Nano-Delivery of a Novel Inhibitor of Polynucleotide Kinase/Phosphatase (PNKP) for Targeted Sensitization of Colorectal Cancer to Radiation-Induced DNA Damage

Abstract

Inhibition of the DNA repair enzyme polynucleotide kinase/phosphatase (PNKP) increases the sensitivity of cancer cells to DNA damage by ionizing radiation (IR). We have developed a novel inhibitor of PNKP, i.e., A83B4C63, as a potential radio-sensitizer for the treatment of solid tumors. Systemic delivery of A83B4C63, however, may sensitize both cancer and normal cells to DNA damaging therapeutics. Preferential delivery of A83B4C63 to solid tumors by nanoparticles (NP) was proposed to reduce potential side effects of this PNKP inhibitor to normal tissue, particularly when combined with DNA damaging therapies. Here, we investigated the radio-sensitizing activity of A83B4C63 encapsulated in NPs (NP/A83) based on methoxy poly(ethylene oxide)-b-poly(α-benzyl carboxylate-ε-caprolactone) (mPEO-b-PBCL) or solubilized with the aid of Cremophor EL: Ethanol (CE/A83) in human HCT116 colorectal cancer (CRC) models. Levels of γ-H2AX were measured and the biodistribution of CE/A83 and NP/A83 administered intravenously was determined in subcutaneous HCT116 CRC xenografts. The radio-sensitization effect of A83B4C63 was measured following fractionated tumor irradiation using an image-guided Small Animal Radiation Research Platform (SARRP), with 24 h pre-administration of CE/A83 and NP/A83 to Luc⁺/HCT116 bearing mice. Therapeutic effects were analyzed by monitoring tumor growth and functional imaging using Positron Emission Tomography (PET) and [¹⁸F]-fluoro-3'-deoxy-3'-L:-fluorothymidine ([¹⁸F]FLT) as a radiotracer for cell proliferation. The results showed an increased persistence of DNA damage in cells treated with a combination of CE/A83 or NP/A83 and IR compared to those only exposed to IR. Significantly higher tumor growth delay in mice treated with a combination of IR and NP/A83 than those treated with IR plus CE/A83 was observed. [¹⁸F]FLT PET displayed significant functional changes for tumor proliferation for the drug-loaded NP. This observation was attributed to the higher A83B4C63 levels in the tumors for NP/A83-treated mice compared to those treated with CE/A83. Overall, the results demonstrated a potential for A83B4C63-loaded NP as a novel radio-sensitizer for the treatment of CRC.

Keywords: DNA damage; DNA repair; PNKP; colorectal cancer; combination therapy; ionizing radiation; nanoparticle; radio-sensitization.

Graphical Abstract

Figure 1

Chemical structure of (A) methoxy poly(ethylene oxide)-b-poly(α-benzyl carboxylate-ε-caprolactone or mPEO-b-PBCL and (B) illustration of encapsulation process of 2-[hydroxy(2-methoxyphenyl)methyl]-6-(naphthalene-1-ylmethyl)-1-[(4-nitrophenyl)amino]-2H, 4aH, 7aH-pyrrolo[3,4-b]pyridine-5,7-dione or A83B4C63. (C) Physicochemical characterization of water-soluble CE, empty NP, A83B4C63-solubilized (CE/A83), and A83B4C63-encapsulated mPEO-b-PBCL (NP/A83) formulations (n = 10). Hydrodynamic diameter and polydispersity index (PDI) of NP/A83 micelles in aqueous medium were obtained using dynamic light scattering (DLS). (D) TEM image of A83B4C63-encapsulated micellar formulation (NP/A83) in aqueous medium. The TEM image was obtained at a magnification of 110,000X at 75 kV. The bar in the bottom left corner of the image indicates a scale of 100 nm. Data from three independent experiments were compared by two-way ANOVA multiple comparison test following Tukey’s method. (****p ≤ 0.0001). The TEM image displayed is a representative of at least three independent experiments.

Figure 2

Formation and repair of double strand breaks of DNA analyzed by γ-H2AX foci formation (H2A.X Ser139) in HCT116 cells. (A) Representative images of γ-H2AX (green) foci and nuclei (blue) were counterstained with DAPI. Inset figures show typical γ-H2AX foci in individual cells. (B) Quantitative analysis for the number of foci in each treated cell. 24 h prior to 3 Gy γ-irradiation, cells on the coverslips were treated with 10 µM CE/A83 and NP/A83. At 40 min or 6 h after irradiation, cells were fixed, permeabilized, and stained for foci to be visualized under the microscope. MetaXpress 6 software was used to take images and to quantify the number of foci in each cell. Data from three independent experiments were compared by two-way ANOVA multiple comparison test following Tukey’s method. Differences were considered significant if (**p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001). Micrographs displayed are representative of at least three independent experiments; scale bar = 40 μm.

Figure 3

(A) Schematic experimental design for evaluating the anticancer activity of A83B4C63 as CE and NP formulations in female NIH-III nude mice following IV administration (n = 6 or 7). Colorectal Luc⁺/HCT116 cells were inoculated and grown as subcutaneous tumor xenografts in the right flank of the mice. When tumors became palpable based on the tumor measurement by calipers the treatments started. The in vivo live imaging system (IVIS^®) was also used before and after treatment to follow tumor growth. A total of 25 mice were divided into 4 groups (6 + 6 + 6 + 7), which were intravenously injected with (i) control empty NPs, (ii) control empty NP plus 3 x 5 Gy IR, (iii) CE/A83 (A83B4C63 formulated with the aid of CE) plus 3 x 5 Gy IR, and (iv) NP/A83 (A83B4C63-encapsulated mPEO₁₁₄-b-PBCL₂₆ micelles) plus 3 x 5 Gy IR three times with a one day interval at a dose of 25 mg/kg. (B) Average tumor volume growth curves for mice in each treatment group for Luc⁺/HCT116 CRC xenograft. (C) The average tumor volumes obtained from the treated groups on day-12 post injection. Using digital calipers, the length (L) and width (W) of the tumor mass were measured 2 times per week and the tumor volume (TV) was calculated according to the following formula, TV = (L × W²)/2. (D) Images of representative tumors from (B). (E) The average percentage for the change in body weight of mice bearing Luc⁺/HCT116 xenografts. Differences were considered significant if (***P < 0.001).

Figure 4

(A) Representative bioluminescence images from the tumor-bearing mice on days-2 and 12 for evaluating the radio-sensitizing anticancer activity of A83B4C63 as CE and NP formulations in female NIH-III nude mice following IV administration (n = 6 or 7). 0.5 × 10⁶ colorectal Luc⁺/HCT116 cells were inoculated and grown as subcutaneous tumor xenografts in the right flank of the female athymic NIH-III nude mice. When tumors became palpable, a total of 25 mice were randomly assigned into 4 groups (6 + 6 + 6 + 7), which were intravenously injected with (i) control empty NPs, (ii) control empty NP plus 3 x 5 Gy IR, (iii) CE/A83 (A83B4C63 formulated with the aid of CE) plus 3 x 5 Gy IR, and (iv) NP/A83 (A83B4C63-encapsulated mPEO₁₁₄-b-PBCL₂₆ micelles) plus 3 x 5 Gy IR three times with a one day interval at a dose of 25 mg/kg. The mice were imaged for luciferase intensity 2 days before the treatment started. Radiation therapy was administered using an image-guided SARRP platform. (B) Quantitative analysis for the average radiance (photons per s per cm² per square) bioluminescence signal for the four treatment groups of mice on day -2 (2 days prior to start treatment) and day 12 (termination day). To show tumor growth, the tumor radiance at day -2 (two days before treatment) is subtracted from tumor radiance at day 12 from the same mouse. Differences were considered significant if *p ≤ 0.05. ns stands for not significant.

Figure 5

(A) Static [¹⁸F]FLT-PET images after 60 min post injection of female athymic NIH-III nude mice (one representative image from each treatment group) post treatment (day 10) with empty NP, CE/A83, and NP/A83 with a fractionated 3 x 5 Gy dose of radiation. The control mice received empty NP without radiation. The white arrows indicate the xenograft CRC. (B) The quantitative data for the analyzed SUV _mean values of the [¹⁸F]FLT tumor uptake. Differences were considered significant if *p ≤ 0.05, and ***p ≤ 0.001 following two-way ANOVA followed by Tukey’s method. Data are shown as mean ± SEM from n experiments.

Figure 6

(A) The experimental schedule for determining the bio-fate of A83B4C63 intravenously delivered via CE and NP formulations in CRC tumor-bearing mice. (B–N) The biodistribution profile of A83B4C63 in wild-type HCT116 CRC xenograft bearing NIH-III female nude mice (n = 3) 4, 24, and 48 h after tail vein administration of CE/A83 and NP/A83 formulations. Mice were inoculated with HCT116 CRC cells. 21 days following tumor cell inoculation, the mice received CE/A83 and NP/A83 formulations intravenously at a dose of 25 mg/kg three times with a one-day interval. The control mice received empty NPs, equivalent to the amounts used in the test groups. 4, 24, and 48 h after the last IV injection, all mice were euthanized to collect blood plasma by cardiac puncture. Then, tumors and other organs including kidney, liver, lung, heart, and spleen were collected, snap frozen in liquid nitrogen, and stored at -80°C for later use. Drug concentration was quantified using LC/MS/MS (mean ± SD). (B) A83B4C63 plasma concentration versus time curves of CE/A83 and NP/A83 formulations in HCT116 xenograft tumor-bearing mice. (C, E, G, I, K, M) represent A83B4C63 concentrations obtained from the excised tumor, kidney, liver, lung, heart, and spleen, respectively, after administration of CE/A8 and NP/A83. (D, F, H, J, L, N) represents the ratio of tissues (tumor, kidney, liver, lung, heart, and spleen, respectively) to plasma concentration of CE/A83 and NP/A83-treated xenograft mice. Differences were considered significant if *p ≤ 0.05, **p ≤ 0.01, and ****p ≤ 0.0001 following two-way ANOVA followed by Tukey’s test.