Gemcitabine interacts with carbonate apatite with concomitant reduction in particle diameter and enhancement of cytotoxicity in breast cancer cells.

Substantial amount of research has been done in recent decades for the development of nanoparticle systems to selectively deliver drugs to cancer cells for concurrently enhancing and reducing anti-cancer and off-target effects, respectively. pH-sensitive carbonate apatite (CA) was originally developed for efficient and targeted delivery of DNA, siRNA and proteins to various cancer cell lines. Recently, the CA particles were employed to deliver anti-cancer drugs, cyclophosphamide, doxorubicin and methotrexate to cancer cells. Here, we report on the fabrication and characterization of gemcitabine- loaded CA particles, followed by the evaluation of their roles in enhancement of cytotoxicity in two human and one murine breast cancer cell lines. HPLC was performed to measure binding efficiency of the drug to the apatite particles whereas particle size and zeta potential were evaluated to characterize drug/apatite complex. Depending on the initial doses of the drug, its bind binding affinity towards the particles varied from 3.85% to 4.45%. The particle size was found to surprisingly decrease with an increase of the initial drug concentration. In vitro chemosensitivity assay revealed that apatite/drug nanoparticle complexes presented significantly higher cytotoxicity to breast cancer cells compared to free drugs, which could be correlated with the enhanced cellular uptake of the small size drug-loaded particles through endocytosis compared to the passive diffusion of the free drug.

Intracellular delivery of potential therapeutic genes: prospects in cancer gene therapy.

Conventional therapies for malignant cancer such as chemotherapy and radiotherapy are associated with poor survival rates owing to the development of cellular resistance to cancer drugs and the lack of targetability, resulting in unwanted adverse effects on healthy cells and necessitating the lowering of therapeutic dose with consequential lower efficacy of the treatment. Gene therapy employing different types of viral and non-viral carriers to transport gene(s) of interest and facilitating production of the desirable therapeutic protein(s) has tremendous prospects in cancer treatments due to the high-level of specificity in therapeutic action of the expressed protein(s) with diminished off-target effects, although cancer cell-specific delivery of transgene(s) still poses some challenges to be addressed. Depending on the potential therapeutic target genes, cancer gene therapy could be categorized into tumor suppressor gene replacement therapy, immune gene therapy and enzyme- or prodrug-based therapy. This review would shed light on the current progress of delivery of potentially therapeutic genes into various cancer cells in vitro and animal models utilizing a variety of viral and non-viral vectors.

pH-sensitive carbonate apatite as an intracellular protein transporter.

The transfer of specific proteins into living cells to enable the regulation of cell function or the tracking of the intracellular distribution of proteins is a desirable objective for offering a potential alternative to gene therapy. Here, protein/carbonate apatite complexes were successfully fabricated for intracellular delivery of functional proteins since the carbonate apatite being highly water solubility under an acidic condition could easily be dissolved in endosomes following endocytosis, thus releasing the electrostatically associated proteins in cytoplasm. In this study, we characterized protein/carbonate apatite complexes as an intracellular protein delivery system and we checked intracellular delivery of proteins by carbonate apatite nanoparticles in vitro. Fluorescently-labeled bovine serum albumin as a model protein was effectively delivered into nearly 100% of HeLa cells by the simple addition of protein/carbonate apatite complexes to the cells. Confocal microscopic imaging suggested the endosomal release of protein delivered with carbonate apatite. And intracellularly delivered ss-galactosidase did not lose its enzymatic activity. These results suggested that intracellular delivery system of protein using pH-sensitive carbonate apatite carrier with a very simple procedure will be a highly effective method to the biological and clinical researches.

High performance mRNA transfection through carbonate apatite-cationic liposome conjugates.

mRNA instead of DNA provides a new and attractive approach for gene therapy and genetic vaccination. Delivery of mRNA can bypass nuclear localization step enabling protein expression directly in cytoplasm through transcription. Current technologies for mRNA delivery are predominantly based on cationic liposomes with low activity for transfection. We, previously reported that applying inorganic nano-particles of carbonate apatite onto cationic liposome of DOTAP {N-[1-(2,3-dioleoloxy)propyl]-N,N,N-trimethyl ammonium chloride} resulted in high transfection potency for luciferase mRNA both in mitotic and non-mitotic cells. In this paper, we expanded the previous work and performed in detail study especially on two important parts, evaluating the image of the complex and analyzing the steps of gene delivery to detect the determinant factor for enhanced transfection potency. Transmission electron microscopic (TEM) observation clearly indicated the presence of inorganic carbonate apatite particles on mRNA-liposome complex and demonstrated the structure of the new hybrid carrier material. Due to apparently higher gravitational force of absorbed inorganic nano-particles, cellular contact and internalization of hybrid-particle-associated mRNA were significantly enhanced compared to DOTAP. This analysis indicates rather than downstream steps, initial steps of cell membrane binding and subsequent way of internalization could be the determinant factor for final protein expression. Moreover, we compared transfection efficiency of mRNA and pDNA in Human Umbilical Vein Endothelial cell (HUVEC) to demonstrate advantages of mRNA delivery.

The influence of the cell-adhesive proteins E-cadherin and fibronectin embedded in carbonate-apatite DNA carrier on transgene delivery and expression in a mouse embryonic stem cell line.

Stem cells have the potential to be differentiated to a specific cell type through genetic manipulation and therefore, represent a new and versatile source of cell replacement in regenerative medicine. However, conventional ways of gene transfer to these progenitor cells, suffer from a number of disadvantages particularly involving safety and efficacy issues. We have recently reported on the development of a bio-functionalized DNA carrier of carbonate apatite by embedding fibronectin and E-cadherin chimera on the carrier, leading to its high-affinity interactions with embryonic stem cell surface and accelerated transgene delivery for subsequent expression. Here, we show the molecular basis of synthesizing highly functional composite particles utilizing DNA, cell-adhesive proteins and inorganic crystals, and finally establish a superior transfection system for a mouse stem cell line having potential applications in cell-based therapy.

Bio-functional inorganic materials: an attractive branch of gene-based nano-medicine delivery for 21st century.

Treatment of a physiological disorder in the genetic level (gene therapy) and induction of a specific immunity by means of a genetic material (genetic vaccination), are considered two revolutionary approaches for clinical medicine. The implementation strategies for these basic concepts demand a vehicle for nucleic acid delivery. Viral delivery systems, although highly efficient, possess severe limitations in terms of life safety and thus non-viral synthetic systems have become increasingly desirable. Intensive efforts for the last 3 decades enabled the development of a lot of synthetic devices, most of which belong to cationic lipids, peptides and other polymers, but comparatively little attention was paid to inorganic materials. This is the first article aimed at reviewing the dramatic progress of non-viral gene delivery research focusing on the functional inorganic materials. Both biodegradable and non-biodegradable inorganic particles have been fabricated in the nano-scale with the attributes of binding DNA, internalizing across the plasma membrane and finally releasing it in the cytoplasm for final expression of a protein. Some in vivo trials also brought highly satisfactory results demonstrating their potential applications in the clinical medicine.