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. 2021 Feb 9;11(2):438.
doi: 10.3390/nano11020438.

Effect of Matrix-Modulating Enzymes on The Cellular Uptake of Magnetic Nanoparticles and on Magnetic Hyperthermia Treatment of Pancreatic Cancer Models In Vivo

Felista L Tansi  1 , Filipp Fröbel  1 , Wisdom O Maduabuchi  1 , Frank Steiniger  2 , Martin Westermann  2 , Rainer Quaas  3 , Ulf K Teichgräber  4 , Ingrid Hilger  1
Affiliations

Effect of Matrix-Modulating Enzymes on The Cellular Uptake of Magnetic Nanoparticles and on Magnetic Hyperthermia Treatment of Pancreatic Cancer Models In Vivo

Felista L Tansi et al. Nanomaterials (Basel). .

Abstract

Magnetic hyperthermia can cause localized thermal eradication of several solid cancers. However, a localized and homogenous deposition of high concentrations of magnetic nanomaterials into the tumor stroma and tumor cells is mostly required. Poorly responsive cancers such as the pancreatic adenocarcinomas are hallmarked by a rigid stroma and poor perfusion to therapeutics and nanomaterials. Hence, approaches that enhance the infiltration of magnetic nanofluids into the tumor stroma convey potentials to improve thermal tumor therapy. We studied the influence of the matrix-modulating enzymes hyaluronidase and collagenase on the uptake of magnetic nanoparticles by pancreatic cancer cells and 3D spheroids thereof, and the overall impact on magnetic heating and cell death. Furthermore, we validated the effect of hyaluronidase on magnetic hyperthermia treatment of heterotopic pancreatic cancer models in mice. Treatment of cultured cells with the enzymes caused higher uptake of magnetic nanoparticles (MNP) as compared to nontreated cells. For example, hyaluronidase caused a 28% increase in iron deposits per cell. Consequently, the thermal doses (cumulative equivalent minutes at 43 °C, CEM43) increased by 15-23% as compared to heat dose achieved for cells treated with magnetic hyperthermia without using enzymes. Likewise, heat-induced cell death increased. In in vivo studies, hyaluronidase-enhanced infiltration and distribution of the nanoparticles in the tumors resulted in moderate heating levels (CEM43 of 128 min as compared to 479 min) and a slower, but persistent decrease in tumor volumes over time after treatment, as compared to comparable treatment without hyaluronidase. The results indicate that hyaluronidase, in particular, improves the infiltration of magnetic nanoparticles into pancreatic cancer models, impacts their thermal treatment and cell depletion, and hence, will contribute immensely in the fight against pancreatic and many other adenocarcinomas.

Keywords: collagenase; hyaluronic acid; hyaluronidase; magnetic hyperthermia; magnetic nanoparticles; pancreatic cancer; tumor microenvironment.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Characterization of magnetic nanoparticles. (A) Schematic presentation of the FluidMAG/C11-D particle core composed of Magnetite-C11 and starch coating. (B) Transmission electron micrographs of the nanoparticles revealing the particle core of approximately 15 and 50 nm diameter for the FluidMAG/C11-D and RCL01 (Dex-MNP), respectively. The white arrow points at a Dex-MNP lying perpendicular to the plane. Scale bars 100 nm. (C) Characteristic properties of the nanoparticles depicting hydrodynamic diameter, zeta potential, and polydispersity indices (PDI) deduced by dynamic light scattering; the iron concentration deduced by atomic absorption spectroscopy (AAS); and the specific absorption rate (SAR) and intrinsic loss power (ILP) determined with an AC frequency f = 1.048 MHz and field amplitude Ho = 8.49 kA/m for the FluidMAG/C-11D.
Figure 2
Figure 2
Matrix-modifying enzymes cause increased cellular uptake of MNP in pancreatic cancer cells. (A) Representative light microscopic images of Prussian blue-stained MNP in pancreatic cancer cells pretreated with 40 µg/mL of the indicated hyaluronidases or 20 µg/mL of collagenase in serum-free medium for 2 h, before incubation with 10 µg Fe/mL MNP in growth media containing FCS for the indicated duration. Collagenase caused a higher rounding of the cells from the culture vessels (green arrows) than the hyaluronidases. (B) Comparative levels of iron per cell after exposure to MNP without or with pretreatment with hyaluronidase IV-S (Hya-IV-S), n = 3/S.D., p = 0.09 for MNP versus Hya-IV-S. (C) Representative light microscopic images of Prussian blue-stained 3D spheroid slices showing characteristic blue stain of MNP-based iron without or with pretreatment with 40 µg/mL of hyaluronidases or 20 µg/mL of collagenase in serum-free medium for 4 h before exposure to 50 µg Fe/mL of the MNP in complete growth media for 24 h. MNP alone shows no loosening of the spheroid stroma and MNP only on the spheroid surface. The hyaluronidases caused moderately dissociated stroma and very high MNP uptake by cells lining the spheroid surface and a few in the spheroid center (yellow arrows), whereas collagenase caused strongly dissociated stroma and deep MNP-infiltration. Scale bars: upper images 50 µm and lower images 20 µm.
Figure 3
Figure 3
Influence of matrix-modulating enzymes on magnetic hyperthermia treatment of pancreatic cancer cells. (A) Representative heat curves of cells during exposure to an alternating magnetic field (AMF). (B) Cells treated with MNP alone (MH) reveal slower increase in temperature upon exposure to an AMF and lower overall heat dose (CEM43) after 60 min than those pretreated with hyaluronidase-IS (Hya-IS/MH), hyaluronidase-IVS (Hya-IVS/MH), or collagenase (Coll/MH) before incubation with MNP n = 3/S.D. (C) Relative vitality of cells grown for 24 and 48 h after magnetic hyperthermia treatment. * p < 0.05 signifies lower cell viability (higher cell death) for Hya-IS-/MH- and Coll-/MH-treated cells as compared to MH-treated cells. ** p ≤ 0.036 signifies higher cell viability (proliferation) for 24 and 48 h collagenase (coll)-treated cells as compared to control untreated Panc-1 cells. Each bar depicts the mean of n = 4/S.D. for control cells (Ctrl) and n = 3/S.D. for the treatment groups. (D) Representative pictures of well plates showing cells monitored for colony formation for 2 weeks after magnetic hyperthermia treatment. 5 × 103 cells were seeded per well. Enzyme-/hyperthermia-treated cells show higher (p < 0.05) cell death and inability to form colonies than cells treated with MNP and hyperthermia (MH) alone.
Figure 4
Figure 4
Influence of hyaluronidase on magnetic hyperthermia treatment of pancreatic cancer models in mice. All mice received comparable concentrations (0.5 mg Fe/100 mm3 tumor volume) of intratumoral Dex-MNP. (A) Setup of mice in the alternating magnetic field (AMF). (B) Representative temperature curve of tumor and mouse body showing increase and maintenance of heat above 43 °C in the tumor when the AMF is on and the decrease when the AMF is switched off. (C) Representative thermographic images of mice treated with the first magnetic hyperthermia (MH1) and the second hyperthermia (MH2) 7 days after. (D) Mean and standard deviations of the calculated heat doses (CEM43) per treatment group (n ≥ 3/S.D.).
Figure 5
Figure 5
Representative micro-CT images of mice showing Dex-MNP deposits after intratumoral application and after hyperthermia treatment. All mice received comparable concentrations (0.5 mgFe/100 mm3 tumor volume) of intratumoral Dex-MNP, which reveal characteristic X-ray densities indicated with yellow circles. (A) Representative mouse of the 24 h group showing Dex-MNP at 24 h after application (d1) and at days 6 and 29 (d6/d29) after the first hyperthermia treatment. (B) Representative mouse of the hyaluronidase treatment group showing Dex-MNP immediately after (d0) and 24 h (d1) after injection, and at days 6 and 29 (d6/d29) after the first hyperthermia treatment. Hyaluronidase I-S (40 µg/100 mm3 tumor volume) was injected 2 h prior to Dex-MNP injection. (C) Representative mouse of the 72 h group showing Dex-MNP immediately after (d-3) and 72 h (d1) after injection, and at days 6 and 29 (d6/d29) after the first hyperthermia treatment.
Figure 6
Figure 6
Representative images showing tumor growth inhibition after magnetic hyperthermia treatment alone or combined with hyaluronidase pretreatment. (A) Representative pictures of mice showing reduction in tumor volumes over time. (B) Relative tumor volumes of the different treatment groups over time. Each bar represents the mean of tumor volumes as percentage of the starting tumor volumes. n = 4/standard error of the mean (SEM) for the 24 h and Hya/24 h groups and n = 5/SEM for the 72 h group. * p < 0.05 for tumor growth differences between 24 and 72 h groups at day 6, 20, and 30. ** p < 0.04 for tumor growth differences between Hya/24 h versus 72 h group at day 6 and for Hya/24 h versus 24 h group at day 27. (C) Photographs of residual tumors excised on the final day (d30) of treatment monitoring substantiate the size of remaining tumor volumes determined by caliper measurements.
Figure 7
Figure 7
Ultrathin transmission electron micrographs of tumor tissues. The tumor tissue from the 24 h mice group show damaged tumor stroma and many invasive cells infiltrating the muscle. Likewise, tumor tissue from the hyaluronidase-treated Hya/24 h group show many intact tumor cells but a degraded and relaxed stroma with fragmented collagen fibers (green arrows). In contrast, the regrown tumor tissue from the 72 h mice group shows compactly arranged cells and stroma (green arrow). Some of the tumor and stromal cells in all groups show vesicles containing partly intact Dex-MNP (asterisks), which is especially high in the 72 h group.
Figure 8
Figure 8
Comparative analysis of blood components of mice at day 30 after magnetic hyperthermia treatment without or with pretreatment with hyaluronidase. Each bar depicts the mean of blood components collected from n = 4 mice for the 24 h and Hya/24 h groups and n = 5 for the 72 h group and the respective standard deviations. * p < 0.05 for decreased white blood cells (WBC) and lymphocytes concentration (LYMPH#) in 72 h compared to the 24 h and Hya/24 h group and also increased platelets (PLT), platelet volume (PCT), and eosinophil concentration (EO#) in the 72 h versus Hya/24 h group. ** p ≤ 0.02 for lower percent of neutrophils (NEUT%) and higher percentage of lymphocytes (LYMPH%) in the 24 h compared to the Hya/24 h and 72 h groups. ° p < 0.01 for higher concentration and percentage of eosinophils (EO# and EO%) in the Hya/24 h compared to the 24 h group.
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