Assembly of bio-nanoparticles for double controlled drug release

Abstract

A critical limiting factor of chemotherapy is the unacceptably high toxicity. The use of nanoparticle based drug carriers has significantly reduced the side effects and facilitated the delivery of drugs. Source of the remaining side effect includes (1) the broad final in vivo distribution of the administrated nanoparticles, and (2) strong basal drug release from nanoparticles before they could reach the tumor. Despite the advances in pH-triggered release, undesirable basal drug release has been a constant challenge under in vivo conditions. In this study, functionalized single walled carbon nanohorn supported immunoliposomes were assembled for paclitaxel delivery. The immunoliposomes were formulated with polyethylene glycol, thermal stable and pH sensitive phospholipids. Each nanohorn was found to be encapsulated within one immunoliposome. Results showed a highly pH dependent release of paclitaxel in the presence of serum at body temperature with minimal basal release under physiological conditions. Upon acidification, paclitaxel was released at a steady rate over 30 days with a cumulative release of 90% of the loaded drug. The drug release results proved our hypothesized double controlled release mechanism from the nanoparticles. Other results showed the nanoparticles have doubled loading capacity compared to that of traditional liposomes and higher affinity to breast cancer cells overexpressing Her2 receptors. Internalized nanoparticles were found in lysosomes.

Figure 1. Size distribution of different nanoparticles.

PEGylated DSPC lipsome (black), SWNH(-CH₂-CH₂-COOH)_x (red), paclitaxel loaded SWNH(-CH₂-CH₂-COOH)_x (green), DSPC NsiL (blue), and DSPC NsiL without paclitaxel (light blue). Sizes are shown in diameters (mean ± S.D., n = 45).

Figure 2. Morphology study of different nanoparticles by TEM.

a, PEGylated DSPC liposomes; b, SWNH(-CH₂-CH₂-COOH)_x; c and d, DSPC NsL; e, an intermediate step of a SWNH(-CH₂-CH₂-COOH)_x surrounded by multiple liposomes before the final one nanohorn one liposome structure. Immediately before TEM tests, samples were deposited onto carbon coated cupper grids and negatively stained with 2% phosphotungstic acid.

Figure 3. Paclitaxel release profile of different carriers.

a, in buffer paclitaxel release from PEGylated DOTAP liposome, SWNH(-CH₂-CH₂-COOH)_x and DOTAP NsiL; b, in serum paclitaxel release from PEGylated DOTAP liposome, SWNH(-CH₂-CH₂-COOH)_x and DOTAP NsiL; c, in serum paclitaxel release from PEGylated DSPC liposome at pH 4.6; d, in serum paclitaxel release from PEGylated DSPC liposome at pH 7.2 and 6.5. All liposomes were PEGylated. All SWNHs were functionalized. Numbers in the legends indicate the pH.

Figure 4. Structure and proposed drug release mechanism of NsiL.

a, in serum; b, in tumor cells.

Figure 5. Study of NPs cytotoxicity by cell viability test.

a, SK-BR-3 cells; b, BT-20 cells. Cells were treated with 640, 320, 160 and 80 µg/ml NPs (in lipid concentration). NsLDO and LipoDO are DOTAP NsL and PEGylated DOTAP liposomes, respectively. NsLDS and LipoDS are DSPC NsL and PEGylated DSPC liposomes, respectively. SWHN indicates SWNH(-CH₂-CH₂-COOH)_x.

Figure 6. Cell binding affinity of different NPs.

Different concentrations of particles were incubated with cell cultures, 40 µg/ml, 20 µg/ml, 10 µg/ml, 5 µg/ml, 2.5 µg/ml, 1.25 µg/ml, 0.625 µg/ml and blank control. A: NsiL with SK-BR-3; B: NsL with SK-BR-3; C: Herceptin NsiL with SK-BR-3; D: NsiL with BT-20; E: NsiL with BT-20; F: Herceptin NsiL with BT-20. NPs were labeled with rhB. Fluorescence emissions at 645 nm were measured.

Figure 7. Cell internalization observed by confocal laser microscopy.

a, stained SK-BR-3 cells with unstained NsiL; b, unstained SK-BR-3 cells with stained NsiL; c and d, co-stained NsiL in SK-BR-3 cells.