Research ArticleHEALTH AND MEDICINE

Drug-encapsulated carbon (DECON): A novel platform for enhanced drug delivery

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Science Advances  14 Aug 2019:
Vol. 5, no. 8, eaax0780
DOI: 10.1126/sciadv.aax0780
  • Fig. 1 Prophylactic, neutralization, and therapeutic efficacy of HPAC.

    (A) Fluorescence imaging of green fluorescent protein (GFP)–HSV-1– and GFP–HSV-2–infected human corneal epithelial cells (HCEs) or HeLa cells treated with HPAC (1 mg/ml) prophylactically. (B) GFP HSV-1 and HSV-2 viruses were neutralized with HPAC (1 mg/ml) before its application to HeLa cells, and the viral entry was measured. Blue, 4′,6-diamidino-2-phenylindole; red, phalloidin staining of actin; green, GFP virus. (C) HCEs and HeLa cells were infected with HSV-1 and HSV-2, respectively, for a period of 2 hours before the addition of mock phosphate-buffered saline (PBS) or HPAC at 1 mg/ml. Twenty-four hours after infection, fluorescence images were taken to understand the extent of viral spread in HPAC-treated samples compared to mock. Green, 17 GFP HSV-1 or HSV-2 333 GFP virus. Viral entry for prophylactic (D) and neutralization treatments (E) was quantified using a β-galactosidase reporter virus. (F) Intracellular viral load for HPAC therapeutic treatment was quantified using a plaque assay. PFU, plaque-forming units. Representative immunoblots of HSV-1 (G) or HSV-2 gB (H) protein and human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from infected HCEs or HeLa cells treated with varying concentrations of HPAC. (I) Toxicity of HPAC in vitro was evaluated by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay after the incubation of multiple concentrations of HPAC with HCEs, HeLa cells, VK2s (vaginal epithelial cells), and HFFs (human foreskin fibroblasts) for 24 hours. (J) Optical density measurements were performed by serially diluting HPAC in a 96-well plate and measuring the optical absorbance at 650 nm. Lower absorbance corresponds to a clearer solution. Data are presented as means ± SD. Significance to mock-treated cells was determined by one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparisons test (n = 3 replicates). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

  • Fig. 2 ACV release from DECON is triggered by the addition of virus.

    ACV (100 μg) dissolved in dimethyl sulfoxide (DMSO) was added to 1 ml of HPAC (1 mg/ml) and incubated overnight to estimate drug loading. (A) ACV standard curve generated by ultraviolet (UV) absorbance at 252 nm. (B) ACV release from HPAC was measured by dispersing DECON in minimum essential medium (MEM) at 1 mg/ml dilution for 7 days, and readings were taken every day to estimate the amount of ACV release from DECON. (C) DECON was dispersed in seven individual tubes containing MEM at 1 mg/ml dilution and incubated at 37°C. Each day for 7 days, a single tube was taken and centrifuged at 14,000g for 5 min. Two microliters of the supernatant was used to estimate ACV concentration via a UV spectrometer. Five hundred microliters of the supernatant was overlaid on HSV-1–infected HCEs to test the antiviral efficacy. DECON pellet was washed and dispersed in fresh 1-ml MEM before adding it to the HSV-1–infected cells. Representative fluorescence images of HSV-1–infected cells treated with the supernatant (top) or DECON pellet (bottom) on days 2, 4, and 7. Green represents 17 GFP HSV-1 virus. Scale bar (similar for all images), 100 μm. (D) Flow cytometry was conducted on the aforementioned experiment. Briefly, HCEs infected with HSV-1 17 GFP virus were overlaid with either released ACV or ACV-loaded DECON taken on either day 2, 4, or 7. Twenty-four hours after infection, cells were washed, resuspended, and fixed with 4% paraformaldehyde before they were analyzed through a BD Accuri C6 flow cytometer. Noninfected (GFP-negative) cells were used as negative control and infected-nontreated (GF-positive) cells were used as positive control. The panel on the right side indicated in green color represents the number of cells infected in each treatment group. X-axis indicates fluorescence. Cells to right of dashed line show fluorescence exceeding that of negative controls. (E) Representative immunoblots (top) and quantification (bottom) from samples treated with supernatant or DECON pellet on respective days. ***P < 0.001; ****P < 0.0001. (F) Varying concentrations of purified virus and cell debris were added to fresh DECON (1 mg/ml) to estimate whether they triggered ACV release from DECON. Samples were incubated at 37°C for 15 min before they were centrifuged, and UV absorbance readings were recorded at 252 nm on the supernatants to estimate ACV release from DECON. (G) Burst and sustained release profiles for DECON. DECON dispersed at 1 mg/ml in MEM was either sonicated, heated at 90°C for 5 min, or incubated with purified virus or cell debris. ACV release was estimated using the supernatants from these samples for a period of 24 hours.

  • Fig. 3 Prophylactic or therapeutic use of DECON protects from herpes infections in vitro.

    (A) Fluorescent images showing extent of HSV-1 infection (green) in HCEs treated with either ACV-loaded DECON, DMSO-loaded DECON, HPAC alone, mock DMSO, prophylactically added ACV, or therapeutically added ACV. Scale bar (similar for all images), 100 μm. (B) Flow cytometry conducted on the samples at 24 hpi showing the extent of cells infected with HSV-1 GFP. Cells to right of dashed line show fluorescence exceeding that of negative controls. (C) Representative immunoblots (top) and quantification (bottom) for samples showing HSV-1 gB protein in comparison with GAPDH for HSV-1–infected HCEs at 24 hpi. (D to F) Plaque assay data showing extent of virus inhibition by DECON in comparison to ACV, HPAC, and mock DMSO for HSV-2, PRV, and BHV for intracellular virus collected 24 hpi. Data are presented as means ± SD. Significance to mock-treated cells was determined by one-way ANOVA followed by Dunnett’s multiple comparisons test (n = 3 replicates).

  • Fig. 4 Alternate-day ocular dosing with DECON curbs HSV-1 in a murine model of ocular infection.

    C57BL/6 mice (four groups; n = 5) were infected with HSV-1 McKrae on day 0. ACV-loaded DECON, TFT, HPAC, or DMSO was topically administered on alternate days for a period of 11 days. (A) Stereoscope images of the ocular region taken on days 0, 7, and 14 for mice infected and treated as stated above. (B) Representative image of the DECON concentration used for this set of experiments (top) is shown. (C) Ocular washes were collected on days 2, 4, and 7 and analyzed for the presence of virus through plaque assays. (D) Kaplan-Meier survival curves for the infected and treated mice. (E) Disease scores (0 to 4; 4 being severe) taken on days 2, 4, 7, 10, 14, and 21 were scored in a blinded fashion. (F) Mice were euthanized on day 21, and their eyes were frozen in OPT (Optimal Cutting Temperature) medium for histology. Ten-micrometer sections of the eye for all the groups were taken and stained with hematoxylin and eosin stain. (G and H) Draining lymph nodes isolated from mice either mock-infected or HSV-1–infected and either mock-treated or DECON-treated were photographed and weighed. (I) Corneal sensitivity measured using a manual esthesiometer in mice (n = 5 per treatment group). Lower scores indicate loss in corneal sensitivity. (Photo credit: Tejabhiram Yadavalli, University of Illinois at Chicago)

  • Fig. 5 Topical vaginal application of DECON on alternate days is as effective as daily systemic ACV dosing.

    Naïve BALB/c mice (three groups; n = 5 per group) were primed with medroxyprogesterone before intravaginal HSV-2 infection. Starting at day 1 after infection, mice were treated with either topical DECON (alternate days) or systemic ACV (5 mg/kg; every day) by intraperitoneal (IP) injections. (A) Representative stereoscope images of the murine genital region 0 and 7 days after infection. (B) Vaginal swabs collected on days 2, 4, and 7 were overlaid on Vero cells to estimate the extent of virus production through plaque assays. (C) Disease scores were given in a blinded fashion from 0 to 4, with 4 being severe. (D) Kaplan-Meier survival curves for the infected and treated mice. (E) Draining lymph nodes for the infected and treated mice were collected on day 21, washed, photographed, and weighed (F). (Photo credit: Tejabhiram Yadavalli, University of Illinois at Chicago)

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/8/eaax0780/DC1

    Fig. S1. HPAC strongly binds to HSV-1 GFP virus.

    Fig. S2. Extracellular virus–based plaque assay for HPAC therapy.

    Fig. S3. HPAC does not cause a loss in visual acuity by blocking light.

    Fig. S4. HPAC does not elevate interferons in HCEs.

    Fig. S5. HPAC does not elevate interferons in HFFs.

    Fig. S6. DECON is effective when added 24 hours after infection.

    Fig. S7. DECON protects the murine cornea from HSV-1 infection.

    Fig. S8. Graphical abstract showing DECON protecting cells against viral infection.

    Fig. S9. Full-length blots for the Western blot shown in Fig. 1 (G and H).

    Fig. S10. Full-length blots for the Western blot shown in Fig. 3D.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. HPAC strongly binds to HSV-1 GFP virus.
    • Fig. S2. Extracellular virus–based plaque assay for HPAC therapy.
    • Fig. S3. HPAC does not cause a loss in visual acuity by blocking light.
    • Fig. S4. HPAC does not elevate interferons in HCEs.
    • Fig. S5. HPAC does not elevate interferons in HFFs.
    • Fig. S6. DECON is effective when added 24 hours after infection.
    • Fig. S7. DECON protects the murine cornea from HSV-1 infection.
    • Fig. S8. Graphical abstract showing DECON protecting cells against viral infection.
    • Fig. S9. Full-length blots for the Western blot shown in Fig. 1 (G and H).
    • Fig. S10. Full-length blots for the Western blot shown in Fig. 3D.

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