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. 2024 Nov 5;14(48):35286-35304.
doi: 10.1039/d4ra06197g. eCollection 2024 Nov 4.

Macroporous chitosan/alginate hydrogels crosslinked with genipin accumulate and retain glioblastoma cancer cells

Lauriane Parès  1 , Sahar Naasri  2 , Lisa Delattre  1 , Hélène Therriault  2 , Benoît Liberelle  3 , Gregory De Crescenzo  3 , Marc-Antoine Lauzon  4 , Nathalie Faucheux  4 , Benoit Paquette  2 , Nick Virgilio  1
Affiliations

Macroporous chitosan/alginate hydrogels crosslinked with genipin accumulate and retain glioblastoma cancer cells

Lauriane Parès et al. RSC Adv. .

Abstract

Grade IV multiforme glioblastoma (GBM) is an aggressive cancer that remains incurable due to the GBM cells invading and proliferating in the surrounding healthy tissues, even after tumor resection. A new therapeutic paradigm to treat GBM is to attract and accumulate GBM cells in a macroporous hydrogel inserted in the surgical cavity after tumor resection, followed by a targeted high dose of radiotherapy. This work presents a molding-based method to prepare macroporous hydrogels composed of sodium alginate and chitosan, homogeneously mixed in solution using sodium bicarbonate, and subsequently crosslinked with genipin and calcium chloride. The gels display a blue color, the result of chitosan crosslinking with genipin, fully interconnected pores with an average diameter of 180 μm (and tunable over a wide range), with a compression modulus of 10 kPa, close to the value of brain tissues. The gels are stable in cell culture media and keep their integrity after radiation doses comparable to current GBM treatment levels. Finally, F98 GBM cells accumulate relatively homogeneously and are retained within the gels.

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Conflict of interest statement

There are no conflicts to declare.

Figures

Fig. 1
Fig. 1. (A) Chitosan (CHI) reaction with sodium bicarbonate, (B) regeneration of primary amines when the pH decreases again, and (C) interactions with sodium alginate (SA) chains once CHI recovers its amino groups.
Fig. 2
Fig. 2. Crosslinking reaction of CHI chains by GNP in acidic (A) or alkaline (B) media.
Fig. 3
Fig. 3. Chitosan (CHI) crosslinking kinetics using genipin (GNP) monitored by UV-vis spectroscopy. (A) Absorbance as a function of time t and temperature T, at [CHI] = 0.75% w/v and [GNP] = 0.05% w/v; (B) absorbance as a function of GNP composition and reaction time, at [CHI] = 0.75% w/v and 37 °C; (C) absorbance as function of CHI concentration at [GNP] = 0.025% w/v and T = 37 °C. Three samples were tested per condition (N = 3), the dotted lines are guides for the eyes. After 22 h, the absorbance remained constant in all cases (data at longer times are not shown). Absorbance values (%) are normalized using the maximum value obtained for a given experiment. Pictures of well-plates containing CHI hydrogels at various conditions are displayed with the graphics.
Fig. 4
Fig. 4. Kinetics of sodium alginate (SA)/chitosan (CHI) solutions crosslinked using genipin (GNP) and monitored by UV-vis spectroscopy. Absorbance as a function of GNP concentration and time, for both pure CHI (0.75% w/v), and SA 1% CHI 0.75% w/v starting solutions. For all experimental points, N = 3, and dotted lines are guides for the eyes. Some of the plate wells have been filled with water to prevent gel drying. After 22 h, gels were fully formed, and data at longer times are not shown.
Fig. 5
Fig. 5. (A) Storage (G′) and loss (G′′) moduli for pure CHI gels (0.75% w/v) crosslinked with GNP, at a composition ranging from 0.0125% to 0.05% w/v, at 37 °C; (B) tan δ (=G′′/G′) as a function of time, from data in (A), compared to a solution composed of SA (1% w/v), CHI (0.75% w/v) and GNP (0.05% w/v); (C) gelation time (tgel), and storage modulus (G′) after 5 h of reaction time, and at equilibrium (at reaction completion), as a function of temperature T, for SA 1% CHI 0.75% GNP 0.05% w/v solutions; (D) gelation time (tgel), storage modulus (G′) after 5 h of reaction time, and at equilibrium at reaction completion, as a function of GNP concentration for SA 1% CHI 0.75% w/v at 37 °C.
Fig. 6
Fig. 6. Impact of SA (1% w/v) addition on the crosslinking of CHI (0.75% w/v) with GNP (0.05%) at 37 °C. (A) G′ and G′′ as a function of time, with and without the addition of SA; (B) impact of SA addition on tgel and final G′ value.
Fig. 7
Fig. 7. Cross-sections of porous PLA molds obtained by microCT for (A) 5, (B) 10, (C) 20, (D) 30, (E) 45 and (F) 60 min of quiescent annealing time (PLA: white domains; pores resulting from selective PS extraction: black domains). Note the difference between the scales of (A) and (B), compared to (C to F).
Fig. 8
Fig. 8. Extracted data from microCT analysis: PS and PLA domains sizes (dPLA and dPS), and specific interfacial area (S) between the PS and PLA phases. The domain size at a given annealing time corresponds to the average of the size distribution, with the bars representing the width of the distribution (±1 standard deviation, modeled with a Gaussian distribution). The domain size distributions are provided in Fig. S6.
Fig. 9
Fig. 9. Injected mold (A to C) and resulting porous gel (D to F), (B, E) and (C, F) are 2D and 3D microCT reconstructions, and the extracted microstructural data are presented in (G).
Fig. 10
Fig. 10. Appearance (A) and compression modulus (B) of porous hydrogels as a function of average pore size. (A) PLA molds filled with hydrogels (top) and resulting porous SA/CHI/GNP gels (bottom) after PLA extraction; (B) compression modulus as a function of pore size, for SA 1% CHI 0.75% GNP 0.05% w/v gels.
Fig. 11
Fig. 11. Evolution of compression modulus of porous gels (average pore size of 180 μm) in PBS medium: (A) effect CHI composition at constant SA and GNP contents (1% and 0.025% w/v, respectively); (B) effect of GNP composition at constant SA and CHI contents (1% and 0.75% w/v, respectively). (C) Evolution of compression modulus in DMEM medium, for 1% w/v SA, (0.5% or 0.75% w/v) CHI, 0.025% w/v GNP. Tests were realized at 37 °C, and solutions were changed after each measurement.
Fig. 12
Fig. 12. Accumulation and retention of F98 mCherry cells in hydrogels formulated with SA 1% CHI 0.75% GNP 0.05% w/v (in black) and SA 1% CHI 0.5% GNP 0.05% w/v (in grey).
Fig. 13
Fig. 13. (A) Number of cell clusters within levels L1 to L4 in hydrogels formulated with SA 1% CHI 0.5% GNP 0.05% w/v, and in SA 1% CHI 0.75% GNP 0.05% w/v; (B) mean surface of cell clusters in levels L1 to L4, in hydrogels formulated with SA 1% CHI 0.5% GNP 0.05% w/v, and in SA 1% CHI 0.75% GNP 0.05% w/v.
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