Development of an Injectable Hydrogel for Histotripsy Ablation Toward Future Glioblastoma Therapy Applications.
Authors: Khan ZM, Zhang J, Gannon J, Johnson BN, Verbridge SS, Vlaisavljevich E
Glioblastoma (GBM) is the most common and malignant type of primary brain tumor. Even after surgery and chemoradiotherapy, residual GBM cells can infiltrate the healthy brain parenchyma to form secondary tumors. To mitigate GBM recurrence, we recently developed an injectable hydrogel that can be crosslinked in the resection cavity to attract, collect, and ablate residual GBM cells. We previously optimized a thiol-Michael addition hydrogel for physical, chemical, and biological compatibility with the GBM microenvironment and demonstrated CXCL12-mediated chemotaxis can attract and entrap GBM cells into this hydrogel. In this study, we synthesize hydrogels under conditions mimicking GBM resection cavities and assess feasibility of histotripsy to ablate hydrogel-encapsulated cells. The results showed the hydrogel synthesis was bio-orthogonal, not shear-thinning, and can be scaled up for injection into GBM resection mimics in vitro. Experiments also demonstrated ultrasound imaging can distinguish the synthetic hydrogel from healthy porcine brain tissue. Finally, a 500 kHz transducer applied focused ultrasound treatment to the synthetic hydrogels, with results demonstrating precise histotripsy bubble clouds could be sustained in order to uniformly ablate red blood cells encapsulated by the hydrogel for homogeneous, mechanical fractionation of the entrapped cells. Overall, this hydrogel is a promising platform for biomaterials-based GBM treatment.
Introduction
Purpose
Drug delivery WITHOUT BBB opening
Study Objective
To evaluate whether an injectable thiol-Michael hydrogel synthesized under conditions mimicking GBM resection cavities can be distinguished by ultrasound and reliably ablated with intrinsic-threshold histotripsy to mechanically destroy cells entrapped within the hydrogel.
Animal model / Human subject
Porcine (pig), strain: not specified, age: not specified, sex: not specified
Disease model
Glioblastoma (GBM)
MRI or image guidance method
Ultrasound imaging (used to distinguish the synthetic hydrogel from brain tissue and to guide focused ultrasound/histotripsy targeting)
Targeted brain region(s)
Gbm Resection Cavity
Cargo name and characteristics
Injectable synthetic thiol–Michael addition hydrogel — bio-orthogonally crosslinked polymer hydrogel (non–shear-thinning, scalable) engineered to present CXCL12 for GBM cell chemotaxis and to entrap cells (tested with encapsulated red blood cells); designed for injection into resection cavities and compatible with histotripsy (500 kHz focused ultrasound) ablation.
Route of administration
Injection into the tumor resection cavity (local intracranial injection) Intra-resection cavity injection
Outcomes and Safety
Summary of Outcomes
An injectable thiol–Michael addition PEGDA hydrogel crosslinked in situ (crosslinking ≈41 s) was bio-orthogonal, not shear‑thinning, and scalable for injection into resection‑mimic cavities; swelling (24 h in PBS) softened the gel slightly. The hydrogel was clearly distinguishable from surrounding porcine brain tissue by ultrasound imaging (appearing hypoechoic), including through a cranial implant. Focused ultrasound histotripsy (500 kHz transducer) produced confined, sustained cavitation bubble clouds inside the hydrogel with intrinsic cavitation thresholds of ~31.7 ± 1.3 MPa (non‑swelled) and ~32.5 ± 3.6 MPa (swelled) — values similar to agarose/brain controls. Swelled gels produced larger individual bubbles and higher pulse‑to‑pulse persistence of residual nuclei (correlation ~0.73 vs 0.53), but cavitation remained spatially confined and effective. Mechanical fractionation/ablation of an entrapped red‑blood‑cell layer showed rapidly advancing, well‑defined lesions that matched the bubble cloud: ~65% ablation within the first 50 pulses, ~90% by 300 pulses, and near‑complete ablation by 500 pulses at 48 MPa (0.5 Hz PRF). Overall, the hydrogel can attract/entrap GBM cells (from prior CXCL12 work), is imageable, and is permissive to precise, uniform histotripsy-mediated mechanical ablation of entrapped cells in vitro, supporting its potential as a platform for biomaterials‑based GBM recurrence control.
Duration of biological effect
Near-complete RBC ablation observed after 500 pulses at 0.5 Hz (~1000 s, ~16.7 min); ~90% ablation by 300 pulses (~600 s, ~10 min); ~65% ablation by 50 pulses (~100 s, ~1.7 min). This is probably the duration of a single FUS session, not the biological effect
Safety-related matter
Mentions include: hydrogel synthesis was bio-orthogonal, crosslinked within 41 s without introducing bubbles or clogging the nozzle (implying procedural safety); ultrasound imaging could distinguish hydrogel from brain tissue and cavitation bubble clouds were confined to the focal region with no cavitation observed outside the central cloud (indicating precision and minimal off-target cavitation); histotripsy produced sharply demarcated lesion areas that matched the bubble cloud and achieved near-complete RBC ablation (tissue/ cell ablation and mechanical fractionation observed); presence of persistent residual cavitation nuclei (cavitation memory effect) in swelled hydrogels could reduce ablation efficiency (potential adverse effect on treatment efficacy); histotripsy can release proinflammatory cytokines and damage-associated molecular patterns (DAMPs) and may elicit systemic anti-tumor immune responses (indicates tissue damage and immune activation); limitation noted that RBC phantoms may underrepresent resistance of ECM-adhered GBM cells so GBM cell ablation could require longer treatment (risk of incomplete ablation); impact of histotripsy on hydrogel stability/material toughness and potential ablation-induced structural damage was not assessed and requires future study (possible material/tissue-damage concern); cranial implant caused some ultrasound attenuation but imaging and treatment remained feasible.
Brain Region
Ultrasound Parameters
Ultrasound instrument
500 kHz transducer (no model or manufacturer specified; transducer aperture/diameter not reported)
FUS Frequency
500 kHz
FUS Intensity
Not reported; only frequency reported (500 kHz)
FUS Pressure
Not specified in the provided text 31.7+- 1.3MPa for non swelled hydrogel and 32.5+- 3.6 Mpa for swelled
FUS Mode
pulsed
Pulse duration
not specified in text
Duration of a single FUS session
Not specified in the provided text. Cavitation threshold test: 100 pulses; RBC ablation: 500 pulses
Focal Characteristics
No focal size, depth, or beam diameter reported in the text. The study used a 500 kHz transducer for focused ultrasound histotripsy and sustained precise bubble clouds, but focal dimensions, focal depth, and beam diameter were not specified. Transducer focus: 75mm; Bubble cloud axial length is 0.2-5mm
Treatment frequency
single session
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