The primary limitation for the successful treatment of osteosarcoma is the development of MDR against various chemotherapeutic agents. Adaptation of cells to drug exposure involves activation of drug efflux pumps, increased cellular detoxification, and up-regulation of repair mechanisms, all of which lead to the development of multidrug resistant character in cancer cells 
. Overexpression of the ATP binding cassette transporters such as ABCB1 (MDR1) has been directly implicated in resistance to a broad spectrum of chemotherapeutic agents in vitro including anthracyclines, paclitaxel, and the Vinca alkaloids 
. For several types of cancer, ABCB1 overexpression may be the predominant factor in limiting the efficacy of chemotherapeutic agents. Overexpression of the ABCB1 gene within drug-sensitive cells or mice to produce transgenic animals confers resistance to the agents described 
The objective of this study was to investigate the ability of MDR1 siRNA loaded dextran based nanoparticles to overcome P-gp mediated drug resistance in osteosarcoma. We developed a biocompatible and safe nanoparticulate system for the delivery of MDR1 siRNA, similar to the design reported recently by us for self-assembled nanosystems for encapsulating doxorubicin 
. For this purpose, we first incubated the MDR1 siRNA with a thiol-modified dextran derivative to entrap the siRNA in the dextran network. This was followed by a sequential addition of a stearylamine modified dextran derivative (to form an interpenetrating dextran hydrogel network) and a thiolated PEG derivative to stabilize the nanoparticles. The combination of several dextran-lipid derivatives and dextran-thiol polymers and their concentrations were arrived at after several testing of the formulation for their ability to form stable nanoparticles as assessed by dynamic light scattering. Also the cytotoxicity testing of various dextran derivatives were performed to assess their safe use (Fig S1
). This selection approach resulted in the development of a method for formation of stable nanoparticles with good siRNA loading/entrapment, supported by the dynamic light scattering data on the size and charge of the nanoparticles.
We used PEG in our formulation to prepare the nanoparticles because it is well known that PEGylated liposomes and nanoparticles can impart stealth characteristics to the nanoparticles, increasing the plasma residence time, and protecting the drug payload from degradation during circulation 
. PEGlyated nanoparticles also have the ability to passively target tumor tissues in vivo by exploiting the inherent abnormalities of the leaky tumor vasculature 
. Further, tumor tissues have impaired lymphatic drainage/recovery system and greatly increased production of a number of vascular permeability mediators 
. This phenomenon is called the enhanced permeability and retention (EPR) effect 
. It has been reported that accumulation of nanoparticle bound drugs are 45–250 times higher in the site of tumors compared to other vital organs such as the liver, kidney, lung, spleen, or heart 
As a next step for development of nanoparticle delivery system for overcoming drug resistant osteosarcoma, we tested our formulation for siRNA silencing effects in vitro. Our results showed that by treating drug resistant osteosarcoma cell lines with MDR1 siRNA nanoparticles at concentrations of 30 nM or higher, the expression of P-gp was effectively suppressed in both the KHOSR2 and the U-2OSR2 cells. In addition, MDR1 siRNA loaded nanoparticles were able to suppress the expression of P-gp for a longer period of time compared to MDR1 siRNA transfected with commercially available agents (96 hr compared to 48 hr).
Another major obstacle when inhibiting P-gp is that it is not only present in malignant cells but is also an important constituent of various normal tissues such as peripheral blood cells and hemopoietic progenitors found in normal human bone marrow and the blood brain barrier 
. In these physiologically normal tissues, P-gp is important in the transport of steroids, the efflux of toxic molecules, the production of bile, and is an important component of cellular defense and protection 
. Although P-gp expression in these tissues is relatively low, it may play an important role in protecting rapidly dividing cells from toxicity after exposure to anticancer drugs 
. Consequently, treatment related morbidity and mortality and increased marrow toxicity associated with chemotherapeutics and biologicals that target P-gp limit the clinical application of these agents. To optimize the application of these agents, it is clear that a delivery system that achieves higher specificity for target tissue than use of the agents alone is necessary. Such a system can improve the therapeutic index of siRNA, increasing the potential for clinical application.
Nevertheless, measuring a reduction in total cellular P-gp content does not indicate that the MDR phenotype has been reversed as only the cell surface-bound portion of P-gp is responsible for drug efflux 
. To confirm the reversal of the MDR phenotype, the Vybrant™ multidrug resistance assay was performed. KHOSR2
cells treated with MDR1 siRNA loaded nanoparticles showed increased accumulation of the P-gp substrate calcein-AM by enhancing the uptake and/or decreasing the efflux of these compounds. There is a possibility that off-target effects of the mixed siRNAs used in this study might had some effect on our results, and further investigations using other sequences of MDR1 siRNA is currently underway to confirm the functional assay results. The actual accumulation inside cells, preservation of drug antitumor activity/stability, and subcellular trafficking are important determinants of the efficacy of an anticancer agent. For anthracyclines in MDR cells, it has been reported that in addition to a decrease in drug accumulation, the intracellular distribution of the drug is rearranged in human myeloma 
, myeloid 
, lung tumor cells 
, ovarian carcinoma cells 
, and epidermoid carcinoma cells 
. Anthracycline fluorescence is found mainly in the nucleus of sensitive cells and in the cytoplasm of cells with relatively high levels of resistance 
. The question then is whether distribution of doxorubicin will change when drug resistant cells are treated with MDR1 siRNA loaded nanoparticles. Using immunofluorescense microscopy, doxorubicin primarily accumulated in the nucleus of drug sensitive cell lines, and in the cytoplasm of MDR cell lines. When doxorubicin was applied to MDR osteosarcoma cells treated with MDR1 siRNA loaded nanoparticles, drug distribution mimicked that of sensitive cells. The observations indicate that co-treatment with the MDR1 siRNA loaded nanoparticle formulation, by inhibiting P-gp, leads to higher intracellular doxorubicin concentration which allowed drugs to accumulate in the nucleus of MDR cells, emulating the behavior of doxorubicin treatment alone in drug sensitive cells.
Finally, in the treatment of osteosarcoma patients, dose limitation poses significant problems. After treatment with nanoparticles loaded with low concentrations of MDR1 siRNA, drug resistant osteosarcoma cell lines are re-sensitized to doxorubicin as demonstrated by the results of MTT assay and by a higher intracellular accumulation of the drug in the nucleus; demonstrating an uptake and distribution pattern that is comparable to drug sensitive cells. This evaluation suggests that low dosages of doxorubicin/MDR1 siRNA therapy show promise in reducing the systemic lethal side effects which are frequently encountered in the clinical setting.
In conclusion, this report demonstrates the feasibility of using MDR1 siRNA loaded dextran nanoparticles to specifically and effectively modulate MDR. Dextran nanoparticles may be a successful platform to overcome the current delivery limitation of siRNA for the treatment of osteosarcoma.