pH-sensitive Carbonate Apatite Research Articles

Targeting Cell Adhesion Molecules via Carbonate Apatite-Mediated Delivery of Specific siRNAs to Breast Cancer Cells In Vitro and In Vivo.

While several treatment strategies are applied to cure breast cancer, it still remains one of the leading causes of female deaths worldwide. Since chemotherapeutic drugs have severe side effects and are responsible for development of drug resistance in cancer cells, gene therapy is now considered as one of the promising options to address the current treatment limitations. Identification of the over-expressed genes accounting for constitutive activation of certain pathways, and their subsequent knockdown with specific small interfering RNAs (siRNAs), could be a powerful tool in inhibiting proliferation and survival of cancer cells. In this study, we delivered siRNAs against mRNA transcripts of over-regulated cell adhesion molecules such as catenin alpha 1 (CTNNA1), catenin beta 1 (CTNNB1), talin-1 (TLN1), vinculin (VCL), paxillin (PXN), and actinin-1 (ACTN1) in human (MCF-7 and MDA-MB-231) and murine (4T1) cell lines as well as in the murine female Balb/c mice model. In order to overcome the barriers of cell permeability and nuclease-mediated degradation, the pH-sensitive carbonate apatite (CA) nanocarrier was used as a delivery vehicle. While targeting CTNNA1, CTNNB1, TLN1, VCL, PXN, and ACTN1 resulted in a reduction of cell viability in MCF-7 and MDA-MB-231 cells, delivery of all these siRNAs via carbonate apatite (CA) nanoparticles successfully reduced the cell viability in 4T1 cells. In 4T1 cells, delivery of CTNNA1, CTNNB1, TLN1, VCL, PXN, and ACTN1 siRNAs with CA caused significant reduction in phosphorylated and total AKT levels. Furthermore, reduced band intensity was observed for phosphorylated and total MAPK upon transfection of 4T1 cells with CTNNA1, CTNNB1, and VCL siRNAs. Intravenous delivery of CTNNA1 siRNA with CA nanoparticles significantly reduced tumor volume in the initial phase of the study, while siRNAs targeting CTNNB1, TLN1, VCL, PXN, and ACTN1 genes significantly decreased the tumor burden at all time points. The tumor weights at the end of the treatments were also notably smaller compared to CA. This successfully demonstrates that targeting these dysregulated genes via RNAi and by using a suitable delivery vehicle such as CA could serve as a promising therapeutic treatment modality for breast cancers.

SiRNAs targeting multidrug transporter genes sensitise breast tumour to doxorubicin in a syngeneic mouse model

Chemotherapy, the commonly favoured approach to treat cancer is frequently associated with treatment failure and recurrence of disease as a result of development of multidrug resistance (MDR) with concomitant over-expression of drug efflux proteins on cancer cells. One of the most widely used drugs, doxorubicin (Dox) is a substrate of three different ATP-binding cassette (ABC) transporters, namely, ABCB1, ABCG2 and ABCC1, predominantly contributing to MDR phenotype in cancer. To silence these transporter-coding genes and thus enhance the therapeutic efficacy of Dox, pH-sensitive carbonate apatite (CA) nanoparticles (NPs) were employed as a carrier system to co-deliver siRNAs against these genes and Dox in breast cancer cells and in a syngeneic breast cancer mouse model. siRNAs and Dox were complexed with NPs by incubation at 37 °C and used to treat cancer cell lines to check cell viability and caspase-mediated signal. 4T1 cells-induced breast cancer mouse model was used for treatment with the complex to confirm their action in tumour regression. Smaller (∼200 nm) and less polydisperse NPs that were taken up more effectively by tumour tissue could enhance Dox chemosensitivity, significantly reducing the tumour size in a very low dose of Dox (0.34 mg/kg), in contrast to the limited effect observed in breast cancer cell lines. The study thus proposes that simultaneous delivery of siRNAs against transporter genes and Dox with the help of CA NPs could be a potential therapeutic intervention in effectively treating MDR breast cancer.

Carbonate apatite nanoparticles carry siRNA(s) targeting growth factor receptor genes egfr1 and erbb2 to regress mouse breast tumor

Cancer cells lose their control on cell cycle by numerous genetic and epigenetic alterations. In a tumor, these cells highly express growth factor receptors (GFRs), eliciting growth, and cell division. Among the GFRs, epidermal growth factor receptor-1 (EGFR1) (Her1/ERBB1) and epidermal growth factor receptor-2 (EGFR2) (Her2/ERBB2) from epidermal growth factor (EGF) family and insulin-like growth factor-1 receptor (IGF1R) are highly expressed on breast cancer cells, thus contributing to the aggressive growth and invasiveness, have been focused in this study. Moreover, overexpression of these receptors is related to suppression of cell death and conferring resistance against the classical drugs used to treat cancer nowadays. Therefore, silencing of these GFRs-encoding genes by using selective small interfering RNAs (siRNAs) could be a powerful approach to treat breast cancer. The inorganic pH sensitive carbonate apatite nanoparticles (NPs) were used as a nano-carrier to deliver siRNA(s) against single or multiple GFR genes in breast cancer cells as well as in a mouse model of breast carcinoma. Silencing of egfr1 and erbb2 simultaneously led to a reduction in cell viability with an increase in cell death signal in the cancer cells and regression of tumor growth in vivo.

Passive Targeting of Cyclophosphamide-Loaded Carbonate Apatite Nanoparticles to Liver Impedes Breast Tumor Growth in a Syngeneic Model.

Despite being widely used for treating cancer, chemotherapy is accompanied by numerous adverse effects as a result of systemic distribution and nonspecific interactions of the drugs with healthy tissues, eventually leading to therapeutic inefficacy and chemoresistance. Cyclophosphamide (Cyp) as one of the chemotherapeutic pro-drugs is activated in liver and used to treat breast cancer in high dose and in combination with other drugs. In an attempt to reduce the off-target effects and enhance the therapeutic efficacy, pH-sensitive carbonate apatite nanoparticles that had predominantly and size-dependently been localized in liver following intravenous administration, were employed to electrostatically immobilize Cyp and purposely deliver it to the liver for activation. Cyp-loaded particles formed by simple 30 min incubation at 37ºC of the DMEM (pH 7.4) medium containing CaCl2 and Cyp, enhanced in vitro cytotoxicity at different degrees depending on the cell types. The size of the particles could be tightly controlled by the amount of CaCl2 required to prepare the particles and thus the bio-distribution pattern inside different organs of the body. Unlike the small particles (~ 200 nm), the large size particles (~ 600 nm) which were more efficiently accumulated in liver, significantly reduced the tumor volume following intravenous injection in 4T1-induced murine breast cancer model at a very low dose (0.17 mg/Kg) of the drug initially added for complex formation, thus shedding light on the potential applications of the Cyp-loaded nano-formulations in the treatment of breast cancer.

PH-sensitive carbonate apatite nanoparticles as DNA vaccine carriers enhance humoral and cellular immunity

To demonstrate the potential of pH-sensitive carbonate apatite (CO3Ap) nanoparticles as DNA vaccine carriers to enhance vaccination efficacy, we examined the humoral and cellular immune responses of C57BL/6 mice immunized with the plasmid expression vector pCI-neo encoding the full-length soluble ovalbumin (OVA) (pCI-neo-sOVA), pCI-neo-sOVA/CO3Ap complexes, or pCI-neo/CO3Ap complexes as a control. Mice immunized with a low dose of pCI-neo-sOVA-loaded CO3Ap (10μg) produced ex vivo splenocyte proliferation after stimulation with CD8 T-cell but not CD4 T-cell epitopes and a delayed-type-hypersensitivity reaction more efficiently than mice in the other groups. Furthermore, mice receiving this immunization generated the same levels of OVA-specific antibodies and interferon (IFN)-γ secretion after CD8 T-cell and CD4 T-cell epitope challenges as those in mice treated with 100μg of free pCI-neo-sOVA, whereas mice injected with a high dose of pCI-neo-sOVA-loaded CO3Ap (100μg) or with control plasmids produced negligible levels of OVA-specific antibodies or IFN-γ. Therefore, our results showed that 10μg of pCI-neo-sOVA delivered by CO3Ap strongly elicited humoral and cellular immune responses. This study is the first to demonstrate the promising potential of CO3Ap nanoparticles for DNA vaccine delivery.

Intracellular delivery of NF-κB small interfering RNA for modulating therapeutic activities of classical anti-cancer drugs in human cervical cancer cells

Cervical cancer is the second most common cancer and fourth leading cause of cancer-related deaths among women. Advanced stage of the disease is treated with radiation therapy and chemotherapy with poor therapeutic outcome and adverse side effects. NFκB, a well-known transcription factor in the control of immunity and inflammation, has recently emerged as a key regulator of cell survival through induction of antiapoptotic genes. Many human cancers, including cervical carcinoma, constitutively express NF-κB and a blockade in expression of its subunit proteins through targeted knockdown of the gene transcripts with small interfering RNAs (siRNA) could be an attractive approach in order to sensitize the cancer cells towards the widely used anti-cancer drugs. However, the inefficiency of the naked siRNA to cross the plasma membrane and its sensitiveness to nuclease-mediated degradation are the major challenges limiting the siRNA technology in therapeutic intervention. pH-sensitive carbonate apatite has been established as an efficient nano-carrier for intracellular delivery of siRNA, due to its strong electrostatic interaction with the siRNA, the desirable size distribution of the resulting siRNA complex for effective endocytosis and the ability of the endocytosed siRNA to be released from the degradable particles and escape the endosomes, thus leading to the effective knockdown of the target gene of cyclin B1 or ABCB1. Here, we report that carbonate apatite-facilitated delivery of the siRNA targeting NF-κB1 and NF-κB2 gene transcripts in HeLa, a human cervical adenocar- cinoma cell line expressing NF-κB, led to a synergistic effect in enhancement of chemosensitivity to doxorubicin, but apparently not to cisplatin or paclitaxel.

English

in the presence of anti-cancer drugs to MCF-7 cells whereas the 3-(4, 5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide assay and Western blot were performed to assess the cell viability and detect the phosphorylation levels of mitogenactivated protein kinase and AKT1, respectively. Results Intracellular delivery of the siRNA targeting c-ROS1 gene transcript in MCF cells which constitutively express the gene, dose-dependently enhanced chemosensitivity to cisplain and paclitaxel, while demonstrating no significant enhancement in cell death with doxorubicin irrespective of its doses following intracellular delivery of the siRNAs. Discussion pH-sensitive carbonate apatite has recently been developed as an efficient device to deliver siRNA into the mammalian cells by virtue of its high affinity interaction with siRNA, the desirable size distribution of the resulting siRNA-apatite complex for effective cellular endocytosis and the ability of the internalised siRNA to escape the endosomes resulting in the effective knockdown of the target gene, such as cyclin B1 or ABCB1 transporter gene. The synergistic effect of c-ROS1 siRNA and paclitaxel on cell death as reported in this paper could be correlated with the inhibition of ERK 1/2 phosphorylation in mitogen- activated protein kinasepathway. Conclusion Thus, ROS1 has evolved as a potential target for gene knockdown in cisplatin- and paclitaxel-based chemotherapy of human breast cancer.

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.

Knockdown of PLC-gamma-2 and calmodulin 1 genes sensitizes human cervical adenocarcinoma cells to doxorubicin and paclitaxel

BackgroundRNA interference (RNAi) is a powerful approach in functional genomics to selectively silence messenger mRNA (mRNA) expression and can be employed to rapidly develop potential novel drugs against a complex disease like cancer. However, naked siRNA being anionic is unable to cross the anionic cell membrane through passive diffusion and therefore, delivery of siRNA remains a major hurdle to overcome before the potential of siRNA technology can fully be exploited in cancer. pH-sensitive carbonate apatite has recently been developed as an efficient tool to deliver siRNA into the mammalian cells by virtue of its high affinity interaction with the siRNA and the desirable size distribution of the resulting siRNA-apatite complex for effective cellular endocytosis. Moreover, internalized siRNA was found to escape from the endosomes in a time-dependent manner and efficiently silence gene expression.ResultsHere we show that carbonate apatite-mediated delivery of siRNA against PLC-gamma-2 (PLCG2) and calmodulin 1 (CALM1) genes has led to the sensitization of a human cervical cancer cell line to doxorubicin- and paclitaxel depending on the dosage of the individual drug whereas no such enhancement in cell death was observed with cisplatin irrespective of the dosage following intracellular delivery of the siRNAs.ConclusionThus, PLCG2 and CALM1 genes are two potential targets for gene knockdown in doxorubicin and paclitaxel-based chemotherapy of cervical cancer.

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

BackgroundMultidrug 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 resultsWe 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.ConclusionThe 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.

Fabrication and intracellular delivery of siRNA/carbonate apatite nano-composites for effective knockdown of cyclin B1 gene

<p>Gene therapy through intracellular delivery of a functional gene or a gene-silencing element is a promising approach to properly treat critical human diseases like cancer. The ability of synthetically designed small interfering RNA (siRNA) to effectively silence genes<em> </em>at post-transcriptional level has made them attractive options in targeted therapeutics. However, naked siRNA being unable to passively diffuse through cellular membranes, poses difficulty in fully exploiting the potential of the technology. pH-sensitive carbonate apatite has been developed as an efficient tool to deliver siRNA into the mammalian cells by virtue of its high affinity interaction with the siRNA and effective cellular endocytosis. Moreover, internalized siRNA has been found to escape from the endosomes in a time-dependent manner and effectively silenced reporter gene expression. Knockdown of cyclin B1 gene with only 10 nM of siRNA delivered by carbonate apatite has resulted in significant death of cervical cancer cells. Moreover, delivery of siRNA against cyclin B1 gene has led to the sensitization of the cancer cells to both cisplatin and doxorubicin at a particulat drug concentration. Thus, the new method of siRNA delivery is highly promising for pre-clinical and clinical cancer therapy using siRNA therapeutics.</p>

Fabrication and intracellular delivery of siRNA/carbonate apatite nano-composites for effective knockdown of cyclin B1 gene

Gene therapy through intracellular delivery of a functional gene or a gene-silencing element is a promising approach to properly treat critical human diseases like cancer. The ability of synthetically designed small interfering RNA (siRNA) to effectively silence genes at post-transcriptional level has made them attractive options in targeted therapeutics. However, naked siRNA being unable to passively diffuse through cellular membranes, poses difficulty in fully exploiting the potential of the technology. pH-sensitive carbonate apatite has been developed as an efficient tool to deliver siRNA into the mammalian cells by virtue of its high affinity interaction with the siRNA and effective cellular endocytosis. Moreover, internalized siRNA has been found to escape from the endosomes in a time-dependent manner and effectively silenced reporter gene expression. Knockdown of cyclin B1 gene with only 10 nM of siRNA delivered by carbonate apatite has resulted in significant death of cervical cancer cells. Moreover, delivery of siRNA against cyclin B1 gene has led to the sensitization of the cancer cells to both cisplatin and doxorubicin at a particulat drug concentration. Thus, the new method of siRNA delivery is highly promising for pre-clinical and clinical cancer therapy using siRNA therapeutics.

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 10 nM 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 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.