Injectable Hydrogels for Localized Chemotherapy and Radiotherapy in Brain Tumors

Abstract

Overall survival of patients with newly diagnosed glioblastoma (GBM) remains dismal at 16 months with state-of-the-art treatment that includes surgical resection, radiation, and chemotherapy. GBM tumors are highly heterogeneous, and mechanisms for overcoming tumor resistance have not yet fully been elucidated. An injectable chitosan hydrogel capable of releasing chemotherapy (temozolomide [TMZ]) while retaining radioactive isotopes agents (iodine, [¹³¹I]) was used as a vehicle for localized radiation and chemotherapy, within the surgical cavity. Release from hydrogels loaded with TMZ or ¹³¹I was characterized in vitro and in vivo and their efficacy on tumor progression and survival on GBM tumors was also measured. The in vitro release of ¹³¹I was negligible over 42 days, whereas the TMZ was completely released over the first 48 h. ¹³¹I was completely retained in the tumor bed with negligible distribution in other tissues and that when delivered locally, the chemotherapy accumulated in the tumor at 10-fold higher concentrations than when delivered systemically. We found that the tumors were significantly decreased, and survival was improved in both treatment groups compared to the control group. Novel injectable chemo-radio-hydrogel implants may potentially improve the local control and overall outcome of aggressive, poor prognosis brain tumors.

Keywords: glioblastoma; hydrogels; injectable; localized chemotherapy; localized radiotherapy.

Figure 1:. Chemical characterization of crosslinked chitosan hydrogels

A) Chemical structure of chitosan, glutaraldehyde (GA), and crosslinking reaction showing the nucleophilic attack of the amine group of the chitosan to the positively charged aldehyde group of GA forming an imine group in the chitosan hydrogels crosslinked with GA. (ChemDraw Professional 15.1 was used for the chemical drawings). B) Physical appearance of chitosan hydrogels crosslinked with increasing GA concentrations i) 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 1%, 2.5%, and 5%; ii) quantification of crosslinking based on GA concentrations measured by the absorption of the hydrogels at 360 nm (n=3). C) FTIR spectra of crosslinking based on GA concentrations.

Figure 2:. Characterization of viscosity, injectability and swelling of crosslinked chitosan hydrogels

A) i) Design of machine used to determine the injectable properties where a constant mass is applied on top of a filled syringe and the time required to release the fractions of hydrogel from the syringe is measured. ii) The injectability of the chitosan hydrogels crosslinked with increasing GA concentrations represented as the fraction of the hydrogel in the syringe in function of the shear force*time needed for emptying the syringe (n=3), and iii) image of the injectability properties of chitosan hydrogel crosslinked with 0.4% GA as an example of the best injectability properties. B) Viscosity measurements of chitosan crosslinked hydrogels with increasing GA concentration (n=3). C) Swelling kinetics of chitosan hydrogels crosslinked with 0.4% GA in DDW (n=3).

Figure 3:. Characterization of alginate microparticles

A) Chemical structure of alginate, calcium ions (Ca²⁺), and crosslinking reaction showing the interaction of positive calcium ions (+2) with the carboxylic acid groups of two alginate chains (upper chain in black and lower chain in light gray) in the crosslinked alginate microparticles forming an egg-box structure. (ChemDraw Professional 15.1 was used for the chemical drawings). B) Representative images of the morphology of alginate particles crosslinked with CaCl₂ 0.1M using different alginate concentration: 10 mg/ml, 25 mg/ml, 50 mg/ml, 75 mg/ml, and 100 mg/ml. Scale Bar: 100μm. C) Effect of alginate concentration (10 – 100 mg/ml) in microparticles size (n=10).

Figure 4:. In vitro studies of chemo- radiotherapy

A) Release profile of fluorescence from chitosan hydrogels loaded with free FITC or BSA-FITC, and BSA-FITC encapsulated in alginate microparticles (mAlg) and incorporated in chitosan hydrogels for 42 days (n=3). B) Percent of radioactive leakage of ¹³¹I-HSA out of 75mg/ml alginate microparticles loaded inside ct hydrogels for 42 days (n=3). Note insert that shows minimal (less than 1%) radioactive leakage. C) Drug release profile from chitosan hydrogels loaded with temozolomide (TMZ) for 48 hours (n=3). D) The effect of TMZ concentrations (0 – 100 μM) of free-drug or TMZ-hydrogels for three days on the survival of D54 cells analyzed by MTT (n=3).

Figure 5:. In vivo studies of localized chemo- radiotherapy

A) Design of in vivo subcutaneous brain tumor model, in which D54 GBM cells are injected in combination with matrigel, once tumors were palpable, mice were stratified into treatment groups where Ct hydrogels were implanted on top of the tumors and mice were followed by BLI twice a week, analyzed for radiotherapy and chemotherapy release. B) Radiotherapy bio-distribution in different organs after 3 and 7 days of implantation of radio-hydrogels measured by gamma counter (counts per minute, cpm) (n=3). C) Chemotherapy cellular uptake in different organs after 18 hours of implantation of localized chemo-hydrogels loaded with doxorubicin or i.v. systemic injection of same amount of doxorubicin (5mg/kg) measured by MFI in flow cytometry (n=3).

Figure 6:. Localized chemo- or radio-therapy hydrogels improves survival by inhibiting tumor growth.

Nude mice were implanted subcutaneously with D54 cells stably expressing luciferase mixed with matrigel. After 10 days, mice were imaged with bioluminescence imaging and then stratified into three groups of ten mice each. Mice were treated with localized TMZ-hydrogels (10mg) or ¹³¹I-hydrogels (4Gy) or controlled empty hydrogel. A) Representative BLI pictures of three mice implanted with control (left), chemotherapy (middle) or radiotherapy hydrogels (right) at day 27 after implantation. B) The effect of hydrogel implantation (empty-control, TMZ, or Rx-4Gy) on tumor progression monitored for 27 days using bioluminescent imagining (BLI) and shown as the average of the luminescent signal of ten mice. C) Kaplan-Meyer survival curves following various treatments of mice bearing subcutaneous brain tumors.