Targeted nanoparticle-aptamer bioconjugates for cancer chemotherapy in vivo

Abstract

Targeted uptake of therapeutic nanoparticles in a cell-, tissue-, or disease-specific manner represents a potentially powerful technology. Using prostate cancer as a model, we report docetaxel (Dtxl)-encapsulated nanoparticles formulated with biocompatible and biodegradable poly(D,L-lactic-co-glycolic acid)-block-poly(ethylene glycol) (PLGA-b-PEG) copolymer and surface functionalized with the A10 2'-fluoropyrimidine RNA aptamers that recognize the extracellular domain of the prostate-specific membrane antigen (PSMA), a well characterized antigen expressed on the surface of prostate cancer cells. These Dtxl-encapsulated nanoparticle-aptamer bioconjugates (Dtxl-NP-Apt) bind to the PSMA protein expressed on the surface of LNCaP prostate epithelial cells and get taken up by these cells resulting in significantly enhanced in vitro cellular toxicity as compared with nontargeted nanoparticles that lack the PSMA aptamer (Dtxl-NP) (P < 0.0004). The Dtxl-NP-Apt bioconjugates also exhibit remarkable efficacy and reduced toxicity as measured by mean body weight loss (BWL) in vivo [body weight loss of 7.7 +/- 4% vs. 18 +/- 5% for Dtxl-NP-Apt vs. Dtxl-NP at nadir, respectively (mean +/- SD); n = 7]. After a single intratumoral injection of Dtxl-NP-Apt bioconjugates, complete tumor reduction was observed in five of seven LNCaP xenograft nude mice (initial tumor volume of approximately 300 mm3), and 100% of these animals survived our 109-day study. In contrast, two of seven mice in the Dtxl-NP group had complete tumor reduction with 109-day survivability of only 57%. Dtxl alone had a survivability of only 14%. Saline and nanoparticles without drug were similarly nonefficacious. This report demonstrates the potential utility of nanoparticle-aptamer bioconjugates for a therapeutic application.

Fig. 1.

Development of Dtxl-encapsulated pegylated PLGA NP-Apt bioconjugates. (A) Schematic representation of the synthesis of PLGA-PEG-COOH copolymer and strategy of encapsulation of Dtxl. We developed Dtxl-encapsulated, pegylated NPs by the nanoprecipitation method. These particles have a negative surface charge attributable to the carboxylic acid on the terminal end of the PEG. The NPs were conjugated to amine-functionalized A10 PSMA Apt by carbodiimide coupling chemistry. (B) Representative scanning electron microscopy image of resulting Dtxl-encapsulated NPs is shown. EDC, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide; NHS, N-hydroxysuccinimide.

Fig. 2.

MTT assay to determine the differential cytotoxicity of Dtxl-encapsulated NP-Apt bioconjugates (Dtxl-NP-Apt), Dtxl-encapsulated NPs lacking the A10 PSMA Apt (Dtxl-NP), control NP-Apt bioconjugates without Dtxl (NP-Apt), and control NPs without Dtxl (NP) after incubation with LNCaP prostate epithelial cells. NPs were incubated with cells for 30 min (Left) or 2 h (Right), and the cells were subsequently washed and incubated in media for a total of 72 h before assessing cell viability in each group. ∗, Significance by ANOVA at 95% confidence interval.

Fig. 3.

Comparative efficacy study in LNCaP s.c. xenograft nude mouse model of PCa. (A) PCa was induced in mice by implanting LNCaP prostate epithelial cells s.c. in the flanks of nude mice and allowing the tumors to develop to appreciable size over 21 days (≈300 mm³). The comparative efficacy study of single intratumoral injection (day 0) of (i) saline (black); (ii) pegylated PLGA NP without drug (NP, brown); (iii) emulsified Dtxl (Dtxl, green), 40 mg/kg; (iv) Dtxl-encapsulated NPs (Dtxl-NP, red), 40 mg/kg; or (v) Dtxl-encapsulated NP-Apt bioconjugates (Dtxl-NP-Apt, blue), 40 mg/kg was evaluated over 109 days and demonstrated that targeted NPs are significantly more efficacious in tumor reduction as compared with other groups. Data represent mean ± SEM of seven mice per group. ∗, Data points for the Dtxl-NP-Apt group that were statistically significant compared with all other groups by ANOVA at 95% confidence interval. (B) Representative mouse at end point for each group is shown (Left) alongside images of excised tumors (Right). For the Dtxl-NP-Apt group, which achieved complete tumor regression, the scar tissue and underlying skin at the site of injection are shown. Black arrows point to the position of the implanted tumor on each mouse. (C) Plot of outcomes for each of the treatment groups divided into four categories: complete tumor regression (blue), incomplete tumor regression (red), tumor growth (yellow), and mortality (black). Two Dtxl-NP animals experienced >20% weight loss on days 9 and 12 after dosing and were euthanized. One mouse in each of the Dtxl and saline groups was euthanized late in the study for excessive weight loss likely attributable to large tumor load. (D) The Kaplan–Meier survival curve demonstrates that 100% of the Dtxl-NP-Apt group was alive on day 109, whereas the other groups had animals reaching our study end points and were euthanized on various days throughout the study period (end points defined as tumor load of 800 mm³ or BWL >20%).

Fig. 4.

Histological staining of the excised tumors in the (i) saline, (ii) pegylated PLGA NP without drug (NP), (iii) emulsified Dtxl (Dtxl), (iv) Dtxl-encapsulated NPs (Dtxl-NP), or (v) Dtxl-encapsulated NP-Apt bioconjugates (Dtxl-NP-Apt) was evaluated by an independent pathologist. The larger images (Lower) are H&E staining of representative specimens at ×20 magnification. The smaller images (Upper) are H&E (Left) and PSMA (Right) staining of consecutive sections for each group at ×50 magnification. All specimens except those obtained from the Dtxl-NP-Apt-treated mice were positive for PSMA staining [dark brown horseradish peroxidase stain]. The Dtxl-NP-Apt staining confirmed the absence of residual tumor and presence of scar and adipose tissue.

Fig. 5.

Evaluation of treatment toxicity. (A) WBC counts at the experimental end point were within the normal range for all groups and confirmed the absence of persistent hematologic toxicity. (B) Mean BWL after dosing of mouse in each group is shown. ∗, Significant difference by ANOVA at 95% confidence interval.