Injectable in situ cross-linking hydrogels for local antifungal therapy

Abstract

Invasive fungal infections can be devastating, particularly in immunocompromised patients, and difficult to treat with systemic drugs. Furthermore, systemic administration of those medications can have severe side effects. We have developed an injectable local antifungal treatment for direct administration into existing or potential sites of fungal infection. Amphotericin B (AmB), a hydrophobic, potent, and broad-spectrum antifungal agent, was rendered water-soluble by conjugation to a dextran-aldehyde polymer. The dextran-aldehyde-AmB conjugate retained antifungal efficacy against Candida albicans. Mixing carboxymethylcellulose-hydrazide with dextran-aldehyde formed a gel that cross-linked in situ by formation of hydrazone bonds. The gel provided in vitro release of antifungal activity for 11 days, and contact with the gel killed Candida for three weeks. There was no apparent tissue toxicity in the murine peritoneum and the gel caused no adhesions. Gels produced by entrapment of a suspension of AmB in CMC-dextran without conjugation of drug to polymers did not release fungicidal activity, but did kill on contact. Injectable systems of these types, containing soluble or insoluble drug formulations, could be useful for treatment of local antifungal infections, with or without concurrent systemic therapy.

Figure 1

UV-visible absorption spectra of dextran-CHO₃₃-AmB_62.5, dextran-CHO₃₃-AmB_12.5 (normalised), AmB suspension (after centrifugation) in PBS and AmB in methanol.

Figure 2

Antifungal activity of AmB in suspension, dextran-CHO₃₃-AmB_62.5 and dextran-CHO₃₃-AmB_12.5. The lower detectable limit of candidal survival was 0.2 %. (The x-axis shows the concentration of AmB present on polymer conjugate or in suspension)

Figure 3

Antifungal activity over time of 0.1 mg/ml AmB and 1.6 mg/ml dextran-CHO₃₃-AmB_62.5. The lower detectable limit of candidal survival was 0.2 %.

Figure 4

(a) Photograph of cross-linked CMC-dextran hydrogel, (6 mm scale bar shown). (b) SEM of same, lyophilised, (20μm scale bar shown).

Figure 5

(a) UV-visible absorption spectra of dextran-CHO₇₅-AmB_62.5, dextran-CHO₆₆-AmB_62.5 and dextran-CHO₃₃-AmB_62.5 released after 144 hours from CMC-Dextran gel. (b) Release kinetics of dextran-CHO_n-AmB_62.5 from CMC-Dex gel discs into PBS at 37°C; data are means with standad devations (n = 4).

Figure 6

Petri plate assay for killing of Candida albicans. Each of the plates has a confluent lawn of cells except where the drug diffused out of the central gel disc and killed them leaving a clear zone. The gel discs consisted of CMC-dextran gels containing (a) dextran-CHO₃₃-AmB_62.5 (24 mg/ml), (b) dextran-CHO₆₆-AmB_62.5 (24 mg/ml) and (c) a suspension of AmB (1.5 mg/ml).

Figure 7

Antifungal activity of the release media from CMC-Dex gels containing AmB suspension, dextran-CHO₃₃-AmB_62.5 and dextran-CHO₆₆-AmB_62.5, each formulation containing 1.5 mg AmB/mL upon gelation of CMC-Dextran gels. The release media (YNB) was changed and tested each day and stored shaking in darkness at 150 rpm at 37°C between tests. The lower detectable limit of candidal survival was 0.2 %.

Figure 8

Anti-fungal activity of CMC-dextran gels containing AmB suspension, dextran-CHO₃₃-AmB_62.5 and dextran-CHO₆₆-AmB_62.5, each formulation containing 1.5 mg AmB/mL upon gelation of CMC-Dextran gels. The release media (YNB) was changed and tested each day and stored shaking in darkness at 150 rpm at 37°C between tests. The lower detectable limit of candidal survival was 0.2 %.

Figure 9

CMC-Dex gels 11 days after intraperitoneal injection. (a) blank, (b) dextran-CHO₆₆-AmB_62.5 and (c) dextran-CHO₃₃-AmB_62.5. Arrows indicate remaining gel mass.

Figure 10

Photomicrographs of hematoxylin-eosin stained sections of injected CMC- dextran-CHO₆₆-AmB_62.5 11 days after injection A. Inflammatory reaction to a collection of hydrogel in the peritoneal cavity (magnification 200X); most inflammation seen was less than this. B. Intact peritoneal mesothelium (the thin layer of cells overlying the abdominal wall musculature). C. Interface between gel and the outside of the bowel. H: hydrogel, I: inflammation, M: abdominal wall muscle, B: bowel, arrows: nuclei of mesothelial cells.

Scheme 1

Conjugation of AmB to oxidized dextran and the incorporation of the dextran-CHO-AmB into a CMC-Dextran gel.