Development of chitosan graft pluronic^®F127 copolymer nanoparticles containing DNA aptamer for paclitaxel delivery to treat breast cancer cells

Chitosan F-MMW 400 kDa was purchased from Sigma Aldrich (USA). Pluronic^®F127 (PF), paclitaxel (PTX), 3-(4,5-dimethylthiazol-2-yl)-2, 5 diphenyltetrazolium bromide (MTT), McCoy's 5A medium, Dulbecco's modified eagle medium (DMEM), fetal bovine serum (FBS) and trypsin-ethylenediaminetetra-acetic acid (EDTA) were purchased from Life Technologies (Singapore). Slide-A-Lyzer™ dialysis cassettes with molecular weight cut-off (MWCO) of 3.5 kD was purchased from Thermo Fisher Scientific (USA). Oligonucleotide library 5'-TCA CCG GGA GGA GAC CCT GA-N40-GTG GCT TGG TGG TGG TTC AA -3'. Forward primer 5'-TCA CCG GGA GGA GAC CCT GA-3' and reverse primer 3'-TTG AAC CAC CAC CAA GCC AC-5' were synthesized by IDT (Singapore). SK-BR-3 breast cancer cell-line (HTB-30TM) was supplied from ATCC^® (USA). Ceftriaxon was purchased from Bidiphar JSC (Vietnam). Sodium nitrite (NaNO₂), hydrochloric acid (HCl), sodium hydroxide (NaOH), acetone, acetic acid, sodium triphotphate (TPP), succinic anhydride (SA), 4-dimethylaminopyridine (DMAP), triethylamine (TEA), N,N-dimethylformamide (DMF), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), N-hydroxysuccinimide (NHS), 2-mercaptoethanol, diethyl ether, dimethylsulfoxide (DMSO) were all analytical reagent purchased from Merck Milipore Chemical Company (Germany). All other chemicals were of analytical grade and used without further purifiation.

The colon cancer tissues and stomach cancer tissues of Vietnamese patients were kindly provided by the Oncology Hospital in Ho Chi Minh City. NS-VN-67 and HT-VN-26 cell-lines in DMEM containing 10% (v/v) FBS and 0.25 μg ml⁻¹ ceftriaxon at 37 °C, 5% CO₂ and 95% humidity, respectively, were cultured and collected.

SK-BR-3 cell-line was harvested by trypsinization and 1 × 10⁶ cells were recovered in complete media at 37 °C for 30 min. 200 pmol ssDNA pool dissolved in 500 μl of binding buffer (4.5 g l⁻¹ glucose, 5 mM MgCl₂, 0.1 mg ml⁻¹ yeast tRNA, 1 mg ml⁻¹ bovine serum albumin in phosphate buffered saline (PBS)) was denatured by heating at 95 °C for 5 min and cooled on ice for 10 min before binding. Then the ssDNA pool was incubated with a cell monolayer in a T25 flask (target cells) at 4 °C for 45 min. Cells were washed twice with washing buffer, and then the bound RNA aptamers were eluted by heating at 95 °C for 5 min and separated by phenol:chloroform:isoamyl alcohol (Tripure, Roche) and chloroform extraction. The obtained DNA was polymerase chain reaction (PCR)-amplified with primers (25 cycles of 30 s at 94 °C, 30 s at 58 °C, and 30 s at 72 °C, followed by 5 min at 72 °C; the Taq polymerase and deoxynucleotide triphosphate (dNTP) were obtained from Promega. In the first round selection, the amount of initial ssDNA pool was 12 nmol, dissolved in 1 ml of binding buffer; and the counter selection step was eliminated. In order to acquire aptamers with high affinity and specificity, the wash strength was enhanced gradually by extending wash time (from 1 to 10 min), increasing the volume of wash buffer (from 1 to 5 ml) and the number of washes (from 3 to 5). Additionally, 20% FBS and 50–300 fold molar excess 45-mer random DNA library (a completely different library) were added to the incubation solution to reduce the nonspecific binding of the selected pool. After 20 rounds of selection, the selected ssDNA pool was PCR-amplified using unmodified primers.

To obtain a low MW Chi, medium MW Chi was depolymerized according to the method described by Moghaddam et al [1]. Briefly, 1 g Chi (400 kDa) was dissolved in acetic acid (6% v/v). Low MW Chi was obtained after addition of 10 ml of NaNO₂ (7.0 mg ml⁻¹) to the dissolved Chi at room temperature under magnetic stirring. After 1 h, the depolymerized Chi was precipitated by raising the pH to 9.0 by adding NaOH (4 N). Centrifugation was 10 000 rpm for 5 min. The white yellowish solid was filtrated, washed with acetone three times, and dissolved in a minimum volume of 0.1 N acetic acid. Purification was carried out by subsequent dialysis at MW of 12–14 kDa against purified water (D-Tube^TM Dialyzer Maxi, Merck Millipore, USA). The yellowish lyophilized product was then stored at 4 °C until further use.

The preparation of Chi NPs was achieved via the ionic-gelation method by Calvo et al [15]. A Chi solution (0.1% w/v) was obtained by dissolving low MW Chi in 1% v/v acetic acid. Chi NPs were prepared spontaneously upon addition of various concentrations of TPP (0.01%, 0.015%, 0.02%, 0.025%, and 0.03% w/v) to Chi solution under gentle magnetic stirring at room temperature for 1 h. In all cases, the volume ratio of Chi-TPP solution was 2:1. PF was incorporated into NPs by adding 0.025% w/v TPP solution to Chi aqueous solution containing different concentrations of PF (10%, 15%, 20% w/w).

A carboxylic acid group was introduced to the chain end of PEO-PPO-PEO (PF, MW = 12 500) by reaction of the terminal hydroxyl group of pluronic^®F127 with succinic anhydride as described in the reference [16]. Briefly, Chi-TPP-PF copolymer (5 g, 0.75 mmol), succinic anhydride (0.56 g, 3 mmol), DMAP (0.5 g), and TEA (0.5 ml) were dissolved in 30 ml anhydrous 1,4-dioxane and stirred overnight at 30 °C. Then the 1,4-dioxane was removed under a centrifuge vacuum. The residue was dissolved in chloroform, precipitated into an excess of diethyl ether, and then filtered to remove un-reacted succinic anhydride, DMAP and TEA. With repeating the process twice, the precipitate, carboxyl-terminated pluronic (about 85% yields), was obtained after filtering and drying in a vacuum for 24 h.

The procedure of the reaction between carboxyl-terminated Chi-TPP-PF copolymer and PTX was as follows: 4 g (0.7 mmol of –COOH groups) of carboxyl-terminated Chi-TPP-PF copolymer was dissolved in 2 ml of N, N-dimethylformamide (DMF) at room temperature. 0.1 g of EDC and 0.2 g of NHS was added in carboxyl-terminated Chi-TPP-PF copolymer solution. After 15 min reaction, 1.5 ml of 2-mercaptoethanol was added to quench the EDC followed by an addition various concentrations of PTX (0.2, 0.4, 0.6, 0.8 and 1.0 mg ml⁻¹) to the activated carboxyl-terminated Chi-TPP-PF copolymer. The activated carboxyl-terminated Chi-TPP-PF copolymer in DMF was reacted with PTX in the presence of triethylamine (1 mg) and stirred at room temperature for 12 h under nitrogen. The resultant was dialysed against DMF by using a dialysis cassette with MWCO of 3.5kD for 24 h and finally lyophilized to gain the product.

The morphological examinations of NPs were made by scanning electron microscopy (SEM) (440, Leica Cambridge Ltd, Cambridge, UK). NPs were dried on an aluminum disk at room temperature. The fixed NPs were coated with gold using a sputter coater (Desk II, Denton Vacuum, Moorestown, NJ).

Transmission electron microscopy (TEM) (JEM 1230, Joel Ltd, Tokyo, Japan) was used to examine and compare the topography of the NPs. Freeze dried NPs were suspended in milli Q water before observation.

Size and zeta potential (surface charge) of NPs were measured at least in triplicate using nano partica SZ-100 (Horiba, Japan). For both of the above measurements, 0.2 mg NPs was suspended in 10 ml milli Q water.

PTX release was determined by incubating the NPs in PBS at 37 ± 0.5 °C. Remaining free PTX in the supernatant was measured for its absorbance at λ = 280 nm by using UV–vis spectrophotometer. For each sample, the release medium was withdrawn at predetermined time intervals at 12 d and replaced by the same medium at the same condition [3]

$$\begin{eqnarray*}&&{\rm{P}}{\rm{T}}{\rm{X}}\;{\rm{r}}{\rm{e}}{\rm{l}}{\rm{e}}{\rm{a}}{\rm{s}}{\rm{e}}{\rm{d}}( \% )=\displaystyle \frac{{\rm{a}}{\rm{m}}{\rm{o}}{\rm{u}}{\rm{n}}{\rm{t}}\;{\rm{o}}{\rm{f}}\;{\rm{P}}{\rm{T}}{\rm{X}}\;{\rm{r}}{\rm{e}}{\rm{l}}{\rm{e}}{\rm{a}}{\rm{s}}{\rm{e}}{\rm{d}}}{{\rm{i}}{\rm{n}}{\rm{i}}{\rm{t}}{\rm{i}}{\rm{a}}{\rm{l}}\;{\rm{a}}{\rm{m}}{\rm{o}}{\rm{u}}{\rm{n}}{\rm{t}}\;{\rm{P}}{\rm{T}}{\rm{X}}}\times 100 \% .\end{eqnarray*}$$

0.5 mg NPs were dissolved in 5 ml milli Q water and centrifuging at 14 000 rpm for 30 min at 15 °C. The amount of PTX released in the supernatant was measured by spectrophotometry at 247–249 nm. Each sample was measured in triplicate. The following equations were used to evaluate the LC and encapsulation efficiency (EE) of the NPs [6]:

$$\begin{eqnarray*}&&{\rm{L}}{\rm{C}}( \% )=\displaystyle \frac{{\rm{t}}{\rm{o}}{\rm{t}}{\rm{a}}{\rm{l}}\;{\rm{P}}{\rm{T}}{\rm{X}}-{\rm{f}}{\rm{r}}{\rm{e}}{\rm{e}}\;{\rm{P}}{\rm{T}}{\rm{X}}}{{\rm{n}}{\rm{a}}{\rm{n}}{\rm{o}}{\rm{p}}{\rm{a}}{\rm{r}}{\rm{t}}{\rm{i}}{\rm{c}}{\rm{l}}{\rm{e}}\;{\rm{w}}{\rm{e}}{\rm{i}}{\rm{g}}{\rm{h}}{\rm{t}}}\times 100 \% ,\end{eqnarray*}$$

$$\begin{eqnarray*}&&{\rm{E}}{\rm{E}}( \% )=\displaystyle \frac{{\rm{t}}{\rm{o}}{\rm{t}}{\rm{a}}{\rm{l}}\quad {\rm{P}}{\rm{T}}{\rm{X}}-{\rm{f}}{\rm{r}}{\rm{e}}{\rm{e}}\quad {\rm{P}}{\rm{T}}{\rm{X}}\quad }{{\rm{t}}{\rm{o}}{\rm{t}}{\rm{a}}{\rm{l}}\quad {\rm{P}}{\rm{T}}{\rm{X}}}\times 100 \% .\end{eqnarray*}$$

The cytotoxicity of PTX NPs and free PTX were performed on SK-BR-3 human breast cancer cell-line, NS-VN-67 colon cancer cells and HT-VN-26 stomach cancer cells of Vietnamese patients. The cell-lines were cultured in Mc'Coy 5A and DMEM supplemented with 10% FBS, L-glutamine, and 0.25 μg ml⁻¹ ceftriaxon solution and incubated at 37 °C at 5% CO₂. Cells were seeded at a density of 12 000 viable cells per well in 96-well tissue culture plates (Corning Incorporated Costar^®) incubated for 24 h to allow cell attachment. The cells were then incubated for another 12, 24 and 48 h with PTX copolymer NPs and free PTX. Cells were then washed in PBS, and 20 μl of MTT solution (5 mg ml⁻¹) was added to each well. The plates were incubated for an additional 4 h and then the medium was discarded. 200 μl DMSO was added to each well, and the solution was vigorously mixed to dissolve tetrazolium dye. The absorbance of each well was measured by PlateReader AF2200 (Eppendorf, Germany) at 570 nm.

Results are shown as mean ± standard deviation and each experiment was measured in triplicate. Statistical data analyzes were performed by applying one-way ANOVA tests and P-value <0.05 was considered significant.

To isolate aptamers with high affinity and specificity to the native, membrane presented form of HER-2, we performed SELEX using a well-known HER-2 overexpressing breast cancer cell line, SK-BR-3. We started the SELEX procedure by using a DNA library of 45 nt randomized region, with 10¹⁴ complexity. We performed positive selection by retrieving aptamers that bind to SK-BR-3 cells. After 20 rounds of positive selection, the binding affinity of DNA library reached saturation (data not shown). After the successful enrichment of aptamers with high affinity to SK-BR-3 cells, individual aptamers were sequenced (table 1).

The NPs were prepared by ionic-gelation upon the addition of TPP to Chi in acetic acid under gentle magnetic stirring at room temperature. The NPs with smaller size have valuable characteristics such as more improved drug delivery, longer circulation in blood, and lower toxicity. The conditions of experiments were carried out at the concentrations of low MW Chi 1% (v/v) in acetic acid and TPP 0.01%, 0.015%, 0.02%, 0.025% and 0.03% (table 2).

Due to dissociation, TPP dissociation in water and hydroxyl ions liberated, the TPP solution exists and the OH⁻ ions and ion ${{\rm{P}}}_{3}{{{\rm{O}}}_{10}}^{5-}$ had been competitive reaction with NH₃⁺ group of Chi. The low MW Chi fragments were participated in the reaction with TPP molecules ionic-gelation, combined with the agitation to form smaller size Chi NPs.

Chi NPs did not generate delamination after 3 d stored in 4 °C, suggesting that NPs formed had been smaller if the larger NP size would have been caused sedimentation and phase separation during storage.

SEM images showed that the shape of Chi original material each large polymer particle size. Chi NPs generated by ionic-gelation method using TPP, with small particle size, relatively uniform (figure 1).

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The results of the particle size distribution showed that the average was 53.2 nm. They were included about 81% particles size of 55.6 nm; 9% ones of 38 nm and larger than 100 nm (figure 2).

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The addition results of pluronic^®F127 (PF) in decreased size which could be attributed to the formation of a rigid gel leading to lesser water uptake. Slighter swelling caused a reduction in the mean diameter of the particles in aqueous medium (table 2). Since there was not significant change in zeta potential by increasing the concentrations of PF (P < 0.028), it could be concluded that PF was mostly incorporated inside the NP matrix, the Chi 1%-TPP 0.025% NPs were mixed in different concentrations of PF (table 3).

Aptamer modified pluronic ^®F127 (PF-Ap) was synthesized by the reaction of carboxylated pluronic ^®F127 with the amino groups at the ends of aptamers (figure 3(a)). PF-Ap containing a scrambled aptamer was obtained by the same method. To confirm the conjugation and determine the maximum conjugation content of Ap on pluronic ^®F127, novex^®12% tris-glycine mini gel electrophoresis was carried out. The samples including free Ap, Chi-TPP-PF, and Chi-TPP-PF-Ap with or without EDC-NHS used in the conjugation reaction were separately subjected to 2% tris borate edta sodium dodecyl sulfide polyacrylamine gel electrophoresis (TBE SDS PAGE). Then the electrophoresis was performed at 125 V for 30 min, and the base pair band on the gel was displayed by SYBR^®green nucleic acid stain (figure 3(b)). The detailed synthesis procedure, chemical compositions, and characterization of the materials were given in supplementary information.

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The general addition of PTX increased the sizes of Chi NPs, but did not affect their zeta potential significantly (P < 0.02). The effect of PTX concentration on particle size is more significant than when the concentration rises 0.2–0.4 mg ml⁻¹ (P < 0.015). The PTX-loaded Chi-PF NPs size did grow significantly at concentrations up to 0.6–1 mg ml, but there was a change in size when the concentration of PTX 0.4 mg ml were increased maximum (EE = 83.28 ± 0.13%; LC = 9.12 ± 0.34%) and mean diameter 86.22 ± 1.45 nm. PTX was a low MW anti-cancer drug. Therefore, it might not be possible to increase the PTX particle diameter severely until it reached its maximum capacity inside the NPs. The PTX concentration did not influence the zeta potential of the prepared NPs (P < 0.04), Chi 1%-TPP 0.025%-PF 15% NPs were loaded in different concentrations of PTX (table 4).

SEM and TEM images of Chi-PF NPs loaded with PTX, showed that particles were spherical and uniform with mean diameter 86.22 nm (figure 4).

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The amount of released PTX was presented as the percentage of cumulative release at 37 °C over a period of 12 d. The drug concentration was determined by a fluorescence spectrophotometer at excitation wavelength of 280 nm. Nanoparticle solution was used for the experiments with concentrations of 5%, 7%, and 10%. In the first 12 h, followed by sustained release of 29%–35% and 85%–93% after 288 h at pH 7.5. This result suggested that there were two phases of PTX release profile. Firstly, the initial burst release of the PTX from the NPs in the first 12 h. Burst release was the phenomenon of drug which a greater amount of initial bulky drug were immediately released prior to arriving at the steady level of the release profile. This direction affected the effective exposure time of nano-carriers. In the next phase, a sustained release of the encapsulated PTX was shown after the 6th day, Chi 1%-TPP 0.025%-PF 15% NPs were loaded with PTX 0.4 mg ml⁻¹ (figure 5).

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Cytotoxicity test was performed on the NPs without PTX, Chi 1%-TPP 0.025%-PF 15% NPs loaded with PTX at concentrations of 0.2, 0.4, 0.6, 0.8 and 1 mg ml⁻¹, using the MTT method on SK-BR-3 cells. The NPs without PTX were shown to have minimum toxic effects and themselves would not cause cellular damage. Their half maximum inhibitory concentration (IC₅₀) is 7.3–9.5 mg ml⁻¹ (figure 6(a)).

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The toxic cell death of NPs without PTX did not change much after 6, 12, 24, 36 and 48 h. Living cell proliferation did grow up grip bottom flask. Therefore, it could be concluded that the incorporation of PF was not influence the cytotoxicity of the NPs. The effect of free PTX 1 mg ml⁻¹ solution on cell death was also investigated. The relative cell viability after 6, 12, 24, 36 and 48 h was 86.5%, 72.77%, 65.82%, 32.8% and 22.55%, respectively.

The IC₅₀ of free PTX against SK-BR-3 cell was 1 mg ml⁻¹ which was about five times lower than NPs loaded PTX. On the other hand, the NPs non-loaded PTX processing lyophilized drug stored when reconstituted in the milli Q water, they were not toxic to cells.

With the effect of NPs Chi 1%-TPP 0.025%-PF 15%-PTX 0.4 mg ml⁻¹, the SK-BR-3, NS-VN-67 and HT-VN-26 were dead 91%–95%, 52%–56% and 26%–28%, respectively, after 6 h. The partition was destroyed and led to broken cells after 6 h. PTX was a hydrophilic anti-cancer drug that needed membrane transporters to enter the cells. Cells could uptake NPs by endocytosis mediated by a clathrin-mediated process. Therefore, the NPs could act as drug delivery systems that facilitated drug entrance into the cells. With the effect of free PTX 1 mg ml⁻¹ the cell dead proportion was 13%–14%. It did not affect the cells efficiently. The above-mentioned results of the in vitro cytotoxicity test showed that the NPs were biocompatible and used as anti-cancer drug carriers.