Gelatinase‑Responsive Nanoparticles Amplify MRI Targeting of Oral Squamous Cell Carcinoma with Omniscan

Abstract

We engineered gelatinase‑responsive nanoparticles (NPs) loaded with the clinically approved MRI contrast agent Omniscan (Omn) via a double‑emulsion technique. The resulting Omn‑NPs displayed a uniform size (~120 nm), high encapsulation efficiency (~45 %), and remained stable for at least six days. In vitro, collagenase IV triggered rapid NP disassembly and release of Omn. In a human oral squamous cell carcinoma (OSCC) xenograft model, Omn‑NPs produced a tumor‑to‑background ratio (TBR) of 2.23 ± 0.10 at 30 min, surpassing free Omn (1.48 ± 0.01 at 5 min) and extending contrast persistence to 180 min. These results demonstrate that Omn‑NPs offer superior specificity and prolonged circulation, underscoring their potential as next‑generation, tumor‑targeted MRI contrast agents.

Introduction

Oral squamous cell carcinoma (OSCC) is the most prevalent malignancy of the oral and maxillofacial region. Early, accurate imaging is crucial for individualized surgical planning and improved prognosis. MRI, free of ionizing radiation, provides high‑resolution soft‑tissue contrast and is the imaging modality of choice for head‑and‑neck cancers. However, current gadolinium (Gd) chelates lack tumor specificity; their small size leads to rapid renal clearance and limited tumor retention.

Nanoparticle (NP) platforms—particularly those based on FDA‑approved polymers such as methoxy‑poly(ethylene glycol) (mPEG) and poly(ε‑caprolactone) (PCL)—have emerged as promising carriers for imaging agents. Their hydrophilic shell prolongs circulation via the enhanced permeability and retention (EPR) effect, while biodegradable cores minimize long‑term toxicity. In prior work, we incorporated a gelatinase‑cleavable peptide (PVGLIG) between mPEG and PCL, enabling tumor‑specific release of payloads in the presence of matrix metalloproteinases (MMP‑2/9). Here, we extend this strategy to load Omn, creating a tumor‑targeted MRI contrast system that leverages both EPR and enzymatic activation.

Materials and Methods

Materials

mPEG‑NHS (Mn 5000), PVGLIG peptide, Omniscan, and collagenase IV were sourced from commercial suppliers. All reagents were used as received.

Synthesis of Omn‑Loaded Gelatinase‑Responsive NPs

mPEG‑Pep‑PCL copolymers were prepared by ring‑opening copolymerization. Omn‑NPs were fabricated by a double‑emulsion (w/o/w) solvent‑evaporation method: 10 mg of copolymer was dissolved in 1 mL dichloromethane; 0.1–0.3 mL Omn was added, followed by sonication in 3 mL 3 % PVA to form the primary emulsion, then a secondary emulsion in 5 mL 0.5 % PVA. Solvent evaporation and filtration yielded the final product. Blank‑NPs and non‑cleavable Con‑Omn‑NPs (mPEG‑PCL) were prepared identically without peptide or Omn, respectively.

Characterization

Dynamic light scattering (DLS) assessed size and polydispersity index (PDI) over 6 days. Transmission electron microscopy (TEM) examined morphology. Drug loading and encapsulation efficiency were quantified by ICP‑AES after nitric‑acid digestion. Stability was monitored at room temperature.

Enzymatic Response

Omn‑NPs and Con‑Omn‑NPs were incubated with Hank’s solution containing 0.34 mg mL⁻¹ collagenase IV at 37 °C for 24 h. Macroscopic transparency and TEM imaging evaluated NP disassembly.

In Vitro Cellular Uptake

HSC3 OSCC cells were incubated with coumarin‑6‑loaded mPEG‑Pep‑PCL NPs (12.5 µg mL⁻¹). After 0.5 and 1 h, cells were fixed, washed, and imaged by confocal microscopy to assess NP internalization.

Animal Studies

All procedures complied with NIH guidelines and were approved by the Nanjing Stomatological Hospital Ethics Committee. Female BALB/c nude mice (5–6 weeks) were inoculated subcutaneously with 1 × 10⁶ HSC3 cells. Tumors reached ~0.4–0.5 cm before imaging.

In Vivo MRI

Mice received a single tail‑vein injection of Omn‑NPs or free Omn (0.025 mmol kg⁻¹ Gd³⁺). A Bruker 7.0 T scanner acquired T1‑weighted images at baseline and 5–180 min post‑injection. Tumor‑to‑background ratios were quantified from signal intensity line scans.

IHC for MMP‑2/9

Post‑imaging, tumor and major organ tissues were fixed, paraffin‑embedded, sectioned, and stained for MMP‑2 and MMP‑9. Semi‑quantitative scoring (−, +, ++) evaluated enzyme expression.

Statistical Analysis

Data are presented as mean ± SD. Student’s t‑test determined significance (p < 0.05).

Results and Discussion

NP Characterization

DLS revealed Omn‑NPs had an average diameter of 118 ± 8 nm and a low PDI (0.12 ± 0.03), indicating narrow size distribution. Blank‑NPs were larger (≈145 nm) and exhibited higher PDI. TEM images confirmed spherical morphology and internal dark cores corresponding to encapsulated Omn.

Drug Loading

Encapsulation efficiency peaked at 45 % with 0.1 mL Omn, while 0.3 mL yielded higher loading (~60 %) but lower efficiency (~30 %). The 0.1 mL formulation was chosen for in vivo studies to balance loading and efficiency.

Enzymatic Disassembly

In the presence of collagenase IV, Omn‑NPs became turbid and TEM showed fragmentation, confirming peptide cleavage and NP disassembly. Con‑Omn‑NPs remained intact, underscoring the specificity of the gelatinase‑responsive design.

Cellular Uptake

Confocal images displayed strong green fluorescence within HSC3 cytoplasm after 1 h, indicating efficient NP internalization via endocytosis.

In Vivo MRI Performance

Omn‑NPs produced a maximum TBR of 2.23 ± 0.10 at 30 min, whereas free Omn peaked at 1.48 ± 0.01 at 5 min. The contrast persisted for 180 min with Omn‑NPs compared to 30 min for free Omn, reflecting prolonged tumor retention and reduced renal clearance. Statistical analysis confirmed significant improvements (p < 0.05).

MMP‑2/9 Expression

IHC demonstrated strong (++), extracellular matrix‑bound staining of MMP‑2/9 in tumor tissue, while normal organs exhibited negligible expression (−). This validates the tumor‑specific enzymatic trigger.

Conclusions

We have successfully fabricated gelatinase‑responsive, Omniscan‑loaded NPs that enhance MRI tumor contrast in OSCC models. The system offers superior tumor-to-background contrast, extended retention, and minimal off‑target accumulation. Its composition of biocompatible PEG, biodegradable PCL, and clinically approved Omn positions it for rapid translational potential. Future work will focus on optimizing loading efficiency and exploring therapeutic payload co‑delivery.

Availability of Data and Materials

Data supporting these findings are available from the corresponding author upon request.

Abbreviations

NPs

Nanoparticles

Omn

Omniscan

OSCC

Oral squamous cell carcinoma

MMP

Matrix metalloproteinase

Gd

Gadolinium

mPEG

Methoxy‑poly(ethylene glycol)

PCL

Poly(ε‑caprolactone)

Pep

Peptide

Omn‑NPs

Omniscan‑loaded mPEG‑Pep‑PCL NPs

Con‑Omn‑NPs

Omniscan‑loaded mPEG‑PCL NPs

MRI

Magnetic resonance imaging

EPR

Enhanced permeability and retention

DCM

Dichloromethane

PVA

Polyvinyl alcohol

DLS

Dynamic light scattering

PDI

Polydispersity index

TEM

Transmission electron microscope

ICP‑AES

Inductively coupled plasma‑atomic emission spectrometry

DMEM

Dulbecco’s modified Eagle’s medium

FBS

Fetal bovine serum

IHC

Immunohistochemistry

PBS

Phosphate‑buffered saline