Cationic dendritic starch as a vehicle for photodynamic therapy and siRNA co-delivery

https://doi.org/10.1016/j.jphotobiol.2017.02.013Get rights and content

Highlights

  • Cationic enzymatically synthesized glycogen (cESG) charge condensed TPPS for PDT.

  • cESG-TPPS improved light-induced cell death response, compared to unconjugated TPPS.

  • cESG-TPPS mediates PDT at dosages as low as 0.16 μM TPPS in vitro.

    siRNA co-delivered with cESG-TPPS resulted in protein knockdown and efficient PDT

Abstract

Cationic enzymatically synthesized glycogen (cESG) is a naturally-derived, nano-scale carbohydrate dendrite that has shown promise as a cellular delivery vehicle owing to its flexibility in chemical modifications, biocompatibility and relative low cost. In the present work, cESG was modified and evaluated as a vehicle for tetraphenylporphinesulfonate (TPPS) in order to improve cellular delivery of this photosensitizer and investigate the feasibility of co-delivery with short interfering ribonucleic acid (siRNA). TPPS was electrostatically condensed with cESG, resulting in a sub-50 nm particle with a positive zeta potential of approximately 5 mV. When tested in normal ovarian surface epithelial and ovarian clear cell carcinoma cell culture models, encapsulation of TPPS in cESG significantly improved cell death in response to light treatment compared to free drug alone. Dosages as low as 0.16 μM TPPS resulted in cellular death upon illumination with a 4.8 J/cm2 light dosage, decreasing viability by 96%. cESG-TPPS was then further evaluated as a co-delivery system with siRNA for potential combination therapy, by charge-based condensation of an siRNA directed at reducing expression of manganese superoxide dismutase (Sod2) as a proof of principle target. Simultaneous delivery of TPPS and siRNA was achieved, reducing Sod2 protein expression to 48%, while maintaining the photodynamic properties of TPPS under light exposure and maintaining low dark toxicity. This study demonstrates the versatility of cESG as a platform for dual delivery of small molecules and oligonucleotides, and the potential for further development of this system in combination therapy applications.

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).

References (39)

  • B. Chauvin

    Plasma distribution of tetraphenylporphyrin derivatives relevant for photodynamic therapy: importance and limits of hydrophobicity

    Eur. J. Pharm. Biopharm.

    (2013)
  • S. Sortino

    Nanoparticles of cationic amphiphilic cyclodextrins entangling anionic porphyrins as carrier-sensitizer system in photodynamic cancer therapy

    Biomaterials

    (2006)
  • S. Hirohara

    Synthesis, photophysical properties and sugar-dependent in vitro photocytotoxicity of pyrrolidine-fused chlorins bearing S-glycosides

    J. Photochem. Photobiol. B

    (2009)
  • Z. Rice et al.

    Adsorption characteristics of a cationic porphyrin on nanoclay at various pH

    J. Colloid Interface Sci.

    (2009)
  • S. Xiang

    Uptake mechanisms of non-viral gene delivery

    J. Control. Release

    (2012)
  • L.P.F. Aggarwal et al.

    Effects of NaCl upon TPPS4 triplet state characteristics and singlet oxygen formation

    J. Photochem. Photobiol. A Chem.

    (2007)
  • T.J. Dougherty

    Photodynamic therapy

    Photochem. Photobiol.

    (1993)
  • R. Prosst et al.

    Photodynamic therapy for esophageal diseases: a clinical update

    Endoscopy

    (2003)
  • M. Dilkes

    Treatment of primary mucosal head and neck squamous cell carcinoma using photodynamic therapy: results after 25 treated cases

    J. Laryngol. Otol.

    (2003)
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