Improving Liposome Delivery with Macroporous Granular Hydrogels Synthesized through Freezing‐Facilitated Secondary Crosslinking of Macromonomers

The integration of liposomes into hydrogels through direct soaking presents advantages such as facile formulation, enhanced drug stability, and controlled release kinetics. However, the feasibility of this approach is impeded by the difficulty of developing matching porous structures within hydrogels and achieving appropriate affinity with liposomes. To address these challenges, an alternative method is proposed for synthesizing macroporous hydrogels using thiolated hyaluronic acid and hyperbranched polyethylene glycol macromonomers in combination with a freezing‐induced secondary crosslinking process. This approach allows the locking of macropores formed by ice crystals during freezing and enables easy modification of the hydrogels with cholesterol groups to promote liposome association. Furthermore, these macroporous hydrogels can be crushed into granular hydrogels, which offer numerous benefits such as high loading capacity, injectability, self‐healing properties, and convenience in storage, dosing, and administration. Therefore, a liposome‐in‐hydrogel system can be formulated without any gelation or pre‐treatment, providing valuable insights for the further development of liposome‐based formulations for medical and cosmetic applications.


Introduction
Liposomes, highly adaptable structures that can be customized in terms of size, surface charge, and membrane composition to significantly impact their stability, biodistribution, and targeting capabilities, have been extensively investigated as a drug delivery system in various medical fields, including cancer therapy, [1][2][3] gene therapy, [4,5] and vaccines, [6,7] while also finding applications in cosmetics, food science, and biotechnology, [8][9][10] since their discovery in the 1960s.However, despite the advantages of liposome drug delivery, such as their ability to penetrate cell membranes and reach targets, they also have certain drawbacks, including premature drug release, reduced efficacy due to instability, rapid elimination from the body due to their small size, and potential toxicity from synthetic liposomes, highlighting the importance of carefully considering these limitations and drawbacks in the development of new liposome delivery systems.[13] It allows controlled drug release, localized drug delivery, and reduced systemic toxicity, while the hydrogel barrier protects the liposomes from degradation and clearance by the body, leading to improved drug efficacy and patient compliance.However, incorporating liposomes into hydrogels can be challenging and requires specific methods tailored to the liposome and hydrogel components.Traditional methods, such as mixing liposomes with hydrogel precursor solutions and crosslinking, can damage the liposomes during preparation or affect their mobility, thus reducing drug efficacy.Furthermore, these methods require strict control of the gelation process, limiting their practical application.Soaking liposomes into a preformed hydrogel scaffold has emerged as a promising method, but it can result in uneven drug distribution and low drug loading due to the limited accessibility and affinity of the hydrogel matrix.
To address these issues, the development of macroporous hydrogels with appropriate affinity to liposomes is crucial.However, the methods for making macroporous hydrogels, such as gas foaming, [14] freeze-drying, [15] templating, [16,17] and photolithography, [18] are complex and labor-intensive, and the resulting hydrogel backbone lacks modifiability, making it challenging to effectively load a large number of liposomes and achieve controlled release.In this study, we present a green and scalable approach for creating macroporous hydrogels by utilizing thiolated hyaluronic acid (SH-HA) and hyperbranched PEG (HB-PEGDA) macromonomers in conjunction with a freezinginduced secondary crosslinking process.This method makes use of the many reactive groups present in the macromonomers, enabling the "locking" of macropores by ice crystals during freezing and facilitating the incorporation of cholesterol groups for enhanced liposome association.In addition to improving liposome stability and controlling release kinetics, macroporous hydrogels offer additional benefits when crushed into granular form.These advantages include high loading capacity, injectability, selfhealing properties, and ease of storage, dosing, and administration.This makes it feasible to create a liposome-in-hydrogel system without the need for prior gelation or pre-treatment, which is highly encouraging for medical and cosmetic uses.

Preparation of Macroporous Hydrogels via Freezing-Induced Deep Crosslinking
A two-step process was used to create macroporous hydrogels.In the first step, HB-PEGDA and SH-HA macromonomers were copolymerized under physiological conditions, resulting in the formation of a hydrogel with a dense matrix (D-Gel).However, due to the steric hindrance of the large-sized macromonomers, some of the reactive groups, including thiol and double bonds, were not utilized after the initial crosslinking (Figure S1, Supporting Information).In the second step, the D-Gel was exposed to sub-zero temperatures, causing the formation of ice crystals and the separation of water molecules from the hydrogel matrix, which created space around the ice crystals. [19]As a result, the residual reactive groups underwent spatial rearrangement and formed a more densely cross-linked structure around the ice crystals via a subsequent Michael-type addition reaction, which is referred to as the second crosslinking.Upon thawing, a hydrogel (P-Gel) with a dense polymer wall and an interconnected, macroporous network, was formed, as demonstrated in Figure 1.
[22] The selection of SH-HA and HB-PEGDA offers several advantages, including the remarkable biocompatibility of the macromonomer system [23] and the high reactivity facilitated by the abundance of functional groups in the hyaluronic acid backbone or at the periphery of the hyperbranched PEG molecule. [24,25]This high reactivity allows for rapid gelation of the pre-gel under neutral conditions, with gel formation occurring within minutes.
The transparency of the hydrogel was observed to decrease gradually with prolonged freezing treatment time, as shown in Figure 2a, which is attributed to the formation of ice crystals that damage the hydrogel's structure, resulting in imperfections such as voids and cracks that reduce its transparency. [26,27]Additionally, the two-step crosslinking process of P-Gel may increase its crosslinking density, further compromising transparency.Furthermore, SEM analysis of the P-Gel revealed the presence of macroporous structures, with macropore sizes ranging from tens to hundreds of microns, as demonstrated in Figure 2b.It is important to consider that the microstructure of the hydrogel may have been affected by the freeze-drying method used in the SEM sample preparation.In order to gain a better understanding of how freezing treatment affects hydrogel structure, O-fluorescein was integrated into the HB-PEGDA and co-polymerized with SH-HA, enabling the direct visualization of the hydrogel backbone using confocal microscopy.The structure of the hydrogel was analyzed using 3D scanning confocal images before and after being frozen at −20 °C for 72 h (Figure 2c,d, respectively).The analysis revealed that the hydrogel initially had a dense network structure but transformed into a 3D porous structure after freezing.Additionally, the stability of the P-Gel structure was evaluated by immersing it in phosphate-buffered saline (PBS, 1X) and incubating it at 37 °C, which showed that the hydrogel structure remained locked for over 2 weeks.It can be seen that the synthesis process of P-Gel is relatively simple, involves only precursor mixing and freezing and thus can be easily scaled up, which makes it a practical option for large-scale macroporous hydrogel production.
Moreover, an inquiry was carried out to determine the source of durability in the pore structure of P-Gel.The investigation aimed to ascertain whether it was due to hydrogen bond interactions between the polymer backbones, as in polyvinyl alcohol (PVA), or the secondary cross-linking of unreacted functional groups in D-Gel.To accomplish this, acrylate HA (A-HA) and HB-PEGDA macromers were employed in an attempt to create a macroporous hydrogel using the same two-step crosslinking process (shown in the bottom panel of Figure 3a).The A-HA and HB-PEGDA macromers were easily able to form a hydrogel through photoinitiated free radical polymerization.During the subsequent freezing step, ice crystal structures were observed within the hydrogel when it was not thawed.However, upon thawing, the pore structure disappeared.Given that the molecular structure of A-HA is quite similar to SH-HA, and the only difference is that A-HA and HB-PEGDA cannot undergo secondary crosslinking due to the lack of photoinitiation conditions, it can be concluded that the stability of the microporous structure of P-Gel does not stem from hydrogen bonding or polymer crystallization but rather from the secondary crosslinking of unreacted functional groups.It is worth emphasizing that the hydrogels produced from A-HA and HB-PEGDA were initially neither transparent nor robust when exposed to freezing, in contrast to the SH-HA and HB-PEGDA systems.This may be due to an unregulated reaction rate during the free radical polymerization process, resulting in a reduction in the polymer's overall effectiveness.
Furthermore, we investigated how the freezing time and process affect the mechanical properties of P-Gel. Figure 3b displays the results of amplitude sweep experiments conducted on P-Gels fabricated using different freezing periods, presenting variations in dynamic storage modulus (G′) and loss modulus (G″) at different shear strains ().These tests spanned a broad range of strains encompassing both low and high amplitude deformation domains, providing insights into the linear viscoelastic (LVE) and non-linear viscoelastic (NLVE) behavior of the P-Gels.The consistent plateau values observed at low strain imply that the P-Gels exhibit solid-like elastic behavior within this range.Nonetheless, with an increase in the deformation amplitudes, the value of G′ declines as the hydrogel structure fractures under high strain.Additionally, we found that the linear viscoelastic (LVE) range of P-Gel decreases as the duration of the freezing process increases.This is because the Michael addition reaction, which increases crosslinking, occurs more slowly at lower temperatures.As a result, hydrogels that undergo shorter freezing times have a more flexible network of polymer chains that can stretch and deform over a wider range of strains.Conversely, longer freezing times result in higher crosslinking levels, making the network more rigid and less susceptible to deformation, ultimately leading to a smaller LVE region.
In addition, we also adjusted our freezing process by reducing the duration of the single freezing time and increasing the freezing cycles.Specifically, our new process involves freezing the P-Gel at −20 °C for 6 h per cycle, allowing the sample to equilibrate at room temperature for 1 h after each cycle.Results presented in Figure 3d,e show that when the cumulative freezing time is fixed, the modulus of P-Gel obtained through cyclic freezing is lower than that obtained through continuous freezing for short freezing times (12 h, 24 h).However, when the cumulative freezing time exceeds 72 h, the modulus of P-Gel obtained through cyclic freezing becomes significantly higher than that obtained through continuous freezing.These observations suggest that the secondary crosslinking process in cyclic freezing may not be sufficient when the cumulative freezing treatment time is short due to the presence of a thawing process in the middle.However, when the cumulative freezing treatment time is longer, both cyclic and continuous freezing methods produce sufficient secondary crosslinking.Moreover, the cyclic freezing process can continuously reset the position of polymer chain segments, potentially allowing unreacted functional groups to fully react with each other, resulting in a higher degree of crosslinking and a higher modulus of the resulting P-Gel.Therefore, it can be concluded that cyclic freezing is a more effective method for producing P-Gel with high mechanical strength.Furthermore, the influence of freezing duration on the mechanical properties of hydrogels made from A-HA and HB-PEGDA macromers was also explored.As depicted in Figure 3f,g, since a secondary crosslinking reaction could not be performed without additional photoinitiation, extending the freezing time did not significantly affect the modulus of the hydrogels.This further supports the notion that the key to producing a stable macroporous structure lies in the polymer surrounding the ice crystal template's ability to undergo a secondary crosslinking reaction.Apart from analyzing the mechanical properties, the impact of freezing time and process on the pore characteristics of the resulting P-Gel was also examined using confocal microscopy, as shown in Figure S2 (Supporting Information).The results indicate that the P-Gel created through cyclic freezing exhibits slightly smaller pore structures when compared to the hydrogel obtained through a single prolonged freezing process, even though the cumulative freezing time is the same.However, the pore features of the cyclically frozen P-Gel are more consistent with those observed in the hydrogel obtained through a single short freezing process.

Degradation and Biocompatibility of Macroporous Hydrogels
To explore the biomedical value of the P-Gel, we first investigated the effect of freezing-facilitated secondary crosslinking on the degradation behavior of the hydrogel.Specifically, we analyzed the degradation behavior of the hydrogel under hydrolysis, enzymatic degradation, and free radical degradation conditions using a weighing method.As shown in Figure S3 (Supporting Information), both D-Gel and P-Gel exhibit long-term stability in PBS buffer, maintaining their gel weight over the course of a month.This is in contrast to previously reported hyaluronic acid hydrogels, which typically degrade within couple of weeks. [28,29]These results indicate that the inclusion of HB-PEGDA significantly enhances the stability of hyaluronic acid.It is worth noting that although there were no significant changes in the mass of D-Gel during this period, it did become increasingly transparent after two weeks, suggesting that some degree of hydrolytic change had occurred in its microstructure.In contrast, when exposed to the same concentrations of hyaluronidase or free radicals, both D-Gel and P-Gel degraded faster (Figure 4a,b).
However, in both cases, P-Gel exhibited significantly enhanced stability compared to D-Gel (Figure S4, Supporting Information).
To ensure the safety of P-Gel for biomedical use, we conducted a thorough investigation into its cytotoxicity using the MTT assay, as shown in Figure 4c.According to ISO 10993-5 guidelines, a medical material's 100% extract that causes less than a 30% reduction in cell viability should be considered non-toxic.To evaluate the cytotoxicity, we used the leaching solution of 1 mg mL −1 freeze-dried P-Gel.The results showed that after 24 h of incubation with the 100% extracts, there was only a slight reduction in L929 and HeLa viability to ≈85%.Moreover, cells exposed to extracts below 50% showed ≈100% viability throughout the entire experiment.We also confirmed the extracts' non-toxicity to L929 and HeLa cell lines using live/dead staining (Figure 4d).The cells exposed to extracts exhibited normal morphology, were well spread, and displayed only green fluorescence, indicating high viability.

Liposome Loading and Drug Release Kinetics of Macroporous Hydrogels
In order to demonstrate the versatility of P-Gel as a delivery system for bioactive substances, P-Gel was utilized as carriers for drug-loaded liposome delivery, as depicted in Figure 5a.Thiolated cholesterol was incorporated into the hydrogel preparation process to produce cholesterol-functionalized P-Gel.To increase its surface area and drug loading capacity, the modified P-Gel was crushed through a defined micrometer-sized stainless-steel mesh to create hydrogel microparticles (HMPs).The resulting HMPs were then immersed in dispersions of liposomes loaded with glabridin and concentrated through centrifugation to generate liposome encapsulated granular hydrogel composites.In this process, the HMPs of P-Gel acted like water-absorbing microsponges, effectively adsorbing the liposomes in water, as shown in Figure 5b.The liposome adsorption capacity of P-Gel with porous features was significantly higher compared to D-Gel.Furthermore, cholesterol-modified P-Gel exhibited a significantly increased adsorption capacity compared to unmodified P-Gel, and the adsorption capacity was positively correlated with cholesterol content due to hydrophobic interactions between cholesterol and the liposome bilayer.The hydrogel composites incorporating liposomes demonstrated controlled drug release of the hydrophobic glabridin, as depicted in Figure 5c.Notably, the porous P-Gel exhibited a more efficient drug release from the liposomes.This phenomenon can be attributed to the larger pores present in the hydrogel, which facilitate enhanced diffusion of drugs and consequently lead to a more effective release mechanism.Thus, these results highlight the potential of P-Gel as a versatile and effective delivery system.Further, to investigate whether the drug glabridin or the glabridin loaded liposomes were being released, an additional experiment was conducted using two different release media: deionized water and PBS buffer containing 1% Tween-80.After a 3-h incubation period, the release media were analyzed using dynamic light scattering (DLS).The results showed that when liposome-loaded P-Gel was exposed to a pure water release medium, the liposome particle sizes matched the initial size of the added liposomes.However, when using PBS buffer containing 1% Tween-80 as the release medium, the presence of Tween-80 caused the DLS results to exhibit a multi-peak pattern, indicating the presence of different-sized nanoparticles (data not shown).Furthermore, HPLC analysis was employed to measure the drug concentration in the release media.Glabridin was not detected in the pure water release medium, indicating its extremely low concentration.In contrast, in the release medium of PBS buffer containing 1% Tween-80, the drug concentration aligned with the results mentioned in Figure 5c.These findings suggest that the liposomes remained stably adsorbed to the P-Gel and did not detach even after prolonged immersion.The hydrophobic drug encapsulated within the liposomes gradually diffused into the release medium due to concentration gradients.However, this release process required addition of surfactants such as Tween-80 or SDS to ensure sink conditions were met.
Additionally, the granular hydrogel-based delivery platform we describe here exhibits shear-thinning behavior, enabling injection through small diameter needles (inset of Figure 5d).Shearthinning behavior was measured by applying steady shear flow sweeps from low to high shear rates, and a 3-order of magnitude decrease in viscosity was observed with increasing shear rates across the range tested (Figure 5d).The granular hydrogel has physical jamming interactions between HMPs that are disrupted when it is injected and sheared through a needle.However, after injection, the jamming interactions rapidly reform, leading to complete self-healing behavior.This makes it an ideal material for delivery applications where facile delivery through injection and rapid formation of a strong and resilient material are desirable.In specific, the self-healing behavior was further measured by imposing alternating intervals of high and low strains, resulting in a quick drop in G′ when high strains were applied, followed by complete and rapid recovery when low strains were applied (Figure 5e).G′ dropped quickly once high strains were applied, and rapidly (<5 s) recovered once low strains were applied.Furthermore, the G′ value is similar to that of human soft tissues (Figure S5, Supporting Information), making it suitable for use as a carrier for various drugs or active ingredients in medical or cosmetic applications, and as scaffolds for tissue regeneration.

Conclusion
This study presents a green and scalable approach to produce macroporous hydrogels using SH-HA and HB-PEGDA macromonomers via a freezing-induced secondary crosslinking process.The hydrogels can be easily customized by incorporating thiolated biopolymers during the hydrogel formation process, allowing tailored properties for specific applications.The abundance of reactive groups in the macromonomers stabilizes the macropores formed by ice crystals during freezing, ensuring pore structure integrity during thawing.The modulus of the hydrogels can be controlled by adjusting freezing time and process.The resulting hydrogels exhibit exceptional biocompatibility, large surface area, and high drug loading capacity.Notably, the production process is gentle, making it potentially compatible with a range of biologically active substances.In summary, the customizable nature of these macroporous SH-HA/HB-PEGDA hydrogels provides a versatile platform for the efficient and controlled delivery of diverse nanomedicines, including gene therapy nanoparticles, liposomes, exosomes, and more.This advancement contributes to the progress of targeted therapeutics and regenerative medicine.

Figure 2 .
Figure 2. a) Effect of different freezing treatment time on the hydrogel transparency.b) A typical SEM image of the hydrogel after being subjected to freezing at −20 o C for 72 h.Confocal images of the bulk hydrogel c) before and d) after freezing at −20 o C for 72 h, respectively.All results were obtained from a bulk hydrogel synthesized using 1.33% w/v SH-HA and 3.33% w/v HB-PEGDA polymers.

Figure 3 .
Figure 3. a) The top and bottom panels demonstrate hydrogels prepared through different polymerization techniques: Michael type addition reaction and photoinitiated free radical polymerization, respectively.b,c) Amplitude and frequency sweep tests of P-Gels prepared with varying freezing times.d,e)Amplitude and frequency sweep tests of P-Gels prepared using multiple short-cycle freezing.f,g) The amplitude and frequency sweep tests were performed on hydrogels synthesized with A-HA and HB-PEGDA using photoinitiated free radical polymerization, followed by different freezing times applied.

Figure 4 .
Figure 4. a) Hydrogel degradation curves are shown under various conditions.b) The degradation process of the P-Gels when immersed in different solutions.From the leftmost column to the rightmost column, the solutions used are PBS buffer, hyaluronidase dispersion (200 U mL −1 ), and a 5 mM H 2 O 2 aqueous solution.c) The leaching solution of 1 mg mL −1 freeze-dried P-Gel was evaluated for cytotoxicity.d) After 24-h exposure to the extracts, live/dead double staining was performed to assess the biological response.

Figure 5 .
Figure 5. a) A schematic illustrates the absorption of liposomes by the cholesterol functionalized P-Gel.b) The effect of cholesterol amount in P-Gel on its ability to absorb liposomes.c) The release profile of glabridin from the liposome-loaded granular hydrogels in a release medium consisting of PBS buffer containing 1% Tween-80.d) Shear viscosity of granular hydrogels with increasing shear rates (0.02-100 s −1 ).Inset image shows macroscopic ejection of liposome-loaded granular hydrogel from a 27-gauge needle.e) Evaluation of self-recovery of the liposome-loaded granular hydrogel under alternating strains of 1% and 1000%.