocratic elution method of 1:1 acetonitrile/25 mM NaH₂PO₄ pH 3.1 buffer at a flow rate of 1 ml/min was used. Absorbance was measured at 265 nm. The concentration of SN-38 from each sample was determined from the peak area observed, based on a calibration curve in the concentration range of 0.2-100 μg/ml (y=79503x + 3907, R²=1).

In vitro cytotoxicity of SN-38-loaded nanoparticles. Cytotoxicity of SN-38-loaded nanoparticles was evaluated on SKOV-3 and OVCAR-8 cell lines using MTT assay. Nanoparticles were dispersed in PBS (pH 7.4) and sonicated for one minute to make a uniform dispersion and suitable dilutions were made in required growth media. As SN-38 is not soluble in water, the stock solution was made in DMSO and suitable dilutions were made in growth media, keeping the final DMSO concentration less than 0.1%. Briefly, 0.6-1×10⁴ cells per well were plated in a 96-well pate. After 24 h of incubation, cells were treated with different concentrations (0.5 nM to 10 μM) of SN-38 alone or nanoparticles loaded with equivalent amounts of SN-38 and incubated for 72 h. Cells were then washed with PBS and 50 μl of 0.5 mg/ml MTT solution in serum-free media was added. After 4 h of incubation, 150 μl of DMSO was added to dissolve the formazan crystals and the optical density was measured using a microplate reader at 570 nm. The results are expressed as the percentage of cell viability as compared to the control and the IC₅₀ was determined using Graphpad prism 5 software (San Diego, CA, USA).

Statistical analysis. Data are represented as means±SD from three separate experiments. Statistical analysis was performed using two-way ANOVA with general linear model (Graph Pad Prism 5 software) followed by multiple comparison. A value of p<0.05 was considered statistically significant.