The effect of poloxamer 188 on nanoparticle morphology, size, cancer cell uptake, and cytotoxicity Nanomedicine: Nanotechnology, Biology, and Medicine xx (2009) xxx–xxx

Background The global incidence and mortality of breast cancer is high, A major barrier to successful cancer treatment is multidrug resistance (MDR) to anticancer agents, Nanoparticles and their use in drug delivery is a far more effective cancer treatment method than conventional chemotherapy, Nanoparticles (& Poloxamers) could reduce the MDR that characterizes many anticancer drugs, Docetaxel, an antitumor chemotherapeutic agent, is widely used in the treatment of solid tumors, particularly of the breast and ovaries, Treatment with docetaxel is associated with a certain degree of toxicity and the treatment with docetaxel can result in the emergence of MDR mediated by P-gp

Aim “In this research we investigated the effect of triblock copolymer poloxamer 188 on ( PLGA) nanoparticle morphology, size, cancer cell uptake, and cytotoxicity to determine if docetaxel-loaded PLGA nanoparticles incorporated with poloxamer 188 could achieve better therapeutic effects than those of the nanoparticles without incorporation of poloxamer 188 in the docetaxel-resistant human breast adenocarcinoma MCF-7 cell line.”

Materials PLGA (50:50, MW 40,000 to 75,000), polyvinyl alcohol (PVA) (30,000 to 70,000 MW) (water soluble synthetic polymer), coumarin-6, Poloxamer 188 (Pluronic F68) (poly(ethylene oxide)/poly(propylene oxide) ≈ 4:1, 8300 MW), Docetaxel, MCF-7 human mammary carcinoma cells, Cell Counting Kit-8 (CCK-8), Solvents,…

Poloxamers Poloxamers are nonionic triblock copolymers composed of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) attached to two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)). Poloxamers are also known by the trade name Pluronics. Because the lengths of the polymer blocks can be customized, many different poloxamers exist that have slightly different properties. For the generic term "poloxamer", these copolymers are commonly named with the letter "P" (for poloxamer) followed by three digits, the first two digits x 100 give the approximate molecular mass of the polyoxypropylene core, and the last digit x 10 gives the percentage polyoxyethylene content, (e.g., P407 = Poloxamer with a polyoxypropylene molecular mass of 4,000 g/mol and a 70% polyoxyethylene content) .

For the Pluronic tradename, coding of these copolymers starts with a letter to define its physical form at room temperature (L = liquid, P = paste, F = flake (solid)) followed by two or three digits. The first digit (two digits in a three-digit number) in the numerical designation, multiplied by 300, indicates the approximate molecular weight of the hydrophobe; and the last digit x 10 gives the percentage polyoxyethylene content, e.g., L61 = Pluronic with a polyoxypropylene molecular mass of 1,800 g/mol and a 10% polyoxyethylene content). In the example given, poloxamer 181 (P181) = Pluronic L61.

Poloxamer HO(C2H4O)a(C3H6O)b(C2H4O)aH (poly(ethylene oxide)/poly(propylene oxide) (see the table)

Journal of Controlled Release 94 (2004) 411–422

PLGA A popular choice as a biodegradable drug carrier and among the most commonly used to prepare nanoparticles for drug delivery, Poly(D,L-lactide-co-glycolide) (PLGA) is approved by the U.S. Food and Drug Administration (FDA), The degradation rates of PLGA and the accompanying release of encapsulated drugs can be controlled by the polymer's physicochemical properties such as molecular weight,hydrophilicity, and the ratio of lactide (LA) to glycolide (GA),

Structure of poly(lactic-co-glycolic acid) Structure of poly(lactic-co-glycolic acid). x= number of units of lactic acid; y= number of units of glycolic acid.

“Zeta potential results showed that both empty and drug-loaded nanosphere formulations exhibited a negative charge with values ranging from -38.9 to -36.0 mV, typically observed for these types of systems. The surface charge of colloidal particles can arise by a number of means, e.g. ionisation of chemical groups on the surface or adsorption of ions. It has been previously reported that the negative charge of PLGA nanoparticles is due to the ionisation of carboxylic-end groups of polymer on the surface. However, carboxylic end groups of the PLGA used in this study were esterified with lauryl groups and hence were not susceptible to ionisation. Thus, the negative charge of prepared nanoparticles can be attributed to the adsorption of anions on the colloidal surface.” Ref. European Journal of Pharmaceutics and Biopharmaceutics 59 (2005) 491–500

Methods Nano particles preparation: docetaxel-loaded nanoparticles prepared by oil-in-water emulsion/solvent evaporation technique. A given amount of docetaxel and PLGA with or without addition of poloxamer 188 were dissolved in dichloromethane (DCM). The formed solution was poured into PVA solution. The mixture was sonicated. The emulsion was then evaporated overnight under reduced pressure to remove the organic solvent.

The particle suspension was centrifuged at 20,000 rpm for 20 minutes and then washed three times to remove the unencapsulated drug and surfactant. The resulting particles were resuspended in water and freeze-dried for 2 days. The fluorescent coumarin-6–loaded PLGA/poloxamer 188 blend nanoparticles were prepared in the same way except 0.05% (wt/vol) coumarin-6 was encapsulated instead of docetaxel.

2. Nanoparticle surface morphology: determined by scanning electron microscopy (SEM)

Results & Discussion The resulting docetaxel-loaded PLGA/poloxamer 188 blend nanoparticles observed by SEM were spherical, had a mean diameter of around 200 nm which much smaller than PLGA nanoparticles, had a rough and porous surface. (Figure 1).

Figure 1

Possible Explanations: Poloxamer 188, a non-ionic emulsifier, may act as a co-emulsifier, resulting in smaller particle size and narrower size distribution. Poloxamer offers additional steric stabilization effect avoiding aggregation of the fine particles in the colloidal system. Due to the hydrophilicity, poloxamer 188 may leach out during the fabrication process, creating a porous structure in the surface of the PCL/poloxamer 188 nanoparticles. Poloxamers adsorbing strongly onto the surfaces of hydrophobic nanospheres via their hydrophobic poly(ethylene oxide) (POP) center block accounted for the relatively rough surface. The adsorption also changes a smooth PLGA surface into a rough one (with extended PEO chains into the solution).

3. Size analysis and zeta potential Zeta potential of nanoparticles is of significance on: Stability in suspension through the electrostatic repulsion between the particles, interaction in vivo with the cell membrane, and judgment of component on the particle surface. The particle size and size distribution were determined by laser light scattering). The particles (about 2 mg) were suspended in deionized water before measurement. Zeta potential of the NP was detected by ZetaPlus zeta potential analyzer. The data were obtained with the average of three measurements.

Results & Discussion: PLGA/ poloxamer 188 NP have smaller mean diameter, and narrower particle size distribution, compared with PLGA NP. Measured zeta potential of drug-loaded NP –23.35 mV: PLGA/poloxamer 188, –41.28 mV: PLGA.

Possible Explanations: The adsorption of poloxamer 188 (non-ionic surfactant) onto the PLGA NP: partially screened the surface charge of the PLGA NP, shifted the shear plane of the diffusive layer to a larger distance, reduced the zeta potential.

4. Fourier transform infrared (FTIR) spectra of poloxame 188 and docetaxel-loaded PLGA/poloxamer 188 NP was recorded by FTIR spectrophotometer using KBr. Results & Discussion: …The presence of poloxamer 188 in the docetaxel loaded PLGA/poloxamer 188 NP was further confirmed by FTIR.

Figure 3. Fourier transform infrared spectroscopy of (A) poloxamer 188; (B) docetaxel-loaded PLGA/poloxamer 188 nanoparticles.

5. Cellular uptake of fluorescent coumarin-6–loaded NP using Human breast cancer cell line. Qualitative and Quantitative experiments were carried out. Results & Discussion: Results showed the cellular uptake efficiency of both PLGA and PLGA/poloxamer 188 NP decreased with increase of the incubated particle concentration. Obviously, there appeared a saturated and limited capability of cellular uptake of the NP. It was demonstrated that there was a size dependent cellular uptake of biodegradable microparticles or NP. PLGA/poloxamer 188 NP showed higher cellular uptake than that of the PLGA NP.

Figure 5. Cellular uptake of courmarin-6–loaded nanoparticles Figure 5. Cellular uptake of courmarin-6–loaded nanoparticles. Data represent mean ± SD (n = 6).

6. In vitro cytotoxicity Measurement of cell viability of the drug-loaded PLGA/poloxamer 188 NP using human breast cancer cell lines. Results & Discussion: Compared with Taxotere, the higher cytotoxicity of the drug formulated in the two NP can be attributed to the: higher cellular uptake, sustained drug release manner, reduction of MDR by a mechanism of NP internalization. The PLGA/ poloxamer 188 NP showed more significant cytotoxicity to the cells, indicating poloxamer 188 could enhance the potency of PLGA NP against MDR. The higher cellular uptake of the NP and the faster drug release from the NP may account for the advantage of PLGA/ poloxamer 188 NP over PLGA NP.

Figure 7. Viability of cells cultured with docetaxel-loaded PLGA nanoparticles (PLGA NP) and docetaxel-loaded PLGA/poloxamer 188 nanoparticles (PLGA/F68 NP) in comparison with that of Taxotere at the same docetaxel dose and empty PLGA/poloxamer 188 nanoparticles (empty PLGA/F68 NP) with the same amount of nanoparticles (n = 6).

7. Differential scanning calorimetry: The physical status of docetaxel inside the NP was investigated by differential scanning calorimetry. Results & Discussion: Figure 2 shows the DSC thermograms of pure docetaxel, docetaxel-loaded PLGA NP, and docetaxel-loaded PLGA/Poloxamer 188 NP. The melting endothermic peak of pure docetaxel appeared at 173°C. No melting peak was detected for both NP formulations. The results showed docetaxel in the NP formulation was in an amorphous or disordered crystalline phase or in the solid solution state.

Figure 2. DSC thermograms of the pure docetaxel and docetaxel-loaded nanoparticles.