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In Vitro and In Vivo Studies on HPMA-Based Polymeric Micelles Loaded with Curcumin

Curcumin-loaded polymeric micelles composed of poly(ethylene glycol)-b-poly(N-2-benzoyloxypropyl methacrylamide) (mPEG-b-p(HPMA-Bz)) were prepared to solubilize and improve the pharmacokinetics of curcumin. Curcumin-loaded micelles were prepared by a nanoprecipitation method using mPEG5kDa-b-p(HPMA-Bz) copolymers with varying molecular weight of the hydrophobic block (5.2, 10.0, and 17.1 kDa). At equal curcumin loading, micelles composed of mPEG5kDa-b-p(HPMA-Bz)17.1kDa showed better curcumin retention in both phosphate-buffered saline (PBS) and plasma at 37 °C than micelles based on block copolymers with smaller hydrophobic blocks. No change in micelle size was observed during 24 h incubation in plasma using asymmetrical flow field-flow fractionation (AF4), attesting to particle stability. However, 22–49% of the curcumin loading was released from the micelles during 24 h from formulations with the highest to the lowest molecular weight p(HPMA-Bz), respectively, in plasma. AF4 analysis further showed that the released curcumin was subsequently solubilized by albumin. In vitro analyses revealed that the curcumin-loaded mPEG5kDa-b-p(HPMA-Bz)17.1kDa micelles were internalized by different types of cancer cells, resulting in curcumin-induced cell death. Intravenously administered curcumin-loaded, Cy7-labeled mPEG5kDa-b-p(HPMA-Bz)17.1kDa micelles in mice at 50 mg curcumin/kg showed a long circulation half-life for the micelles (t1/2 = 42 h), in line with the AF4 results. In contrast, the circulation time of curcumin was considerably shorter than that of the micelles (t1/2α = 0.11, t1/2β = 2.5 h) but ∼5 times longer than has been reported for free curcumin (t1/2α = 0.02 h). The faster clearance of curcumin in vivo compared to in vitro studies can be attributed to the interaction of curcumin with blood cells. Despite the excellent solubilizing effect of these micelles, no cytostatic effect was achieved in neuroblastoma-bearing mice, possibly because of the low sensitivity of the Neuro2A cells to curcumin.

. Characteristics of the mPEG 5kDa -b-p(HPMA-Bz) X block copolymers as determined by 1    The GPC chromatograms of the fluorescently-labeled polymers are shown in Figure S3. The polymer eluted between 10 to 15 min (RI detector), which coincided with the elution time as measured by absorbance at 700 nm (Cy7) and 650 nm (Cy5). The results confirm that the dyes were conjugated to the polymer. On the other hand, free dye eluted at 18 min (UV detector). The fluorescently-labeled polymers did not show absorbance at this elution time, confirming that free dyes were completely removed by dialysis. Figure S3. GPC chromatograms of fluorescently labeled mPEG-b-p(HPMA-Bz) polymers. (A) Cy7labeled polymer: green and blue lines represent absorbance at 700 nm for free Cy7 dye and Cy7-labeled polymer, respectively. Red line is the refractive index (RI) signal of Cy7-labeled polymer under the same chromatography conditions. (B) Cy5-labeled-polymer: green and blue lines are absorbance at 650 nm for free Cy5 dye and Cy5-labeled polymer, respectively. Red line is the RI signal of Cy5-labeled polymer under the same chromatography conditions. Abbreviations: AU, absorbance unit; MV, voltage offset in millivolts.

The effect of dialysis on curcumin-loaded micelles
Curcumin-loaded micelles (polymer 30.0 mg and curcumin 3.0 mg) were prepared using the method described in section 2.5. Dialysis was performed in HBS solution for one day at 4 °C using an 8-kDa dialysis membrane. Samples were taken before and after dialysis and analyzed for polymer and curcumin concentration. Data are presented as mean ± SD (n = 5).

HPLC method validation
The method to extract curcumin from plasma was validated by spiking plasma (100 µL) with known amounts of curcumin-loaded micelles (10 µL). Samples were extracted by addition of 4 volumes of acetonitrile (ACN) to 1 volume of spiked plasma and vortexed for 2 min, followed by centrifugation at 15,000 × g for 10 min. The concentration of curcumin was determined in the supernatant using an isocratic HPLC method described in the Materials and Methods section 2.12 of the main text. The ratio of the measured curcumin concentration to the known curcumin concentration was used to calculate the recovery percentage ( Figure S4 and Table  S4).  Data are presented as mean ± SD (n = 3).

Ratio of HPMA-Bz/curcumin
The exemplary calculation to obtain the ratio of HPMA-Bz/curcumin (mol/mol) for mPEG 5kDab-p(HPMA-Bz) 17.1kDa is provided as an illustration.

Fluorescence spectrum of curcumin-loaded micelles
Curcumin-loaded micelles were prepared at 30 mg/mL polymer and varying curcumin concentrations (0.06-3.0 mg/mL) using the method described in section 2.5. The samples were diluted to a curcumin concentration of 40 µg/mL. The fluorescence spectra were recorded using a Jasco FP8300 spectrofluorometer (Tokyo, Japan). The excitation wavelength was 429 nm.  Figure S8 shows that the fluorescence spectra of curcumin-loaded micelles changed with increasing the curcumin loading. The fluorescence intensity decreased with curcumin loadings > 0.4%. Micelles with a curcumin loading of 9% (w/w) were used for the in vitro cell studies and Figure 8 demosntrates that the curcumin fluorescence was substantially quenched.

Cytotoxicity of Empty Micelles in Human Pancreatic Stellate Cells (hPSC)
The cytotoxicity of mPEG 5kDa -b-p(HPMA-Bz) 17.1kDa micelles was investigated in hPSC as the main precursor of pancreatic cancer-associated fibroblasts. hPSC cells were seeded in 96-well plates at a density of 2500 cells/well. Empty micelles were added to the cells in the range of 0-800 µg/mL. Also, HEPES buffer equivalent to the highest concentration of micelles diluted in the medium was used as control. After 24, 48, and 72 h of incubation, the cells were washed with PBS and AlamarBlue (×1 in cell culture medium) was added to the cells. After 4 h of incubation, the medium was removed and collected in a 96-well plate. The absorbance of the medium was measured with a microplate reader (Tecan Group, Männedorf, Switzerland).     Figure S14. Therapeutic efficacy of curcumin-loaded micelles in Neuro2A tumor-bearing mice. Tumors were grown from cells subcutaneously injected (3 × 10 6 cells/100 µL PBS, pH = 7.4) into the right flank of female A/J mice. Treatment consisted of 10 consecutive intravenous injections (starting 9 days after inoculation) of HEPES-buffered saline (HBS), empty mPEG 5kDa -b-p(HPMA-Bz) 17.1kDa micelles (500 mg/kg, polymer), and curcumin-loaded mPEG 5kDa -b-p(HPMA-Bz) 17.1kDa micelles (500 mg/kg micelles, equivalent to 50 mg/kg curcumin) dispersed in HBS. The injection volume was between 100-200 µL and infusions were performed slowly. Arrows point to the days on which intravenous injections were administered. (A) Mean ± SD tumor size (n = 5). (B) Mean ± SD relative body weight of Neuro2A tumor-bearing mice (n = 5). The body weights were normalized to the animal's body weight on day 9 (considered as 100%).