Tumor vascular permeabilization using localized mild hyperthermia to improve macromolecule transport

Abstract

The abnormal tumor vasculature presents a major challenge to the adequate delivery of chemotherapeutics, often limiting efficacy. We developed a nanoparticle-based technique to deliver localized mild hyperthermia (MHT) used to transiently alter tumor vascular transport properties and enhance transport of macromolecules into tumor interstitium. The strategy involved administering and localizing accumulation of stealth gold nanorods (GNRs, 103 μg of GNRs/g of tumor), and irradiating tumor with a low-photon laser flux (1 W/cm(2)) to generate MHT. The treatment increased vascular permeability within 24 h after treatment, allowing enhanced transport of macromolecules up to 54 nm in size. A mathematical model is used to describe changes in tumor mass transport properties where the rate of macromolecular exchange between interstitial and vascular region (R) and maximum dye enhancement (Ymax) of 23-nm dextran dye is analytically solved. During enhanced permeability, R increased by 200% while Ymax increased by 30% relative to untreated group in pancreatic CAPAN-1 tumors. MHT treatment also enhanced transport of larger dextran dye (54 nm) as assessed by intravital microscopy, without causing occlusive cellular damage. Enhanced vascular transport was prolonged for up to 24 h after treatment, but reversible with transport parameters returning to basal levels after 36 h. This study indicates that localized mild hyperthermia treatment opens a transient time-window with which to enable and augment macromolecule transport and potentially improve therapeutic efficacy. From the clinical editor: In this study, local intra-tumor mild hyperthermia is induced using a nanoparticle-based approach utilizing stealth gold nanorods and irradiating the tumor with low-photon laser flux, resulting in locally increased vascular permeability enabling enhanced delivery of therapeutics, including macromolecules up to 54 nm in size. Similar approaches would be very helpful in addressing treatment-resistant malignancies in clinical practice.

Keywords: Intravital microscopy; Macromolecular enhancement; Mild hyperthermia; Transport barriers; Vascular permeabilization.

Figure 1

Photothermal properties of PEG-coated GNRs used to induce selective and localized hyperthermia; A) schematic representation of functionalization strategy used to make PEG-coated GNRs using 5kDa methyl-PEG-thiol through ligand exchange; B) heating curves of dilute solution GNRs (80 μg/mL) showing selective 25-degree temperature elevation, control, PBS solution; C) thermographic images showing selective heating of s.c. tumor actuated by localized GNRs heating with minimal temperature rise in control group; D) temperature profile at tumor bed during hyperthermia treatment showing elevated and sustained at ~ 42°C which corresponded well with thermographic readings.

Figure 2

Nanoparticle-assisted MHT treatment enhances on tumor perfusion and permeability. A) Graph showing relative perfusion change as function of time, where perfusion rates quickly rise 11-fold compared to initial baseline and 3-fold higher after cessation; B) Amounts of Evans blue dye in tumor homogenate 1 & 3 h after hyperthermia as quantified fluorometrically at 470/680 nm excitation/emission wavelengths, respectively. Both time points demonstrated a significant effect in dye extravasation over the unheated control (2-fold increase at 1 h and 2.5 fold increase at 3 h) with statistical significance denoted by * p <0.02, ** p< 0.004 (n = 5).

Figure 3

Enhanced extravasation of 23-nm dextran in CAPAN-1 pancreatic tumors resulting from MHT treatment as studied by IVM imaging and analyses; (A, B) time lapse IVM images showing increased dextran tumor accumulation in treatment groups 1 & 5 h post heat versus untreated groups; C) mean average fluorescent intensities with distinct R values between the treated and untreated group; D) average intensity acquired over 20 min (1 h post-heat) and fitted to a mass transport model with a higher Y_max and R for treated group; E) comparison of treatment groups (1 & 5 h post-treatment) showed similar average fluorescent intensities and transport enhancement properties with Y_max = 3816 & 3661 fluorescent units (FUs) and (R = 0.325 & 0.458 min⁻¹), respectively; F) Histological analyses confirm that MHT caused little morphological alterations to tumor microenvironment while thermal ablation caused cellular damage.

Figure 4

MHT treatment causes persistent but reversible vascular effects. CAPAN-1 tumors were heated and evaluated at different times after MHT (t = 0 – 36 h) by injecting 70kDa dextran dye and quantifying fluorescence intensity in tumor homogenate and compared to unheated controls. The effects of localized MHT were measureable immediately treatment and persisted for 24 h and but returns to basal levels after 36 h with statistical significance denoted by * for p < 0.025, ** for p < 0.04, *** for p < 0.020, and **** for p < 0.01 (n = 8 per group).

Figure 5

MHT treatment enhances macromolecule extravasation up to 24 h which abates at 36 h after treatment; A) (top panel) IVM images show enhanced dye extravasation (red) in interstitial space at 5 h after MHT and circulation of 2,000kDa dextran dye; (bottom panel) show reduced dye accumulation in unheated control with majority of dye in vascular region with little in interstitial space; FITC dextran dye was injected immediately (~ 1 min) and used as vessel tracer; B) total amount of 2,000kDa dextran in tumor homogenate after MHT treatment and quantified by spectrofluorimetrically at 561/595 nm emission/ excitation wavelengths. Statistical significance is denoted by * p < 0.046, **p < 0.004, ***p < 0.002.

Figure 6

Representative histological tumor specimens demonstrating increased extravasation of 2,000kDa dye after hyperthermia treatment and 5h of circulation compared to untreated group; Top panel) shows extravasated dye away from the vasculature into interstitium (CD31 staining showed in green) enabled by hyperthermia that caused little damage to tumor microenvironment where H&E staining revealed intact cell morphology; bottom panel) shows dye accumulated in the vasculature but hardly any in the tumor interstitium.