Polyester Nanoparticle Encapsulation Mitigates Paclitaxel-Induced Peripheral Neuropathy

Abstract

Chemotherapy utilizing cytotoxic drugs, such as paclitaxel (PTX), is still a commonly used therapeutic approach to treat both localized and metastasized cancers. Unlike traditional regimens in which PTX is administered at the maximum tolerated dose, alternative regimens like metronomic dosing are beneficial by administering PTX more frequently and in much lower doses exploiting antiangiogenic and immunomodulatory effects. However, PTX-induced peripheral neuropathy and lack of patient compliant dosage forms of PTX are major roadblocks for the successful implementation of metronomic regimens. Because of the success of polyester nanoparticle drug delivery, we explored the potential of nanoparticle-encapsulated paclitaxel (nPTX) in alleviating peripheral neuropathy using a rat model. Rats were injected intraperitoneally with 2 mg/kg body weight of PTX or nPTX on four alternate days, and neuropathic pain and neuronal damage were characterized using behavioral assessments, histology, and immunohistochemistry. The reduction in tactile and nociceptive pressure thresholds was significantly less in nPTX-treated rats than in PTX-treated rats over a 16-day study period. Histological analysis showed that the degree of dorsal root ganglion (DRG) degeneration and reduction in motor neurons in the spinal cord was significantly lower in the nPTX group than the PTX group. Further, immunofluorescence data reveals that nPTX-treated rats had an increased density of a neuronal marker, β-tubulin-III, reduced TUNEL positive cells, and increased high molecular weight neurofilament in the spinal cord, DRG, and sciatic nerves compared with PTX-treated rats. Therefore, this work has important implications in improving risk-benefit profile of PTX, paving the way for metronomic regimens.

Keywords: Dorsal root ganglion; motor neurons; nanoparticles; necrosis; neuropathic pain; paclitaxel; sustained release.

Figure 1.

The characteristics of nPTX used in this present study. a) Table showing the formulation characteristics of different batches b) Representative dynamic light scattering (DLS) particle size distribution profile and c) Representative scanning electron micrograph of nPTX. *The difference in the entrapment numbers is not the reproducibility issue, but different volume of the suspension used in the vial for freeze drying.

Figure 2.

nPTX reduces the pain hypersensitivity of rats compared to those treated with PTX. a) Tactile threshold and b) Pressure threshold, time course of the effect of intraperitoneal injections of PTX and nPTX at 2mg/kg administered as indicated by arrows on x-axis. BL is baseline data. We used same rats in a & b experiments. We used same rats in a & b experiments. Comparisons were made between PTX vs nPTX using two-way analysis of variance followed by Tukey’s multiple comparisons test (n=8) Error bars represent the S.E. [a) day 6 ***p < 0.001; day 8 ****p < 0.0001; day 10 ****p < 0.0001; day 12 ***p < 0.001; day 14 ****p < 0.0001; day 16 ***p < 0.001. b) day 4 **p < 0.01; day 6 ***p < 0.001; day 8 ****p < 0.0001; day 10 ***p < 0.001; day 12 **p < 0.01; day 14 **p < 0.01; day 16 ***p < 0.001].

Figure 3.

nPTX prevents dorsal root ganglion (DRG) degeneration. a) H&E-stained sections were prepared from the DRG nerve at the end of the study. b) PTX group showed degeneration of neurons presenting significantly low in DRG numbers compared to nPTX group indicated with bright red arrowheads. c) PTX levels in DRG, the difference between PTX and nPTX groups is not statistically significant (n=4). Error bars represent the S.D., 8–10 images were used and analysis was performed using Student’s t-test, ***p < 0.001.

Figure 4.

nPTX increase motor neuron survival in spinal cord (SC) region. a) Representative images of ventral root horn spinal cords of PTX and nPTX treated rats. b) SC motor neuron counts significantly decreased in PTX groups compared to nPTX. Images were acquired at 10 and 40× original magnification. c) PTX levels in SC, the difference between PTX and nPTX groups is not statistically significant (n=4). The PTX levels between DRG and SC are about 100 orders of magnitude, presumably due to the deficiency in blood-nerve barrier in the former. The yellow arrows mark axons and red are oligodendrocytes. Error bars represent the S.D., 8–10 images were used and analysis was performed using Student’s t-test, ***p < 0.001.

Figure 5.

Double immunofluorescence with ß-tubulin III [TUBB3] (red) and parvalbumin [PVALB] (green) in a) spinal cord showing high TUBB3 and insignificant PVALB staining in nPTX compared to PTX group, images are acquired at 6× magnification under tile scan mode (3×3) b) DRG showing high TUBB3 and PVALB staining in nPTX compared to PTX group, yellow arrow marks myelin sheath in nPTX group that is absent in PTX c) sciatic nerve showing high TUBB3 and insignificant PVALB staining in nPTX compared to PTX group. Quantification of TUBB3, PVALB and TUBB3/PVALB colocalization in a) spinal cord b) DRG and c) sciatic nerve. Error bars represent the S.D., 8–10 images were used and analysis was performed using Student’s t test, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 5.

Double immunofluorescence with ß-tubulin III [TUBB3] (red) and parvalbumin [PVALB] (green) in a) spinal cord showing high TUBB3 and insignificant PVALB staining in nPTX compared to PTX group, images are acquired at 6× magnification under tile scan mode (3×3) b) DRG showing high TUBB3 and PVALB staining in nPTX compared to PTX group, yellow arrow marks myelin sheath in nPTX group that is absent in PTX c) sciatic nerve showing high TUBB3 and insignificant PVALB staining in nPTX compared to PTX group. Quantification of TUBB3, PVALB and TUBB3/PVALB colocalization in a) spinal cord b) DRG and c) sciatic nerve. Error bars represent the S.D., 8–10 images were used and analysis was performed using Student’s t test, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 6.

Double immunofluorescence with GFAP (red) and TUNEL (green) in a) spinal cord showing high TUNEL positive cells and low GFAP staining in PTX compared to nPTX group, images are acquired at 6× magnification under tile scan mode (3×3) b) DRG showing high TUNEL positive cells and insignificant GFAP staining in PTX compared to nPTX group e) sciatic nerve showing high TUNEL positive cells and low GFAP staining in PTX compared to nPTX group. Quantification of TUNEL positive cells and GFAP in a) spinal cord b) DRG and c) sciatic nerve. Error bars represent the S.D., 8–10 images were used and analysis was performed using Student’s t test, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 6.

Double immunofluorescence with GFAP (red) and TUNEL (green) in a) spinal cord showing high TUNEL positive cells and low GFAP staining in PTX compared to nPTX group, images are acquired at 6× magnification under tile scan mode (3×3) b) DRG showing high TUNEL positive cells and insignificant GFAP staining in PTX compared to nPTX group e) sciatic nerve showing high TUNEL positive cells and low GFAP staining in PTX compared to nPTX group. Quantification of TUNEL positive cells and GFAP in a) spinal cord b) DRG and c) sciatic nerve. Error bars represent the S.D., 8–10 images were used and analysis was performed using Student’s t test, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 7.

Double immunofluorescence with CNPase (red) and NF-H (green) in a) spinal cord showing high CNPase and low NF-H staining in PTX compared to nPTX group, images are acquired at 6× magnification under tile scan mode (3×3) b) DRG showing high CNPase and low NF-H staining in PTX compared to nPTX group c) sciatic nerve showing high CNPase and low NF-H staining in PTX compared to nPTX group. High magnification images of a1) spinal cord b1) DRG and c1) sciatic nerve. The CNPase and NF-H fluorescent intensities as well as CNPase/NF-H %colocalization for all the tissues were plotted. Error bars represent the S.D., 8–10 images were used and analysis was performed using Student’s t test, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 7.

Double immunofluorescence with CNPase (red) and NF-H (green) in a) spinal cord showing high CNPase and low NF-H staining in PTX compared to nPTX group, images are acquired at 6× magnification under tile scan mode (3×3) b) DRG showing high CNPase and low NF-H staining in PTX compared to nPTX group c) sciatic nerve showing high CNPase and low NF-H staining in PTX compared to nPTX group. High magnification images of a1) spinal cord b1) DRG and c1) sciatic nerve. The CNPase and NF-H fluorescent intensities as well as CNPase/NF-H %colocalization for all the tissues were plotted. Error bars represent the S.D., 8–10 images were used and analysis was performed using Student’s t test, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.