Dual-Cross-Linked Methacrylated Alginate Sub-Microspheres for Intracellular Chemotherapeutic Delivery

Abstract

Intracellular delivery vehicles comprised of methacrylated alginate (Alg-MA) were developed for the internalization and release of doxorubicin hydrochloride (DOX). Alg-MA was synthesized via an anhydrous reaction, and a mixture of Alg-MA and DOX was formed into sub-microspheres using a water/oil emulsion. Covalently crosslinked sub-microspheres were formed via exposure to green light, in order to investigate effects of crosslinking on drug release and cell internalization, compared to traditional techniques such as ultra violet (UV) light. Crosslinking was performed using light exposure alone, or in combination with ionic crosslinking using calcium chloride (CaCl2). Alg-MA sub-microsphere diameters were between 88 – 617 nm, and zeta-potentials were between −20 and −37 mV. Using human lung epithelial carcinoma cells (A549s) as a model, cellular internalization was confirmed using flow cytometry; different sub-microsphere formulations varied the efficiency of internalization, with UV-crosslinked sub-microspheres achieving the highest internalization percentages. While blank (non-loaded) Alg-MA sub-microspheres were non-cytotoxic to A549s, DOX-loaded sub-microspheres significantly reduced mitochondrial activity after five days of culture. Photo-crosslinked Alg-MA sub-microspheres may be a potential chemotherapeutic delivery system for cancer treatment.

Anticancer Therapeutic Alginate-Based Tissue Sealants for Lung Repair

“…29 Briefly, a 2% (w/v) AMA solution was prepared in DI water, and subsequently supplemented with 0.1% (w/v) DOX (Sigma). 29 Additionally, photo-initiators were added to the mixture, as listed in Section 2.3.…”

“…29 Briefly, a 2% (w/v) AMA solution was prepared in DI water, and subsequently supplemented with 0.1% (w/v) DOX (Sigma). 29 Additionally, photo-initiators were added to the mixture, as listed in Section 2.3. DOX-encapsulated hydrogel sub-microspheres were formed using a water-in-oil emulsion technique and covalently photo-crosslinked using visible green light.…”

“…Emulsion droplets were photo-crosslinked for 5 minutes using visible green light produced by a custom LED-array. 29 Sub-microspheres were washed in isopropanol and DI water, and subsequently freeze-dried for storage. Sub-microsphere size was assessed by dynamic light scattering (DLS, Zetasizer Nano, Malvern), using a dilute suspension in PBS (pH 7.4) at 37°C.…”

“…At 1, 2, 4, 6, 12, 24, 48 hours, 100 μL of supernatant were collected from each well and immediately replaced with 100 μL of fresh PBS. 29 In addition, 1 mg of sub-microspheres was suspended in 1 mL of PBS, incubated at 37°C; the solution was centrifuged, and the supernatant was analyzed. To quantify cumulative drug released at each time point, the absorbance of collected aliquots was assessed at 485 nm wavelength using a plate reader, and compared to a DOX standard curve.…”

“…photo-crosslinking) by free radical polymerization in the presence of a photo-initiator. 24, 29–32 Alginate is inherently non-adhesive; however, oxidation of the backbone structure will elicit functional groups capable of forming crosslinks with extracellular matrix proteins, such as those found on the pleural surface. 33–36 Indeed, the incorporation of functional aldehyde groups has been investigated in several hydrogel biomaterial platforms for use in tissue engineering 37–53 and bio-adhesive applications.…”