Nanoparticle-mediated p53 gene therapy for tumor inhibition

Abstract

The p53 tumor suppressor gene is mutated in 50% of human cancers, resulting in more aggressive disease with greater resistance to chemotherapy and radiation therapy. Advances in gene therapy technologies offer a promising approach to restoring p53 function. We have developed polymeric nanoparticles (NPs), based on poly (lactic-co-glycolic acid), that provide sustained intracellular delivery of plasmid DNA, resulting in sustained gene expression without vector-associated toxicity. Our previous studies with p53 gene-loaded NPs (p53NPs) demonstrated sustained antiproliferative effects in cancer cells in vitro. The objective of this study was to evaluate the efficacy of p53NPs in vivo. Tumor xenografts in mice were established with human p53-null prostate cancer cells. Animals were treated with p53NPs by either local (intratumoral injection) or systemic (intravenous) administration. Controls included saline, p53 DNA alone, and control NPs. Mice treated with local injections of p53NPs demonstrated significant tumor inhibition and improved animal survival compared with controls. Tumor inhibition corresponded to sustained and greater p53 gene and protein expression in tumors treated with p53NPs than with p53 DNA alone. A single intravenous dose of p53NPs was successful in reducing tumor growth and improving animal survival, although not to the same extent as with local injections. Imaging studies showed that NPs accumulate in tumor tissue after intravenous injection; however, further improvement in tumor targeting efficiency of p53NPs may be needed for better outcome. In conclusion, the NP-mediated p53 gene therapy is effective in tumor growth inhibition. NPs may be developed as nonviral vectors for cancer and other genetic diseases.

Fig. 1

Tumor growth inhibition after local treatment with p53NP. a Tumor growth after direct intratumoral injection of p53NP (n = 7), p53DNA (n = 7), CNP (n = 7), and p53(−)NP (n = 6). A second dose of p53NP was administered on day 32. b Tumor volume at day 17 (first animal death) shows significant reduction in tumor volume in the p53NP group compared with p53DNA, CNP, and p53(−)NP controls. c Area under the tumor growth curve by day 17 demonstrates overall reduction in tumor growth in the p53NP group compared with controls. Statistical analysis performed using Student’s t test; *p≤0.05

Fig. 2

Animal survival after local treatment. Kaplan–Meier plot showing significant improvement in animal survival after intratumoral injection of p53NPs compared with that with controls (*p<0.05 by log-rank test)

Fig. 3

Intratumoral expression of p53. p53 transcript levels in the tumor tissues treated with p53NPs and p53DNA at 3, 14, and 30 days posttreatment

Fig. 4

p53 protein expression. Immunohistochemical analysis of p53 expression in tumors treated with p53NPs (a), p53DNA (b), and saline (c) after 14 days. Arrows indicate positive staining of tumor cell nucleus for p53. Original magnification, ×400

Fig. 5

Biodistribution of nanoparticles after intravenous delivery. a Correlation of the amount of NIR dye-loaded NPs with average signal count. b In vivo photobleaching of NIR dye loaded-NPs. c Images from one animal at various time points post-intravenous injection with NPs containing an NIR dye. The arrows indicate the location of tumor; red indicates relative NIR signal from the NPs. d The signal intensity of the NPs in the tumor (T), liver (L), and muscle (M) was determined from ROIs drawn over the area of each organ/tissue. e Quantification of NIR signal in tumor, liver, and muscle (background) over time (n = 3 animals). NIR signal accumulates in the tumor several hours after NP injection and is retained for several days

Fig. 6

Tumor growth inhibition after intravenous treatment. a Tumor growth after a single intravenous injection of p53NP (n = 7), p53DNA (n = 8), p53(−)NP (n = 5), and saline (n = 9). b Tumor volume at day 13 (first animal death, p53DNA group) shows significant reduction in tumor volume in the p53NP group compared to saline and p53(−)NP groups. c Area under the tumor growth curve by day 13 demonstrates overall reduction in tumor growth in the p53NP group compared with controls. Statistical analysis performed using Student’s t test, *p≤0.05

Fig. 7

Animal survival after intravenous treatment. Kaplan–Meier plot shows improvement in animal survival after intravenous injection with p53NPs compared with controls. Log-rank test of p53NP and respective control groups yield the following p values: p53NP vs. p53DNA, p = 0.02; p53NP vs. p53(−)NP, p = 0.0003; p53NP vs. saline, p = 0.06