Ezharul Hoque Chowdhury

Galactosylated chitosan-graft-poly(ethylene glycol) as hepatocyte-targeting DNA carrier.

Lactobionic acid bearing galactose group was coupled with chitosan for liver specificity, and poly(ethylene glycol) (PEG) was grafted to galactosylated chitosan (GC) for stability in water and enhanced cell permeability. Complex formation of galactosylated chitosan-graft-PEG (GCP)/DNA complexes was confirmed by agarose gel electrophoresis. Compared to GC/DNA complex, the stability of GCP/DNA complex could be enhanced. Particle sizes of GCP/DNA complexes decreased as the charge ratio of GCP to DNA increased and had a minimum value around 27 nm at the charge ratio of 5. Conformational change of DNA did not occur after complex formation with GCP compared to conformation of DNA itself. GCP/DNA complexes were only transfected into Hep G2 having asialoglycoprotein receptors (ASGR), indicative of specific interaction of ASGR on cells and galactose ligands on GCP.

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.

Carbonate apatite-facilitated intracellularly delivered siRNA for efficient knockdown of functional genes

Gene therapy through intracellular delivery of a functional gene or a gene-silencing element is a promising approach to treat critical diseases. Elucidation of the genetic basis of human diseases with complete sequencing of human genome revealed many vital genes as possible targets in gene therapy programs. RNA interference (RNAi), a powerful tool in functional genomics to selectively silence messenger RNA (mRNA) expression, can be harnessed to rapidly develop novel drugs against any disease target. The ability of synthetic small interfering RNA (siRNA) to effectively silence genes in vitro and in vivo, has made them particularly well suited as a drug therapeutic. However, since naked siRNA is unable to passively diffuse through cellular membranes, delivery of siRNA remains the major hurdle to fully exploit the potential of siRNA technology. Here pH-sensitive carbonate apatite has been developed to efficiently deliver siRNA into the mammalian cells by virtue of its high affinity interactions with the siRNA and the desirable size of the resulting siRNA/apatite complex for effective cellular endocytosis. Moreover, following internalization by cells, siRNA was found to be escaped from the endosomes in a time-dependent manner and finally, more efficiently silenced reporter genes at a low dose than commercially available lipofectamine. Knockdown of cyclin B1 gene with only 10nM of siRNA delivered by carbonate apatite resulted in the significant death of cancer cells, suggesting that the new method of siRNA delivery is highly promising for pre-clinical and clinical cancer therapy.

pH-sensitive nano-crystals of carbonate apatite for smart and cell-specific transgene delivery

The treatment of a human disease at a genetic level by either providing a cell with a functional gene or a nucleic acid sequence to precisely silence a harmful gene, is a powerful approach that could revolutionise clinical medicine. Despite the existence of both genetically engineered viral vectors and synthetically designed lipid- or polymer-based nanocarriers, an ideal delivery system in terms of safety and efficacy is still lacking. This editorial reports on the development of biocompatible, inorganic nanoparticles of carbonate apatite, which has the unique features essentially required for smart delivery, as well as for the expression of a genetic material in a mammalian cell.

Reversing multidrug resistance in breast cancer cells by silencing ABC transporter genes with nanoparticle-facilitated delivery of target siRNAs

Background Multidrug resistance, a major impediment to successful cancer chemotherapy, is the result of overexpression of ATP-binding cassette (ABC) transporters extruding internalized drugs. Silencing of ABC transporter gene expression with small interfering RNA (siRNA) could be an attractive approach to overcome multidrug resistance of cancer, although delivery of siRNA remains a major hurdle to fully exploit the potential of siRNA-based therapeutics. Recently, we have developed pH-sensitive carbonate apatite nanoparticles to efficiently carry and transport siRNA across the cell membrane, enabling knockdown of the cyclin B1 gene and consequential induction of apoptosis in synergy with anti-cancer drugs. Methods and results We report that carbonate apatite-mediated delivery of the siRNAs targeting ABCG2 and ABCB1 gene transcripts in human breast cancer cells which constitutively express both of the transporter genes dose-dependently enhanced chemosensitivity to doxorubicin, paclitaxel and cisplatin, the traditionally used chemotherapeutic agents. Moreover, codelivery of two specific siRNAs targeting ABCB1 and ABCG2 transcripts resulted in a more robust increase of chemosensitivity in the cancer cells, indicating the reversal of ABC transporter-mediated multidrug resistance. Conclusion The delivery concept of multiple siRNAs against ABC transporter genes is highly promising for preclinical and clinical investigation in reversing the multidrug resistance phenotype of breast cancer.

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.

Nuclear targeting of viral and non-viral DNA

The nuclear envelope presents a major barrier to transgene delivery and expression using a non-viral vector. Virus is capable of overcoming the barrier to deliver their genetic materials efficiently into the nucleus by virtue of the specialized protein components with the unique amino acid sequences recognizing cellular nuclear transport machinery. However, considering the safety issues in the clinical gene therapy for treating critical human diseases, non-viral systems are highly promising compared with their viral counterparts. This review summarizes the progress on exploring the nuclear traffic mechanisms for the prominent viral vectors and the technological innovations for the nuclear delivery of non-viral DNA by mimicking those natural processes evolved for the viruses as well as for many cellular proteins.

Fabrication and Intracellular Delivery of Doxorubicin/Carbonate Apatite Nanocomposites: Effect on Growth Retardation of Established Colon Tumor

In continuing search for effective treatments of cancer, the emerging model aims at efficient intracellular delivery of therapeutics into tumor cells in order to increase the drug concentration. However, the implementation of this strategy suffers from inefficient cellular uptake and drug resistance. Therefore, pH-sensitive nanosystems have recently been developed to target slightly acidic extracellular pH environment of solid tumors. The pH targeting approach is regarded as a more general strategy than conventional specific tumor cell surface targeting approaches, because the acidic tumor microclimate is most common in solid tumors. When nanosystems are combined with triggered release mechanisms in endosomal or lysosomal acidic pH along with endosomolytic capability, the nanocarriers demonstrated to overcome multidrug resistance of various tumors. Here, novel pH sensitive carbonate apatite has been fabricated to efficiently deliver anticancer drug Doxorubicin (DOX) to cancer cells, by virtue of its pH sensitivity being quite unstable under an acidic condition in endosomes and the desirable size of the resulting apatite-DOX for efficient cellular uptake as revealed by scanning electron microscopy. Florescence microscopy and flow cytometry analyses demonstrated significant uptake of drug (92%) when complexed with apatite nanoparticles. In vitro chemosensitivity assay revealed that apatite-DOX nanoparticles executed high cytotoxicity in several human cancer cell lines compared to free drugs and consequently apatite-DOX-facilitated enhanced tumor inhibitory effect was observed in colorectal tumor model within BALB/cA nude mice, thereby shedding light on their potential applications in cancer therapy.

Current Approaches for Drug Delivery to Central Nervous System

Brain, the center of the nervous system in all vertebrate, plays the most vital role in every function of human body. However, many neurodegenerative diseases, cancer and infections of the brain become more prevalent as populations become older. In spite of the major advances in neuroscience, many potential therapeutics are still unable to reach the central nervous system (CNS) due to the blood-brain barrier (BBB) which is formed by the tight junctions within the capillary endothelium of the vertebrate brain. This results in the capillary wall behaving as a continuous lipid bilayer and preventing the passage of polar and lipid insoluble substances. Several approaches for delivering drugs to the CNS have been developed to enhance the capacity of therapeutic molecules to cross the BBB by modifying the drug itself, or by coupling it to a vector for receptor-mediated, carrier mediated or adsorption-mediated transcytosis. The current challenge is to develop drug delivery systems that ensure the safe and effective passage of drugs across the BBB. This review focuses on the strategies and approaches developed to enhance drug delivery to the CNS.