Plasmonic photothermal therapy increases the tumor mass penetration of HPMA copolymers

Abstract

Effective drug delivery to tumors requires both transport through the vasculature and tumor interstitium. Previously, it was shown that gold nanorod (GNR) mediated plasmonic photothermal therapy (PPTT) is capable of increasing the overall accumulation of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers in prostate tumors. In the present study, it is demonstrated that PPTT is also capable of increasing the distribution of these conjugates in tumors. Gadolinium labeled HPMA copolymers were administered to mice bearing prostate tumors immediately before treatment of the right tumor with PPTT. The left tumor served as internal, untreated control. Magnetic resonance imaging (MRI) of both tumors showed that PPTT was capable of improving the tumor mass penetration of HPMA copolymers. Thermal enhancement of delivery, roughly 1.5-fold, to both the tumor center and periphery was observed. Confocal microscopy of fluorescently labeled copolymers corroborates these findings in that PPTT is capable of delivering more HPMA copolymers to the tumor's center and periphery. These results further demonstrate that PPTT is a useful tool to improve the delivery of polymer-drug conjugates.

Fig. 1. GNR characterization and HPMA copolymer schematic

GNRs were synthesized to be 58.6 × 15.4 nm in size (A) with an SPR peak at 800 nm (B). Scale bar, 100 nm. Two HPMA copolymers were synthesized for this study (C). The first was copolymerized with HPMA (i), DOTA to chelate Gd to provide MRI contrast (ii), and a hydrolyzed reactive carboxyl group to enable targeting in future studies using the same copolymer (iv). The other was copolymerized with HPMA (i), APMA-FITC for fluorescent imaging (iii), and a hydrolyzed reactive carboxyl group for the same reasons (iv).

Fig. 2. HPMA copolymer delivery two hours after treatment

Laser treatment of the right tumor in animals previously administered GNRs (PPTT) facilitated significant enhancement of HPMA copolymer delivery in terms of both accumulation and overall tumor distribution (A, top row). Laser alone did not cause any increased delivery (A, bottom row). A 3D surface plot provides better visualization of this effect at a single slice (B).

Fig. 3. Image analysis of HPMA copolymer delivery

Treatment of tumors with PPTT was capable of significantly enhancing the delivery of HPMA copolymers to the tumor’s center and periphery two hours after treatment (A, left). Treatment with laser alone, absence of GNRs, did not increase delivery. Representative ROIs for this analysis is also shown (A, right). A histogram of R1 values of both control and PPTT treated tumors is shown (B). This data shows the capability of PPTT to increase tumor mass distribution. *Indicates a statistically significant difference (p < 0.05) by t-test. Error bars represented as ±standard error of the mean.

Fig. 4. HPMA copolymer delivery over time

The majority of delivery occurs within the first hour of PPTT treatment.

Fig. 5. Fluorescent imaging of HPMA copolymer delivery

Two hours after treatment with PPTT, HPMA copolymer delivery (green) enhancement in the tumor’s periphery and center is observed relative to untreated controls. This effect is most pronounced in the tumor’s periphery. Blood vessel (red) density does not appear to be affected by treating with PPTT. Scale bar = 1 mm.

Fig. 6. Histology of control and PPTT treated tumors

No differences between the tumor periphery of both control and PPTT treated tumors were observed (A–B, 10x objective, scale bar = 100 μm). IHC staining of blood vessels (BV) in the periphery did not provide evidence of damage in either group (C–D, 20x objective, scale bar = 50 μm). The center of tumors treated with PPTT had evidence of cell and tissue damage most likely due to excessive heating in this region (E, 10x objective, scale bar = 100 μm). A higher resolution view of this area shows the presence of capillary blood vessels which appear viable despite surrounding damage (F, 20x objective, scale bar = 50 μm).