Acid-degradable Dextran as an Image Guided siRNA Carrier for COX-2 Downregulation

Purpose: Effective in vivo delivery of siRNA to silence genes is a highly sought-after goal in the treatment of multiple diseases. Cyclooxygenase-2 (COX-2) is a major mediator of inflammation and its effective and specific downregulation has been of major interest to treat conditions ranging from auto-immune diseases to gastric inflammation and cancer. Here we developed a novel and efficient method to produce a multiple imaging reporter labeled cationic dextran nanopolymer with cleavable positive charge groups for COX-2 siRNA delivery. Methods: Small molecules containing amine groups were conjugated to the dextran scaffold through acetal bonds that were cleaved in weak acid conditions. With multiple imaging reporters located on different regions of the nanopolymer, cleavage of acetal bonds was visualized and quantified by imaging, for the first time, in cancer cells and tumors. Results: The biocompatibility of dextran and the rapid cleavage and release of amine groups minimized proinflammatory side effects and COX-2 induction observed with other siRNA carriers, to successfully achieve COX-2 downregulation in cancer cells and tumors. Imaging results confirmed that this nanoplex, consisting of the dextran nanopolymer with COX-2 siRNA, accumulated in tumors, and the amine functional groups were rapidly cleaved in cancer cells and tumors. Along with effective downregulation of COX-2, we also demonstrated, for the first time, effective downregulation of its major product prostaglandin E2 (PGE2). Conclusions: We successfully developed an efficient method to produce an acid-degradable dextran nanopolymer containing cleavable amine groups as the siRNA carrier. Because of its biocompatibility, this degradable dextran delivered COX-2 siRNA within tumors and efficiently downregulated COX-2 expression.


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Compound 3: 40.0 mg compound 2 was dissolved in 2 ml HEPES buffer (pH 8.4), then 1 mg Cy5.5 NHS ester in 100 μL DMSO was added into the dextran solution. The mixture was stirred at room temperature for 2 h. After 2 h stirring, the pH value was adjusted to 7.0 by adding 10% HCl. Then 10 µl acetic acid was added into this solution, keeping the pH between 6.0-7.0. 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) 40 mg was added to form the amide bonds between the acetic acid and the unreacted amines of dextran. This reaction prevented the unreacted amines to be involved in further modifications of the dextran carriers. The Cy 5.5 labeled dextran was purified by centrifugal filter tubes (Amicon ultra-15, 10,000 MW cutoff) for 3 times. After lyophilization, 38.7 mg cyan solid was harvested. Colorimetric assay of Cy5.5 at 670 nm indicated that there were 1.1 Cy5.5 molecules in one dextran molecule. NMR could not characterize Cy5.5 on dextran due to the very low concentration of Cy5.5.
Compound 4: 30 mg of compound 3 was dissolved in 2 ml anhydrous DMSO containing 2 g 4Å active molecular sieves. Then p-toluenesulfonic acid monohydrate (270 mg, 4 eq of compound 1) was added into this mixture. This mixture was stirred at room temperature under nitrogen atmosphere for 4 h to remove water in the solution. Meanwhile 150 mg compound 1 (2 eq of glucose unit) was dissolved in anhydrous DMSO (1 mL) with 1 g 4Å active molecular sieves, and the solution was stirred at room temperature under nitrogen atmosphere for 4 h. Then compound 1 solution was transferred into compound 3 solution containing 4Å active molecular sieves, and this mixture was stirred at 65 ºC under nitrogen atmosphere for 16 h. This reaction was stopped by adding 100 μl triethylamine, and this DMSO solution was dropped into ethyl acetate to precipitate the cyan product. The suspension was centrifuged to obtain the cyan solid precipitate, which was purified by three times re-precipitation in ethyl acetate. After drying, 55.3 mg product was obtained. This product was dissolved in 2.0 ml HEPES buffer (pH 8.2), then 100 μl DMSO containing 0.5 mg rhodamine 6G NHS ester was added to this HEPES buffer. After stirring at room temperature for 2 h, this dextran product was purified by centrifugal filter tubes (Amicon ultra-15, 10,000 MW cutoff). After repeating purification 2 more times and lyophilization, 49.

N/P value dependent stability of siRNA/compound 4 nanoplex
The N/P ratio is crucial for determining the stability and delivery efficacy of the polymer/siRNA nanoplex. Electrophoretic gel mobility shift assay was used to evaluate the N/P ratio between compound 4 and COX-2 siRNA. Compound 4 and COX-2 siRNA were mixed in PBS (pH = 7.4) buffer with N/P values of 0, 1, 5, 10, and 15. After 30 min incubation at room temperature, the resulting nanoplexes were loaded on a 5% agarose gel (1.0 μg COX-2 siRNA per well). After electrophoresis, the nucleotide was stained by ethidium bromide and UV light images were obtained. Figure S2: Electrophoretic gel mobility shift assay of stability of dextran-siRNA nanoplex.

The Stability of Encapsulated siRNA in Fresh Mouse and Human Serum
A nuclease stability assay was conducted to determine the stability of siRNA encapsulated in siRNA/compound 4 nanoplex. The nanoplex was formulated in PBS, pH 7.4 with an N/P ratio of 15 for 20 min. The nanoplex was incubated for 4 and 8 h in 70% fresh serum at 37 °C. At the end of incubation, all encapsulated siRNA was displaced from the nanoplex by adding 2% SDS and loaded to the wells (1 μg/well siRNA) containing 4% agarose gel.

Degradation of compound 4
Compound 4 was incubated in pH 5.5 buffer for 8 h, after which the pH value was changed to pH 7.4 by adding NH3·H2O. After dialysis in pH 7.4 NH3·H2O solution, partially degraded compound 4 solid was collected by lyophilization. This solid was dissolved in D2O for 1 H NMR spectroscopy, or dissolved in PBS pH7.4 buffer for gel permeation chromatography (GPC).

Determination of Cytotoxicity of Compound 4 in vitro
The concentration dependent cytotoxicities of compound 4 in MDA-MB-231 human breast cancer cells and human mammary fibroblasts after 3 days incubation were measured using an MTT assay.
As demonstrated in Figure S6, compound 4 did not show toxicity up to 3 days incubation with concentrations up to 100 g/ml.