Cationic dendritic starch as a vehicle for photodynamic therapy and siRNA co-delivery
Graphical abstract
Introduction
Photodynamic therapy (PDT) is a treatment modality that takes advantage of the activation of photosensitizers by light. Most commonly, light exposure results in oxygen radical production which elicits desired biological effects, most notably cytotoxicity. The first porphyrin photosensitizer for clinical use, Photofrin®, was approved for the treatment of papillary early stage bladder cancer in 1993 [1]. Since then, PDT applications have expanded to include a wide variety of cancers including prostate [2], head and neck [3], [4], [5], [6], gastrointestinal [7], pancreatic [8], lung [9], and nonmelanoma skin cancer [10], [11], [12], [13], as well as non-malignant conditions such as macular degeneration [14], [15] and psoriasis [16]. One obstacle in the clinical implementation of PDT (and other cancer therapies) is chemoresistance due to genetic aberrations and adaptations as a result of expression changes that confer a survival advantage to recurrent tumor populations [17]. Applying gene therapy in conjunction with PDT opens the possibility to tune the treatment to accommodate the genetic profile of the disease and target expression of proteins that may aid in chemoresistance. For example, in mouse xenograft models, knockdown of HIF1α (hypoxia-inducible factor 1-alpha) and VEGF-A (vascular endothelial growth factor A) have proven promising gene targets for improving photodynamic efficacy in head and neck cancer [18], [19] while downregulation of the protein DJ-1 has shown promise for ovarian cancer treatment [20]. Enhancement of PDT treatment by targeting disease-specific genes has also been demonstrated in vitro for urothelial [21] and breast cancer [22], [23], [24].
In previous work, our laboratory investigated the modification of enzymatically synthesized glycogen (ESG) as a cationic delivery vector for short interfering RNA (siRNA) to decrease targeted protein expression [25]. ESG is a naturally-derived, carbohydrate dendrite synthesized using in vitro enzymatic methods, resulting in a 20–40 nm diameter nanoparticle [26]. The dendrimeric glycan is highly branched and composed of α-glucose chains, bound by α1 → 4 glycosidic bonds, with α1 → 6 branching. Quaternary ammonium groups were introduced into cationic ESG (cESG, Fig. 1) via epoxy chemistry to give a positively charged nanoparticle product with a zeta potential of about + 20 mV. Electrostatically condensed cESG-siRNA successfully decreased expression of its target protein, mitochondrial superoxide dismutase (Sod2), in an in vitro ovarian clear cell carcinoma model [25]. We have demonstrated that Sod2 maintains mitochondrial function and is important for ovarian cancer metastasis. Knock-down of this enzyme in ovarian cancer increases both accumulation of the superoxide anion in the mitochondria, and prevents superoxide conversion to H2O2, which abrogates H2O2 mediated signaling and migration [27].
The primary aim of this study was to investigate whether chemically modified ESG can be considered as a delivery platform to load therapeutic modalities beyond siRNA. To test one such application we evaluated loading of the photosensitizer tetraphenylporphinesulfonate (TPPS) into cESG for PDT. In addition, the feasibility of co-delivery with siRNA was tested. TPPS (Fig. 2) is an anionic, hydrophilic porphyrin that was first explored as a photosensitizer for cancer treatment in the 1960s, showing good tumor localization and photodynamic efficiency [28], [29]. However, in vivo studies revealed that systemic administration of TPPS induced neurotoxic effects [30]. Similar functional damage was observed with injection of Photofrin® and Levulan®, but this damage was reversible once they cleared circulation. Increased circulation time of TPPS, attributed to albumin binding, led to irreversible damage and structural changes in the peripheral nervous system [31]. Given this unwanted toxicity, TPPS formulation has, so far, not moved from the bench to the clinic. Electrostatic condensation of anionic TPPS to cargo vehicles has been demonstrated as a feasible mechanism for delivery, as observed in the retention of photobehavior of TPPS in both cationic amphiphilic cyclodextrin [32] and coiled peptides [33]. Thus we hypothesized that charge-based condensation of cESG may be a feasible strategy to encapsulate TPPS and retain functionality. cESG-mediated TPPS delivery was investigated in an ovarian cancer cell line model and improved light-induced death response and reduced dark-toxicity, compared to unconjugated TPPS. The flexibility of cESG as a potential PDT delivery vector was further demonstrated when siRNA was successfully conjugated and co-delivered with TPPS to cells in culture.
Section snippets
Materials
Enzymatically synthesized glycogen (ESG, Bioglycogen™ lot 100526) was purchased from Glico Nutrition Co. Ltd. 5, 10, 15, 20-tetrakis(4-sulfonatophenyl)-21H,23H-porphyrin (TPPS) was purchased from Frontier Scientific. McCoy's 5A media, RPMI media, and Trypsin EDTA 1 × were obtained from ATCC. Hyclone fetal bovine serum was purchased from GE Healthcare. Dulbecco's phosphate buffer saline 1 × was obtained from ThermoFisher Scientific. A 5′ fluorescein 6-FAM-labeled, previously validated [27] siRNA
TPPS Incorporation into cESG
The charge-based encapsulation of TPPS with cESG was investigated using an overnight, room temperature reaction. As TPPS is highly water soluble, dialysis was attempted to remove unbound TPPS and UV–Vis absorbance used to determine the concentration of remaining TPPS. At neutral pH, stacking of the TPPS in solution occurred. Some of these aggregates were larger than the pores of dialysis tubing, resulting in TPPS not associated with cESG to remain in the sample solution. As such, gel permeation
Discussion
In this study, we demonstrated the ability of a cationic dendritic starch nanoparticle, cESG, to bind and deliver a small molecule therapeutic (TPPS photosensitizer). We also demonstrated the ability of cESG to co-deliver siRNA for potential combination therapy. Charge-based binding of TPPS was conducted by an overnight room temperature reaction, with resultant complexes retaining a positive zeta potential. Minor changes in spectroscopic absorbance and fluorescence characteristics of TPPS were
Conflict of Interest Disclosure
The authors declare no competing financial interest.
Acknowledgments
The authors would like to acknowledge the SUNY Research Fund and NIH for funding (NIH/NCI grant R00CA143229 to NH).
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