Prostate-targeted biodegradable nanoparticles loaded with androgen receptor silencing constructs eradicate xenograft tumors in mice

Abstract

Background: Prostate cancer is the major cause of cancer death in men and the androgen receptor (AR) has been shown to play a critical role in the progression of the disease. Our previous reports showed that knocking down the expression of the AR gene using a siRNA-based approach in prostate cancer cells led to apoptotic cell death and xenograft tumor eradication. In this study, we utilized a biodegradable nanoparticle to deliver the therapeutic AR shRNA construct specifically to prostate cancer cells.

Materials & methods: The biodegradable nanoparticles were fabricated using a poly(dl-lactic-co-glycolic acid) polymer and the AR shRNA constructs were loaded inside the particles. The surface of the nanoparticles were then conjugated with prostate-specific membrane antigen aptamer A10 for prostate cancer cell-specific targeting.

Results: A10-conjugation largely enhanced cellular uptake of nanoparticles in both cell culture- and xenograft-based models. The efficacy of AR shRNA encapsulated in nanoparticles on AR gene silencing was confirmed in PC-3/AR-derived xenografts in nude mice. The therapeutic property of A10-conjugated AR shRNA-loaded nanoparticles was evaluated in xenograft models with different prostate cancer cell lines: 22RV1, LAPC-4 and LNCaP. Upon two injections of the AR shRNA-loaded nanoparticles, rapid tumor regression was observed over 2 weeks. Consistent with previous reports, A10 aptamer conjugation significantly enhanced xenograft tumor regression compared with nonconjugated nanoparticles.

Discussion: These data demonstrated that tissue-specific delivery of AR shRNA using a biodegradable nanoparticle approach represents a novel therapy for life-threatening prostate cancers.

Figure 1. ARHP8 nanoparticles induce xenograft tumor eradication in nude mice

(A) Xenograft tumors were established in nude mice with 22RV1, (B) LAPC-4 or (C) LNCaP cells. Once tumors were palpable (~30–50 mm³ in volume), animals were divided into different groups to receive various nanoparticles (n = 8). Nanoparticles were injected via tail vein in a volume of 200 µl that contains 4.0 µg plasmid DNA. A second dose was delivered in 1 week. Animals were monitored for tumor growth for 3 weeks. Data shows the average value of relative tumor volume (%) at the measuring time-point compared with the initial size at the first nanoparticle injection. Error bars represent the standard error of the mean. The vertical axis is in log scale. *Significant difference compared with the control (p < 0.05, Student’s t-test). GFP: Green fluorescent protein.

Figure 2. ARHP8 nanoparticles reduces serum prostate-specific antigen levels in nude mice

Orthotopic xenograft tumors were established in nude mice with LNCaP cells by inoculating 2 × 10⁵ cells in 10 µl volume of culture media. Blood samples were collected through tail incision and serum PSA levels were monitored every week. Once serum PSA levels reached 70–90 ng/ml, animals were divided into three groups (n = 8) to receive different nanoparticles. Nanoparticles were injected via tail vein in a volume of 200 µl that contains 4.0 µg plasmid DNA. A second dose was delivered in 1 week. Animals were monitored for PSA levels for another 3 weeks. (A) Data shows the average value of serum PSA and the standard error of the mean. (B) Mouse prostate loops were harvested at sacrifice and the wet weight was recorded. (C) Total RNAs were extracted from frozen tissue samples of the prostate loops and reverse transcription-PCR analysis was conducted to assess the AR gene expression. *A significant difference compared with the control (p < 0.05, Student’s t-test). AR: Androgen receptor; GFP: Green fluorescent protein; PSA: Prostate-specific antigen.

Figure 3. Nanoparticle-aptamer bioconjugation

(A) PLGA nanoparticles were modified by covalent conjugation with a PEG spacer. The RNA aptamer A10 that recognizes the human PSMA extracellular domain was conjugated onto the PEG spacer. (B) Confirmation of aptamer conjugation onto nanoparticles. Agarose gel image of plasmid DNA and A10 aptamer extracted from different nanoparticles. GFP: Green fluorescent protein; M: Marker; PEG: Polyethylene glycol; PLGA: Poly-dl-lactic-co-glycolic acid; PSMA: Prostate-specific membrane antigen.

Figure 4. Cellular uptake of nanoparticles with or without A10 Aptamer

(A) Different amounts of nanoparticle loaded with Nile-red fluorescent dye were added to LNCaP cell culture media and 1 h later, cells were stained with blue nuclear dye Hoechst 33324. Quantitative data were summarized in panel (B) and error bars represent the standard error of the mean. (C) A total of 3 µl nanoparticles were added in cell culture for the indicated time period and the cell nuclei were visualized with Hoechst 33324 staining. (D) Tumor uptake of nanoparticles in vivo. 22RV1-derived xenografts were established in nude mice, and 150 µl nanoparticles were injected via tail vein. Xenografts were harvested at indicated time points after injection. Frozen sections were prepared and the cell nuclear DNA was stained with 4’,6-diamidino-2-phenylindole. Sections were evaluated under a fluorescent microscope. Magnification ×200. *Significant difference compared with the control (p < 0.01, student’s t-test).

Figure 5. ARHP8-loaded nanoparticle-mediated AR gene silencing

(A) LNCaP or (B) LAPC-4 cells were placed in six-well plates overnight and then different nanoparticles were added into cell culture media at a dose of 2 µg DNA/well. Cells were harvested 7 days later and the total RNAs were extracted for RT-PCR and the S18 gene was used as an internal control. Cell lysates were used in western blot assays and anti-Actin blot served as protein loading control. (C) Total RNAs from panel (A) and (B) were used for quantitative RT-PCR to assess AR gene expression. Error bars represents the standard error of the mean. *Significant difference compared with the control (p < 0.05, Student’s t-test). AR: Androgen receptor; GFP: Green fluorescent protein; RT-PCR: Reverse transcription-PCR.

Figure 6. Pilot experiment for dose determination of ARHP8 nanoparticle-mediated AR gene silencing

Xenograft tumors were established in nude mice with human prostate cancer cells PC-3/AR. Once tumors were palpable (~30–50 mm³ in volume), Nano-ARHP8 nanoparticles were injected via tail vein. A total of five different doses were used. One animal was injected with PBS as a negative control. After 1 week, animals were sacrificed and xenograft tumors, together with various major organs, were harvested for further analysis. (A) Total RNAs were extracted for reverse transcription-PCR analysis for AR mRNA and GFP mRNA expression. S18 was used as an internal control. (B) Protein extracts were prepared from xenograft tumors and probed with anti-AR antibody for AR protein levels. Actin blot served as a loading control. (C) Genomic DNA extracts from xenograft tumor and major organs of the animal were used as templates in the PCR reaction to detect GFP sequence on the ARHP8 shRNA vector for the purpose of nanoparticle distribution. AR: Androgen receptor; GFP: Green fluorescent protein; PBS: Phosphate-buffered saline.

Figure 7. Prostate-specific membrane antigen A10 conjugation increases nanoparticle retention in prostate cancer xenografts

(A) The qPCR standard curve was generated using a serially diluted ARHP8-GFP construct as a template. (B) Genomic DNA samples were extracted from xenograft ssmples derived from 22RV1 and LNCaP cells as described in Figure 1 and used for quantitative PCR analysis of GFP expression. The relative construct copy numbers in the tissue were calculated based on the molecular weight of the ARHP8-GFP plasmid construct. *A significant difference in A10-Nano-ARHP8 groups compared with the nonconjugated Nano-ARHP8 group (n = 4, p < 0.05, Student’s t-test). GFP: Green fluorescent protein; qPCR: Quantitative PCR.