HIFU-triggered burst release of gallic acid from gelatin/polyvinyl pyrrolidone hydrogel network crosslinked with magnesium gallate MOF
Badrinathan Sridharan, Cho Eun Lee, Daehun Kim, Jin Hyeong Park, Wooram Um, Seung Yun Nam, Juhyun Kang, Hae Gyun Lim
TL;DR
A hydrogel crosslinked with magnesium gallate can release gallic acid on demand using high-intensity focused ultrasound, improving its cancer-fighting potential.
Contribution
A novel hydrogel system using magnesium gallate as both crosslinker and drug carrier enables HIFU-triggered burst release of gallic acid.
Findings
HIFU triggered rapid and consistent release of gallic acid from the hydrogel over 90 minutes.
The hydrogel showed enhanced cytotoxicity against cancer cells after HIFU-induced drug release.
The system demonstrated excellent biocompatibility with dermal fibroblast cells.
Abstract
•Magnesium gallate acts as drug carrier and crosslinker for gelatin/PVP hydrogel.•Physicochemical and mechanical characteristics of the hydrogel were demonstrated.•Hydrogel responded to HIFU and exhibited burst and on-demand release of gallic acid.•HIFU-triggered gallic acid release resulted in enhanced and rapid cytotoxicity. Magnesium gallate acts as drug carrier and crosslinker for gelatin/PVP hydrogel. Physicochemical and mechanical characteristics of the hydrogel were demonstrated. Hydrogel responded to HIFU and exhibited burst and on-demand release of gallic acid. HIFU-triggered gallic acid release resulted in enhanced and rapid cytotoxicity. In this study, we report the high-intensity focused ultrasound-triggered burst release of gallic acid from a gelatin-polyvinyl pyrrolidone (PVP) based hydrogel crosslinked with magnesium gallate (Mg-Gal) microparticles. Hydrogel was…
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TopicsHydrogels: synthesis, properties, applications · Tissue Engineering and Regenerative Medicine · Wound Healing and Treatments
Introduction
1
Biomaterials-based drug delivery systems (DDS) such as liposomes, hydrogels, nano/microspheres, nano/microparticles, scaffolds act as drug cargos and are successful for the management of several diseases like cancer, diabetes, arthritis, neurodegenerative disorders etc, that require targeted and optimal drug release without adverse side effects [1], [2], [3], [4]. It is of an utmost importance to choose a suitable DDS for the intended applications [5]. Hydrogels are hydrophilic networks composed of polymers that are chemically crosslinked with inorganic or organic molecules through some of the prominent functional groups in the polymers like carboxylic, hydroxyl, amide, etc. Due to the presence of hydrophilic groups, hydrogels can absorb and retain water for a long time which mimic the natural soft tissues and makes them suitable for various biomedical applications [6], [7], [8]. The polymeric composition of the hydrogel and its interactions with crosslinkers determine the physicochemical properties of hydrogels [9]. On the other hand, mechanical properties of hydrogels formulated as scaffolds, implants, drug carriers or injectables, has been constantly explored for rendering optimal biological activity [10], [11]. Synthetic polymers can provide excellent mechanical strength over natural polymers to withstand varied physiological conditions [12]. Chemical reinforcement plays a vital role in mechanical strength of the hydrogel and crosslinking of polymers with aldehydes, isocyanates and similar economical and readily available crosslinkers, though it helps in facile hydrogel fabrication, is a challenging aspect in terms of biocompatibility [13], [14]. Hydrogels with high rigidity are unfavorable for drug delivery applications as the release of drug molecules will be very slow and will become sub-optimal to render the bioactivity. Though certain disease conditions like, cancer and diabetes require a sustained DDS with prolonged availability of the drug at the target site, chance of delayed onset of drug action, drug resistance, and systemic toxicity are some of the major concerns [15], [16]. Hence, an external device-triggered release of the drug from a physiologically suitable rigid hydrogels are highly advisable.
Ultrasound is considered one of the safest biomedical devices that can influence a material or biological system mechanically, thermally, and chemically with limited to no adverse effects [17], [18], [19]. Mechanical effect rendered by focused ultrasound was studied extensively for drug delivery through microbubbles which involve acoustic cavitation [20], [21] and in the present study as illustrated in Fig. 1, we intended to utilize the high intensity focused ultrasound (HIFU) to mechanically degrade the gelatin and PVP-based hydrogel network crosslinked with Mg-Gal for rapid release of gallic acid for enhanced in vitro anti-cancer activity against 4T1 mouse breast cancer cells. HIFU parameter (applied acoustic power) utilized in this study comply with the FDA approved ultrasound safety criteria (Mechanical Index < 1.9, Thermal Index < 6.0) with controlled temperature condition and negligible cavitation mediated physical and chemical effect on the hydrogels reinforced with stable co-polymeric network [22], [23], [24], [25].Fig. 1. Overview of the study illustrating HIFU induced burst release of Mg-Gal & enhanced anti-cancer property.
Hydrogel (PG) fabricated in our study contains naturally derived gelatin with PVP to improve the crosslinking of the hydrogel with better mechanical properties. This hydrogel was crosslinked with Mg-Gal MOF, which was already reported for its anti-cancer, anti-oxidant and anti-inflammatory properties. MOF based crosslinkers offers unique advantages over conventional small molecules, like high drug loading capacity, biocompatibility, stimuli responsiveness and tunable structural, mechanical and release characteristics. Magnesium based MOF & other biomaterials were explored for several decades because of the stability, cost effectiveness, and facile fabrication process [26], [27]. HIFU has been revolutionizing the medical and cosmetic industries by enhancing several therapeutic and surgical techniques. Pharmacological processes such as drug formulation, targeted delivery and bioavailability were also successfully improved by focused ultrasound techniques, especially HIFU [28], [29]. In the present study, HIFU was utilized as an external trigger force that can be manipulated for either burst release or on-demand release of gallic acid that can provide enhanced cytotoxic activity. Several studies have extensively reported thermal and cavitation-based effects for drug delivery and therapeutic enhancement, but we intended to establish a HIFU mediated process that can enhance the drug release through mechanical disruption of the hydrogel. Establishing burst/on-demand release of gallic acid shows the potential of the hydrogel to offer spatiotemporal control over targeted drug delivery process with enhanced precision that minimizes off-target drug interactions. This study provides the foundation for adapting various other drug molecules-based MOF formulation for multiple disease conditions.
Materials and methods
2
Chemicals and reagents
2.1
Magnesium Chloride, gallic acid, potassium hydroxide, gelatin, polyvinylpyrrolidone (PVP), MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay kit and Live/Dead cell imaging kit, were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). All other materials utilized in this study were of analytical grade. Materials for cell culturing and maintenance such as RPMI medium, sodium pyruvate, fetal bovine serum, antibiotic–antimycotic agent were purchased from Thermo Fisher Scientific Korea Co., Ltd.
Fabrication of magnesium gallate (Mg-Gal) MOF and Mg-Gal crosslinked gelatin/PVP hydrogel (PG-Mg-Gal)
2.2
Microporous magnesium gallate particles were synthesized by the method described by Cooper and coworkers [26], [27]. We first prepared a dispersion of 100 ml containing 1 g of MgCl_2_ and mixing it with 3.8 g gallic acid resulting with pH of around 4. Then the pH was adjusted between 8 and 9 by dropwise addition of 10 M KOH. Brown-coloured clear solution at pH 8 was taken in a sealed glass container and incubated for 24 hrs at 120°C using heating mantle with occasional mixing. The resulting material was washed several times with water, dried at 40°C and stored at room temperature until further experiments.
Fabrication of hydrogel was started with preparing gelatin in DI water in a magnetic stirrer at 60°C (Solution A) and then solution B was prepared by dissolving PVP in DI water. Mg-Gal (0.1%, 0.5% & 1%) was added to solution B and homogenized by ultrasonication. Solution B was then added to Solution A and the contents were homogenized under magnetic stirrer for 1 hr at 60°C. Finally, the molten gel was poured in the 12 well plates and left to solidify for 1 hr at room temperature. Gel was transferred to 4°C and incubated overnight for complete solidification. The fabricated gel was washed by incubating with DI water for 1 hr under mild shaking and this step was repeated three times for complete removal of unreacted materials. The protocol for fabrication of Mg-Gal and PG-Mg-Gal is illustrated in Fig. 2A. The gelation time was observed with different concentration of Mg-Gal and with 0.5% Mg-Gal loaded PVP/Gelatin hydrogel (PG-Mg-Gal) showing the lowest gelation time (Fig. S1) compared to other 2 concentrations (0.1% & 1%). We carried out the further characterization and biological application with freeze dried hydrogels loaded with 0.5% Mg-Gal loaded.Fig. 2. Fabrication and physico-chemical characterization of Mg-Gal & PG-Mg-Gal. (A) – Illustration of Mg-Gal & PG-Mg-Gal fabrication methods; (B & C) – Hydrodynamic size & zeta potential value of Mg-Gal. The size range of Mg-Gal microparticles was observed between 2250 – 2750 nm (Fig. 2B, insert)); (D & E) – Structural characterization of Mg-Gal & PG-Mg-Gal through X-ray diffraction (D) & FT-IR analyses (E); (F − M) – Morphological analysis of Mg-Gal and PG-Mg-Gal. FE-SEM image (F), TEM image (G) & Energy-dispersive X-ray spectroscopy (EDX) pattern of Mg-Gal (H & I), FE-SEM (J & K) & Energy-dispersive X-ray spectroscopy (EDX) pattern of PG-Mg-Gal (L & M).
Characterization of Mg-Gal and PG-Mg-Gal hydrogel
2.3
Dynamic light scattering (DLS) was used to evaluate the hydrodynamic size of Mg-Gal and zeta-potential of the Mg-Gal was utilized to determine its surface charge. Zetasizer Nano-ZS equipment (Malvern, UK) equipped with a He-Ne laser (633 nm) and an Electrophoretic Light Scattering (ELS) controller at 90° for regulating optical data, for measurement of size and zeta potential of Mg-Gal. The Mg-Gal microparticles were used for PG-Mg-Gal hydrogel fabrication and further characterization of PG-Mg-Gal were performed and compared with Mg-Gal microparticles.
The spectroscopy and diffraction techniques provide a comprehensive approach to understand its structural, chemical, and electronic properties. Mg-Gal & PG-Mg-Gal was submitted for powder X-ray diffraction (XRD) analysis using Rigaku (Ultima IV) X-ray diffractometer with 2θ from 10° to 80° and Fourier transform infrared spectrometer (CARY 640, Agilent) for obtaining FT-IR spectra with a wave number between 400 and 4000 cm^−1^. Morphological features of synthesized Mg-Gal microparticles & PG-Mg-Gal were examined by field emission scanning electron microscopy (FE-SEM, Magellan 400, and TESCAN (MIRA 3 LMH In-Beam Detector)) equipped with EDX and transmission electron microscope (TEM, model-JEM-F200, HRP) operated at 200 kV. Samples for TEM was prepared by dropping NPs solutions onto carbon-coated Cu grids and examined on a Hitachi JED-2300 system (Tokyo, Japan).
Physical properties of hydrogel
2.4
Mechanical property
2.4.1
The mechanical property of PG-Mg-Gal was demonstrated through the stress–strain response obtained using universal testing machine (UTM, QMESYS, QM100S) equipped with a 500 N load cell. The freeze-dried hydrogel specimens were prepared as cylindrical blocks 19 mm (diameter) x 10 mm (height) and uniaxial compression was performed where load was applied at a uniform deformation rate of 1 mm/min, until a 50% reduction in each specimen height was accomplished. Stress–strain curves were generated by calculating stress and strain using the formula given below.
where F is the compressive force, A is the area loaded on to the sample, H is the deformed height and H0 is the original height of the hydrogel specimens. Stress–strain curve was utilized to demonstrate several mechanical parameters, including compressive strength, which required the structural stability under applied stress and yield strength, which representing the onset of plastic deformation. Additionally, the shape recovery was evaluated immediately after the test as the ratio of recovered height to the original height. The experiments were replicated 3 times to represent the average mechanical properties of PG-Mg-Gal and PG.
Swelling behavior
2.4.2
Freeze dried PG-Mg-Gal was first weighed (W_0_) and immersed in 1 ml of PBS with different pH (5, 7.4 & 9) & incubated at different temperatures (4°C, 25°C & 37°C). The wet gel was removed at predetermined time points for 24 hrs and weighed (W_s_) by placing in pre-weighed petri dishes after removing excess liquid using filter paper. The difference between W_s_ & W_0_ was represented as “ΔW” (W_s_-W_0_ = ΔW). The swelling ratio was calculated using the formula given below.
Water retention
2.4.3
PG-Mg-Gal after freeze drying was swollen with PBS at different pH (5, 7.4 & 9) & incubated at different temperatures (4°C, 25°C & 37°C) after measuring the initial wet weight (W_0_). The wet gels were then removed and weighed at different predetermined time points (W_t_) for 24 hrs by placing them in the open petri dishes, after removing excess liquid using filter paper. The water retention rate of the hydrogel was calculated using the formula given below.
Analysis of gallic acid release pattern in vitro
2.5
Amount of gallic acid released from PG-Mg-Gal was analyzed after diffusion across the dialysis bag. Predetermined amount of PG-Mg-Gal was enclosed in a 3.5 kDa dialysis membrane of 4 cm length and 1 ml of PBS (pH – 7.4) was added. The dialyzing membrane with hydrogel was submerged in 10 ml of 1x PBS at different pH (5, 7.4 & 9). The contents were incubated at different temperatures (4°C, 25°C & 37°C) for 7 days under constant stirring (200 rpm) in a magnetic stirrer and 1 ml of PBS was withdrawn from the content at predetermined time points and equal amount of PBS at specific pH was added in to the tubes to maintain the volume constant. All the sampling procedures were performed aseptically and sink condition was maintained throughout the study. The samples collected were centrifuged at 12000 rpm for 10 mins and the amount of gallic acid was estimated spectrophotometrically by Folin-Ciocalteu method. Cumulative release of gallic acid was calculated using the formula given below.
In vitro anti-cancer studies
2.6
Maintenance of cell lines
2.6.1
Mouse breast cancer cell line (4T1) and Detroit-551 dermal fibroblast cell lines (D-551) was obtained from Korean Cell bank (Seoul, South Korea). 4T1 cells were cultures in RPMI medium and D-551 cells were cultured in minimum essential medium (MEM). The complete media for both the cell lines were supplied with fetal bovine serum (FBS; 5%), antibiotic/antimycotic (1%), Sodium pyruvate (1 mM), HEPES (14 mM) and incubated at 37°C in an atmosphere containing 5% CO_2_. Growth pattern and morphology of the 4T1 & D-551 cells were routinely monitored before each experiment. Characteristics of D-551 cells like elongated, spindle shaped adherent cells with more than 24 hrs of doubling time (Fig. S2A) and 4T1 cells such as strongly adherent polygonal shaped cells that grows in clusters with a doubling time of ∼20 h were observed and this exhibited the quality of cells (Fig. S2B). Passage number of the cells between 3–5 were used for all the experiments in this study.
Cytotoxic potential of Mg-Gal & PG-Mg-Gal
2.6.2
Cells were seeded in a 96 well plate at a density of 1 × 10^4^ cells/well and incubated for 12 h for attachment to the bottom of the well. Mg-Gal & freeze-dried PG-Mg-Gal hydrogels were added at 8 different concentrations from 25 to 2000 μg/ml and incubated at 37°C in an atmosphere containing 5% CO_2_ for 24 h. At the end of incubation cells were washed with PBS and replaced with fresh media (100 μl) before adding 10 μl of 12 mM MTT reagent to the cells. After incubating the cells for 4 hrs, 100 μl of 0.01 M SDS was added and incubated again for 4 h. Finally, the optical density was measured at 570 nm and the percentage of cell viability was calculated using the formula given below.
Inhibition of cell migration by Mg-Gal & PG-Mg-Gal
2.6.3
Cells were seeded in a 96 well plate at a density of 5 × 10^4^ cells/well and incubated for 12 hrs for attachment to the bottom of the well. At the middle of the well a scratch was carefully made using a sterile 200 μl micropipette and the cells were washed with PBS. Mg-Gal (250 μg/ml) & lyophilized PG-Mg-Gal (100, 250 & 500 μg/ml) were added to wells and incubated at 37°C in an atmosphere containing 5% CO_2_ for 24 h. Migration of cells was observed using the bright-field microscopy at x10 magnification to measure the scratch area and scratch width using ImageJ Software. The percentage of migration was calculated by the formula given below.
Biocompatibility studies
2.7
D-551 cells were seeded in a 96 well plate at a density of 1 × 10^4^ cells/well and incubated for 12 h for attachment to the bottom of the well. Freeze-dried PG & PG-Mg-Gal hydrogels and Mg-Gal were added at 8 different concentrations from 25 to 2000 μg/ml and incubated at 37°C in an atmosphere containing 5% CO_2_ for 24 h. At the end of incubation cells were washed with PBS and replaced with fresh media (100 μl) before adding 10 μl of 12 mM MTT reagent to the cells. After incubating the cells for 4 hrs, 100 μl of 0.01 M SDS was added and incubated again for 4 h. Finally, the optical density was measured at 570 nm and the percentage of cell viability was calculated using the formula given as Eq. (6).
For Live/Dead cells staining assay freeze-dried PG & PG-Mg-Gal hydrogels at different concentration (100, 250 & 500 μg/ml) and Mg-Gal (100 μg/ml) were utilized for this experiment. After seeding the cells in 6-well plates at a density of 5 x 10^4^ with the samples they were incubated in CO_2_ atmosphere at 37°C. At the end of 24 hrs incubation, the cells were stained with Live/Dead staining solution for 15 min (as per the protocol given for LIVE/DEAD™ Cell Imaging Kit (488/570) purchased from Thermo Fisher Scientific, Waltham, USA). Stained cells were imaged with inverted microscope (IX51S1F-3, Olympus, Tokyo, Japan) and the percentage of dead cells were quantified with ImageJ software.
Gallic acid release triggered by HIFU
2.8
Enhanced release of gallic acid was achieved by focused ultrasound produced from MIUP-HIFU instrument (HR Meditech, Incheon, South Korea). Hydrogel samples in the form of cylindrical blocks (similar to the dimensions used for mechanical study) were used for the release study, which was conducted by acoustically coupling them with a fixed transducer. Hydrogel samples were immersed in PBS (coupling medium) and transducer was immersed and placed on top of the wet hydrogels without any gap, which indicates the probe-contact coupling method was adapted in our study. Hydrogel was irradiated with HIFU at intensities 2 & 4 as per the manufacturer’s instructions. The acoustic power generated by the instrument at different intensities was measured using Acoustic Intensity Measurement System (AIMS III, ONDA, Sunnyvale, CA, USA) and in our study we have hydrogels were exposed to HIFU at intensities 2 & 4 which corresponds to acoustic powers of 250 mWatts & 750 mWatts respectively. To establish that temperature is not the primary mechanism of hydrogel disruption, we monitored the temperature variation in the hydrogel after HIFU exposure at the specific acoustic powers (250 & 750 mWatts) and performed SEM imaging for HIFU irradiated hydrogel for demonstrating the disruption of crosslinking network.
The hydrogel was placed in 6 well plate with 5 ml in each well containing 50 mg of the freeze-dried gel and irradiated with HIFU at 250 & 750 mWatts of acoustic power for 30 secs. Cumulative gallic acid release was observed for 90 mins after HIFU exposure, by withdrawing 300 μl of PBS every 10 mins and equal amount of PBS was added in to the wells to maintain the volume constant. On-demand release of gallic acid was observed by irradiating the hydrogel with HIFU for 30 secs and measuring gallic acid release every 10 mins for 30 mins. This cycle was repeated 3 times and the release pattern was observed for 30 mins after each cycle of HIFU irradiation. Similar to cumulative release study, 300 μl of PBS was withdrawn at each time point and replaced with equal amount of PBS to maintain the volume constant. The samples collected were centrifuged at 12000 rpm for 10 mins to remove the hydrogel debris released due to HIFU mediated disruption and the amount of gallic acid was estimated in the dispersion medium spectrophotometrically using Folin-Ciocalteu method. Cumulative and on-demand release of gallic acid was calculated using the formula given below.
Cytotoxicity of HIFU triggered burst release of gallic acid
2.9
Cells were seeded in a 6 well plate at a density of 5 × 10^4^ cells/well and incubated for 12 h for attachment to the bottom of the well. Then cells were divided into untreated control, PG-Mg-Gal treated group, followed by cell treated with HIFU irradiated PG-Mg-Gal and HIFU treated group (negative control). The freeze-dried hydrogels were utilized for this experiment where the concentration of PG-Mg-Gal was maintained at 5 mg/ml and the HIFU was irradiated at 750 mWatts power to obtain burst release of gallic acid. After seeding the cells in 6-well plates at the density of 5 × 10^4^, PG-Mg-Gal was added and irradiated with HIFU, while untreated control with and without PG-Mg-Gal and only HIFU irradiation were the other experimental groups in this study. Cells were then incubated in CO_2_ incubator at 37°C and at the end of 4 hrs and 24 hrs incubation, the cells were stained with Live/Dead staining solution for 15 min (as per the protocol given for LIVE/DEAD™ Cell Imaging Kit (488/570) purchased from Thermo Fisher Scientific, Waltham, USA). Stained cells were imaged with inverted microscope (IX51S1F-3, Olympus, Tokyo, Japan) and the percentage of dead cells were quantified with ImageJ software.
Statistical analysis
2.10
The data were analyzed using Graph Pad Prism 10.1 software and results were expressed as mean ± SD (n = 3). Statistical analysis was performed by One-Way ANOVA followed by Tukey’s multiple comparison test for all the results.
Results and discussion
3
Hydrogels are one of the robust types of biomaterial formulations with strong chemical interactions with drug molecules and tunable physico-chemical properties making it suitable for variety of biological applications [30]. Most of the hydrogels are sensitive to external stimuli such as light, sound, pH etc., which helps in manipulating the drug release pattern according to the targeted ailments and organs [31]. Among the external stimuli, ultrasound triggered drug release has gained significant interest in the recent years due to its radiation free application and deeper penetration in to the system compared to light. Rapid/burst, sustained or on-demand mode of drug release can be achieved using ultrasound irradiation, according to the required application [32]. In our study, we have developed a hydrogel composed of gelatin and PVP as the polymeric network crosslinked by a microporous Mg-Gal MOF. Based on our previous observation on responsiveness of Mg-Gal to acoustic tweezers, we intended to manipulate the release pattern of gallic acid from the hydrogel using focused ultrasound.
Characterization of Mg-Gal & Mg-Gal crosslinked gelatin/PVP hydrogel
3.1
Hydrogels fabricated with 0.1% Mg-Gal showed high gelation time because of the limited concentration of crosslinker (Mg-Gal), while Mg-Gal concentration is 1% hydrogel the gelation time is higher as the optimal polymer concentration is not available for gelation process. Hence, Further studies were carried out with 0.5% hydrogel considering its optimal gelation time and suitability to test the burst/on-demand releasing behavior. The characterization, drug release and in vitro anti-cancer studies were carried out using freeze dried hydrogel loaded with 0.5% of Mg-Gal. The amount of gallic acid loaded onto the hydrogel was observed to be 4.71 µg/mg of hydrogel, which showed around 84.5% of drug loading capacity (Table S1).
Size & zeta potential of Mg-Gal
3.1.1
The hydrodynamic size of Mg-Gal was observed to be in the range of 2250 to 2750 nm (Fig. 2B) with a surface charge of −17.8 mV (Fig. 2C) and is consistent with our previous observation. The particle size distribution width was ranged within 500 nm and the intensity weighted percentile of the particles size demonstrated that 90% of the particles falls between 1905 nm and 2589 nm, which is approximately within 680 nm. Particle size distribution observed with DLS results were supported by SEM based particle size count (Fig. S3). These observations clearly support the moderately narrow size distribution pattern of Mg-Gal microparticles, but the size range of 2000 – 3000 nm may not be optimal for biological applications due to reduced cell penetration. However, their interaction with the cellular membrane could execute the cytotoxic mechanism of Mg-Gal [15], [21]. Electronegative materials play a moderate role in bioactivity than the electropositive materials, but negative surface charge of materials are important for their size stability, enhanced drug loading capability through electrostatic interactions, increased circulation time and lower systemic toxicity which can improve its theranostic applications [33], [34], [35]. The electronegativity of Mg-Gal predominantly contributed by carboxylic groups from gallic acid, which could influence the stable crosslinking of gelatin & PVP [36], [37], [38].
Structural and chemical analysis
3.1.2
X-ray diffraction demonstrated distinct differences between peak obtained from Mg-Gal and PG-Mg-Gal (Fig. 2D). Sharp crystalline peaks were obtained with Mg-Gal materials, whereas PG-Mg-Gal displayed broader pattern of XRD peaks with reduced signal that represents the amorphous nature of the hydrogel due to the polymer compositions. Peaks obtained at 2θ values such as 11.52°, 14.17°, 24.57° & 36.05° represents the signature peaks of Mg-Gal as reported in previous studies [26], [27]. On the other hand, based on previously reported XRD analysis of gelatin/PVP complex, the peak at 20.29° & 10.55° indicates the presence of PVP in the hydrogel and the co-polymeric interaction of PVP & gelatin was represented by the peak at 29.01° [29]. Functional groups responsible for the stable interactions of gelatin & PVP were identified using the FT-IR pattern of the hydrogel compared with pure Mg-Gal (Fig. 2E). We observed that organo-metallic interactions of Magnesium with the other organic molecules (gallic acid, gelatin & PVP), was represented by peaks at 840 cm^−1^ (Mg-Gal) & 829 cm^−1^ (PG-Mg-Gal). Peaks at 1621 cm^−1^ in Mg-Gal microparticles indicates the carbonyl groups (C=O) while there is shift in this peak to 1646 cm^−1^in the hydrogel with increased intensity is due to gelatin containing the amide bonds. Aromatic ring containing C-C interactions were represented in both the materials from 1300 to 1600 cm^−1^ and the aliphatic C-O groups were responsible for the peaks around 1000 – 1300 cm^−1^. Similar to previously synthesized coplymeric hydrogel containing PVP & gelatin specific peaks at 2877 cm^−1^ & 2952 cm^−1^ were observed with PG-Mg-Gal indicating strong interactions between gelatin & PVP through asymmetric C-H stretching [27], [39], [40]. Though the current observation provides significant shift in the FT-IR peaks between Mg-Gal & PG-Mg-Gal, we wanted to support the FT-IR results of the samples with Raman spectroscopy. Fig. S4 shows Raman spectra of pure GA, PVP/gelatin (PG), Mg-Gal, and PG-Mg-Gal composite over 100–2000 cm^−1^. GA spectrum exhibits multiple sharp bands at 1600 cm^−1^ to 1800 cm^−1^, assigned to aromatic ring C=C and C=O vibrations, confirming its highly ordered phenolic structure. PVP/gelatin spectrum shows broader features typical of polymeric amide and CH vibrations, indicating a more amorphous matrix with peaks around (700 cm^−1^ to 900 cm^−1^). Upon Mg^2+^ coordination (Mg-Gal, red), most GA peaks are strongly damped and partially shifted, implying chelation of Mg^2+^ with GA and interaction with gelatin functional groups. PG-Mg-Gal spectrum (green) at bottom is much flatter, showing significant fluorescence background and loss of distinct GA bands, which evidences strong embedding within PVP-gelatin network. The three shaded regions and inserts highlight low-, mid-, and high-wavenumber windows where characteristic GA vibrations broaden in the composites, supporting successful integration of PG-Mg-Gal composite. These current results support the fabrication of PG-Mg-Gal hydrogel with Mg-Gal as crosslinker that stabilizes the hydrogel network with stable interactions that can be disrupted by external forces like ultrasound. Chemical composition and its interactions with in the hydrogel materials play an inherent role in its drug release behaviour and bioactivity [41]. Further, responsiveness of the hydrogel to external force and its advantage primarily depends on the chemical interaction of the elements in the material [2]. Our previous observation on ability of focused ultrasound over Mg-Gal provided solid evidence for this study to proceed with manipulation of Mg-Gal comprising hydrogel using HIFU [27].
Morphological analysis
3.1.3
Morphological characterization of Mg-Gal was performed using FE-SEM and TEM where, highly aggregated crystals of Mg-Gal was observed (Fig. 2F & 2G, respectively). The EDS spectrum supported the presence of magnesium in the MOF (Fig. 2H & 2I). FE-SEM analysis of PG-Mg-Gal showed a highly interconnected network of gelatin & PVP crosslinked by Mg-Gal (Fig. 2J). Smooth surface of the hydrogel was appreciated with particles dispersed in the porous structure indicating the presence of Mg-Gal (Fig. 2K) and supported by crystalline pattern observed in XRD (Fig. 2D). Incorporation of Mg-Gal in the hydrogel was observed by EDS spectrum obtained from the hydrogel which indicated the presence of Magnesium (Fig. 2L & 2M). These findings demonstrate that crosslinking PVP & gelatin with Mg-Gal alters the physicochemical properties of the hydrogel with reduction in amorphous and porous nature of PG-Mg-Gal which could enhance various physical properties suitable for biomedical applications, such as water retention capacity, swelling property and mechanical stability swelling, and drug-loading capacity [42].
Physical properties of hydrogel
3.2
Mechanical properties
3.2.1
The stress–strain curve, compressive strength, yield strength and shape recovery were illustrated in Fig. 3A – 3F. Hydrogel used for mechanical testing and further analyses of physical properties were performed with freeze-dried samples. Though mechanical property of swollen hydrogels was not included in the current study which demonstrate its potential for various physiological application, we primarily focused on establishing strength of the internal crosslinking network of the hydrogel and to demonstrate the properties with respect to its stability and feasibility for external physical force (HIFU) mediated drug release, through analyzing the strength of the hydrogel network which primarily depends on the structural integrity provided by the crosslinker (Mg-Gal). Hydrogel specimens as cylindrical blocks (Diameter × Height: 19 mm × 10 mm) were used for the mechanical testing (Fig. 3A) and the visual observation of mechanical deformation occurred in the hydrogel was showed in Fig. 3B where within the experimental working time of 5 mins the compression occurred in the gel when load was given and the recovery gel dimension after the load was withdrawn were reported. Compressive behaviour was demonstrated in Fig. 3C, which shows that stress values were significantly higher in PG-Mg-Gal compared to PG hydrogel without Mg-Gal. In case of PG-Mg-Gal, at 15.2% of strain, the stress value performed 358.9 kPa which was significantly higher than PG hydrogel, where the stress value was achieved at 117.1 kPa itself when the strain was around 15.0%. The results suggested that complete deformation of the internal structure has occurred only when 358.9 kPa load of stress was exerted on PG-Mg-Gal, which is significantly more than destruction of PG hydrogel that required only 117.1 kPa. The compressive strength of PG-Mg-Gal was approximately 396.3 kPa and it is 2.5 times higher than the PG hydrogel with 157.4 kPa (Fig. 3D). Yield strength depicted in Fig. 3E also showed a similar trend as observed in compressive strength where PG-Mg-Gal (259.3 kPa) showed 3 times higher values than PG hydrogel (82.3 kPa). This indicates the crosslinking of PVP & gelatin by Mg-Gal resulted in denser and mechanically a better stable network than PG. Previous studies on gelatin and PVP as polymeric components have shown strong mechanical property and are suitable for biological applications such as bone tissue engineering, antimicrobial properties, wound healing and etc [43], [44], [45]. The observation on strong mechanical property of PG-Mg-Gal indicates the tightly crosslinked internal structure of PG-Mg-Gal and correlates with its FE-SEM images displayed in Fig. 2J. The intermolecular interactions between Mg-Gal and functional groups of polymers, such as carboxylic, carbonyl groups from gallic acid and co-ordination with Mg^2+^ ions that are predominant from the FT-IR pattern of PG-Mg-Gal (Fig. 2E), plays a vital role in crosslinking of the hydrogel [38], [46].Fig. 3. Mechanical and physical properties of PG-Mg-Gal. (A) – Gross observation of lyophilized PG & PG-Mg-Gal hydrogels; (B) – Experimental set up for analysis of mechanical property of PG & PG-Mg-Gal indicating the compression and recovery images of the gels; (C − F) – Mechanical properties of PG & PG-Mg-Gal reporting Stress-Strain curve (C), Compression strength (D), yield strength (E) & shape recovery percentage (F) of PG & PG-Mg-Gal; (G − J) – Physical property of PG-Mg-Gal. Swelling behaviour (G & I) and water retention property (H & J) of PG-Mg-Gal at different pH & temperature. The results were expressed as mean ± SD (n = 3) and statistically analyzed by One-Way ANOVA with Tukey’s multiple comparison post test. The comparisons are made between the swelling ratio & water retention rate of hydrogels impregnated at different pH & temperature at 24 hrs (1440 mins) as follows: pH 5 Vs 7.4 (), pH 7.4 Vs pH 9 (#), pH 5 Vs pH 9 (), 4°C Vs 25°C (*), 25°C Vs 37°C (#), and 4°C Vs 37°C (). The statistical significance was denoted as p < 0.0001 (), p < 0.001 (), p < 0.01 (**); Not significant (ns).
The shape recovery of both PG-Mg-Gal and PG hydrogels was further evaluated to assess their elastic behaviour where the Mg-Gal crosslinked hydrogel exhibited 37% recovery after deformation, while PG hydrogel resulted in only 32% recovery of the original dimension in Fig. 3B and 3F. These findings further indicate that Mg-Gal plays a role in enhancing the mechanical properties of hydrogel. Overall, the mechanical performance of the hydrogels reflects the stability of the interaction between the polymers and crosslinkers. Ultrasound responsiveness of PG-Mg-Gal is a crucial factor for this study, and the further studies on physical properties and drug release behaviour may provide observation that are vital for HIFU-triggered drug release via mechanical degradation of the hydrogel. In addition, the swelling behaviour and water retention properties may also throw further insights into the pH & temperature stability.
Swelling behavior and water retention
3.2.2
The water absorption capacity of the hydrogel was studied by observing the swelling behavior of the gel and pH plays a vital role in swelling pattern of the gel. In our study, we have selected 3 different pH range (5, 7.4 & 9) and the results showed that at pH 5, the swelling ratio did not increase rapidly and attained a swelling ratio (ΔW/W_0_) of 8.22 after 24 hrs. However, at pH 7.4 & 9, the swelling ratio of 7.81 & 7.87, respectively was reached in the first 10 mins and was consistent for 24 hrs (Fig. 3G). This trend indicated that PG-Mg-Gal possesses optimal crosslinking pattern for rapid absorption of water at pH 7.4 and the results also exhibit the anionic nature of the gel. On the other hand, water retention capacity of PG-Mg-Gal at different pH indicated that at pH 5 & 7.4, the hydrogel showed better ability to hold water molecules close to 19.6% & 15.6% more retention compared to less than 3.06% of water retention at pH 9 (Fig. 3H).
Temperature dependent swelling behavior of PG-Mg-Gal indicated that at 37°C the swelling ratio has reached up to 9 in 24 hrs while gels incubated at 4°C and 25°C showed no significant difference in water absorption property (Fig. 3I). Similar pattern was observed in water retention, where our observation reported that varying temperatures (4°C, 25°C & 37°C) did not influence the water retention capacity of PG-Mg-Gal (Fig. 3J). These results clearly show that swelling and water absorption properties of PG-Mg-Gal at different temperature showed significant difference, but it cannot be inferred that PG-Mg-Gal exhibits temperature responsive hydration properties, because they showed only minor difference in hydration equilibrium, though the statistical difference seems to be significant. Further the swelling ratio shows similar pattern across the three different temperatures and hence, the overall swelling mechanism indicates no appreciable temperature responsiveness., while pH played a vital role in physical behaviour of the hydrogel, specifically at pH 7.4 which is will significantly impact the drug release pattern at physiological conditions. Water acts as the acoustic medium and plays a significant role in conducting the ultrasound throughout the structure of the biomaterials and rapid swelling behaviour and high-water retention capacity for a longer duration indicated the ability of hydrogel to successfully carry out the mechanical degradation effectively through focused ultrasound [47], [48].
Gallic acid release studies
3.3
Gallic acid release from the hydrogel with respect to time at varied pH & temperature was illustrated in Fig. 4. The study was performed with 3.5 k Da dialyzing membrane which is 20 times more than the molecular weight of gallic acid (∼170 Da) to ensure that membrane diffusion was not affecting the release pattern. Further, the experiment was performed under constant agitation and the sink condition was maintained throughout the study (Fig. S5). Amount of gallic acid released in to the medium was calculated using Folin-Ciocalteu method, which is considered as gold standard for estimation of phenolic compounds. Utilization of this method provides the amount of overall phenolic compounds present in the medium and not specific for gallic acid. However, there are no other reducing phenolic species included as a component in the formulation of PG-Mg-Gal and hence, we monitored gallic acid release utilizing Folic-Ciocalteu method. PG-Mg-Gal hydrogel incubated at 37°C in pH 7.4 showed around 50% of gallic acid release while at pH 5, hydrogel showed more than 60.9% of release. This indicated that the acidic pH showed enhanced drug release pattern from the hydrogel than neutral (pH 7.4; 50.3%) and basic (pH 9; 48.6%), which was not significantly different (Fig. 4A & 4B).Fig. 4. Gallic acid release study. (A) – Gross observation of hydrogel submerged in medium at different pH and incubated at 37°C; (B) – Gallic acid release pattern from hydrogel at different pH. The results were expressed as mean ± SD (n = 3) and statistically analyzed by One-Way ANOVA with Tukey’s multiple comparison post test. The comparisons of gallic acid release (%) on day 7 between pH 5, 7.4 & 9 were made and the statistical significance were denoted as p < 0.0001 (); p < 0.01 (**). (C) – Gross observation of hydrogel submerged in medium at pH 7.4 and incubated at different temperature. (D) – Gallic acid release pattern from hydrogel at different temperature. The results were expressed as mean ± SD (n = 3) and statistically analyzed by One-Way ANOVA with Tukey’s multiple comparison post test. The comparisons of gallic acid release (%) on day 7 between 4°C, 25°C & 37°C were made and the statistical significance were denoted as p < 0.0001 (); p < 0.01 (**); Not significant (ns).
Gallic acid release pattern from hydrogels incubated at different temperatures (4°C, 25°C & 37°C) showed that release was almost similar at 25°C (51.3% at 24 hrs) & 37°C (50.3% at 12 hrs) while at 4°C (53.9% at day 4) the release pattern was much slower (Fig. 4C & 4D). The release pattern was observed for 7 days and the maximum release was observed from 12 to 24 hrs. PG-Mg-Gal incubated at 37°C in pH 7.4 showed gradual release for 1 hr and showed a logarithmic release from 1 hr to 12 hrs (8.34% – 50.3%). However, at pH 5 gallic acid release was almost similar to pH 7.4 and the release was much higher from 1 hr (12.05%) to 24 h (60.9%). This logarithmic release pattern showed that acidic pH disrupted the crosslinking between PVP & gelatin achieved by Mg-Gal. Influence of pH and temperature on the chemical interaction between the polymer and crosslinkers plays a vital role in drug release behaviour of hydrogel. Hence, in our study we wanted to establish the role H^+^ and ^–^OH ions in modulating the interaction between gallic acid in Mg-Gal and the polymers [49]. This experiment established the sustained increase in release of gallic acid over a period of 24 hrs while strongly acidic pH slightly increased the release pattern indicating the protonation of carboxylic groups in gallic acid and side chains of gelatin compared to neutral and basic pH where hydroxylation of the materials was not achieved, leading to lower release behaviour [50]. Conversely, there was a considerable reduction in the gallic acid release rate after 24 h, which is due to the depletion of gallic acid concentration in the hydrogel as a function of time. Rapid release of gallic acid at pH 5 clearly indicates the feasibility of pH driven drug release to the highly acidic intracellular compartment. The current observation establishes the release pattern of gallic acid at a wide range of pH condition (pH 5–9) and it indicates PG-Mg-Gal is suitable for drug release under strong acidic environments. The pH responsiveness of hydrogel will be further explored to optimize the hydrogel composition suitable for pH triggered drug release at the tumor microenvironment, intracellular compartment and other physiologically relevant conditions for enhancing the biological applicability of the formulation [51], [52]. Temperature based variation in the gallic acid release PG-Mg-Gal was not significant which indicated the stability of the hydrogel in physiological and atmospheric temperatures. These observations provided an important insight to our following studies with HIFU based release studies as focused ultrasound tend to elevate the temperature of the material and dispersion medium. The physiological temperature did not affect the gallic acid release pattern and at an elevated temperature the hydrogel may be degraded and in turn enhance the drug release.
In vitro anti-cancer studies
3.4
Cytotoxic potential of Mg-Gal & PG-Mg-Gal on 4T1 breast cancer cells was studied by MTT assay (Fig. 5A & 5B). Among several breast cancer cell lines triple negative breast cancer cells (4T1) are highly aggressive and hard to treat, because of its intrinsic resistance. These are also known for its high metastatic potential. Utilizing breast cancer cells in our study is physiologically more relevant because we have chosen hydrogel as the biomaterial formulation and HIFU as external trigger mechanism for localized delivery through topical application or local implantation which are relatively feasible around the breast tissue. Based on the results obtained, the ability of the materials to inhibit the cell migration was demonstrated by scratch assay and the cell migration pattern was depicted with the cell intensity calculation using ImageJ software (Fig. 5C). In case of MTT assay the cytotoxic potential of the materials at increasing concentrations (25–2000 μg/ml) was illustrated in Fig. 5E, while the inhibition of cell migration was represented as percentage of scratch area and width covered by the cell incubated with the materials in comparison to untreated cells (Fig. 5D). Cells incubated with different concentrations of the material and hydrogel showed significant reduction in cell viability from 100 μg/ml. Mg-Gal showed only 42.07% cell viability at 500 μg/ml the cell viability has reduced to less than 20% at 2000 μg/ml (12.5%). On the other hand, PG-Mg-Gal showed significant reduction in cell viability (63.8%) at 500 μg/ml compared to control group of cells. However, the cytotoxicity was saturated at 500 μg/ml and there was no significant reduction in the cell viability up to 2000 μg/ml (54.05%). In comparison with PG-Mg-Gal, cytotoxic potential of Mg-Gal was significantly higher from 500 μg/ml (Fig. 5A & 5E). The cytotoxic property of Mg-Gal against 4T1 breast cancer cells was an obvious observation as gallic acid was previously reported for its in vitro & in vivo anti-cancer effect against varied type of cancer cells through multiple mechanisms such as inhibition of several pro-cancerous pathways, immunomodulatory pathways, apart from epithelial to mesenchymal transition (EMT) pathways [53], [54], [55]. EMT process plays a key role in cancer cell migration and metastasis [56], [57]. In case of cell migration experiment in the present study, we have chosen 250 μg/ml of Mg-Gal and 100, 250 & 500 μg/ml of PG-Mg-Gal, where cell viability was appreciable to exclude the ambiguity between cytotoxicity and anti-migration effects. The results demonstrated that migration of 4T1 cells significantly inhibited in Mg-Gal treated cells. Cells were able to invade only 30.8% of the scratch area and 35.6% of the scratch width (Fig. 5F & 5G) was covered by the cells incubated with Mg-Gal. In case of PG-Mg-Gal the inhibition of cell migration (Fig. 5C & 5D) was not as prominent as observed in Mg-Gal treated cells but area of scratch invaded by the cell has reduced by increase in concentration of the hydrogel, which indicated the inhibition of cell migration has increased with increase in Mg-Gal concentration in the hydrogel in comparison to control cells. Fig. 5C depicts the increase in cell density in control group around the cell scratch area, while the Mg-Gal & PG-Mg-Gal supplemented cells showed reduced cell intensity at the scratch area compared to control group indicated the anti-migration effect of the material & hydrogel. As observed in Fig. 5F & 5G, the scratch area & width in PG-Mg-Gal incubated cells at the concentration of 100 μg/ml was up to 48.6% & 49.7% respectively, while 500 μg/ml of the hydrogel has significantly inhibited the cell migration up to 82.9% & 83.5% (scratch area & width, respectively). Migration of cancer cells was executed by several cellular factors that are responsible for metastasis. Matrix metalloproteinases (MMPs) are the one of the key regulators of cell migration. Our results showed possible influence of Mg & gallic acid on regulating the enzymatic activity of MMPs. Magnesium is believed to influence the cell viability and migration presumably through dissociated Mg^2+^ ions and the 2OH^–^ that can neutralize or alkalinize the tumor microenvironment there by reducing tumor progression [58], [59], [60].Fig. 5. In vitro anti-cancer studies. (A & B) – Morphological observations of the untreated 4T1 cells (A(i) & B(i)) and cells incubated with different concentration of Mg-Gal (A(ii) – 500 µg/ml, A(iii) – 750 µg/ml, A(iv) – 1000 µg/ml & A(v) – 2000 µg/ml) & PG-Mg-Gal (B(ii) – 500 µg/ml, B (iii) – 750 µg/ml, B (iv) – 1000 µg/ml & B (v) – 2000 µg/ml); (C) – Migration of 4T1 cells incubated with Mg-Gal & PG-Mg-Gal (Scale bar − 10 μm); () – Graphical illustration of intensity vs distance curve indicating the cell density at 0 hr & 24 hrs depicting change in cell density at the scratch area of the untreated and Mg-Gal & PG-Mg-Gal treated group (Scale bar − 10 μm); (E) – Graphical representation of the cell viability indicating the cytotoxic potential of Mg-Gal & PG-Mg-Gal; (F & G) − Scartch area invaded (F) & scratch width (G) indicating the anti-migration effect of Mg-Gal & PG-Mg-Gal. The results were expressed as mean ± SD (n = 3) and statistically analyzed by One-Way ANOVA with Tukey’s multiple comparison post test. The comparison made between viability percentage of cells treated with different concentration of Mg-Gal & PG-Mg-Gal was represented as ‘’. The comparison was also made between Mg-Gal & PG-Mg-Gal treated cells at concentration of 2000 μg/ml and their respective untreated cells was represented as ‘#’. The statistical significance was denoted as p < 0.0001 (####). In cell migration assay the comparisons were made between Mg-Gal & PG-Mg-Gal treated groups against untreated cells () and between Mg-Gal and different PG-Mg-Gal treated cells (#). The statistical significance was denoted as p < 0.0001 (**); p < 0.001 (*); Not significant (ns).
Biocompatibility of PG-Mg-Gal
3.5
Successful translation of biomaterials to pre-clinical and clinical set up can be considered for materials that possess excellent biocompatibility. After establishing the anti-cancer potential of PG-Mg-Gal, we wanted to report that the hydrogel formulation and the components does not induce any cytotoxicity to the normal cells (Fig. 6A – 6F). Hence, we performed the study with human dermal fibroblast cells (D-551), and we observed that all the material components were biocompatible and did not elicit appreciable cytotoxicity. MTT assay revealed that Mg-Gal reduced the cell viability of the D-551 cells at the concentration of 250 μg/ml (57.4%), while PG (91.3%) & PG-Mg-Gal (86.3%) showed relatively minimal cytotoxicity up to 1000 μg/ml, where the cytotoxic potential against 4T1 cells were significant (Fig. 6E). Morphological observation clearly suggested that the cytotoxic potential of Mg-Gal was mitigated when it was loaded on to the hydrogel (Fig. 6A – 6C). Since, the crosslinker property of Mg-Gal was exploited, the availability of the gallic acid in the material to interact with the cells were reduced. Further, gallic acid was previously reported for its inhibitory effects over specific oncogenic pathways such as PI3K/Akt/mTOR, MAPK/ERK, NF-κB and several associated signaling pathways, which indicates the variability in cytotoxic potential on 4T1 cells and D-551 cells survived the higher concentrations of hydrogel components due to the intrinsic anti-oxidant potential of gallic acid maintained the redox homeostasis and poor uptake of gallic acid [53], [55], [61], [62]. We have also supported the biocompatibility of PG-Mg-Gal with Live/Dead cells staining assay with corresponding doses utilized in the cell migration studies and the results demonstrated that no significant difference in percentage of dead cells between control (0.35%) and other experimental groups (Fig. 6D). PG-Mg-Gal showed only 0.35% of dead cells at high concentration (500 μg/ml), which has no significant difference compared to PG (0.24%) at the same concentration (Fig. 6F). This biocompatibility studies supports selective anti-cancer property of PG-Mg-Gal and a localized rapid release at the tumor microenvironment using an external trigger (HIFU) may enhance the cytotoxic activity of gallic acid at par with the Mg-Gal, with minimal non-specific toxic effects.Fig. 6. Biocompatibility of Mg-Gal, PG-Mg-Gal & PG. (A – C) – Morphological observation of control D-551 cells (A(i) B(i) & C(i)) and cells incubated with Mg-Gal (A(ii) – 250 µg/ml, A(iii) – 500 µg/ml, A(iv) – 750 µg/ml & A(v) – 1000 µg/ml), PG (B(ii) – 250 µg/ml, B (iii) – 500 µg/ml, B (iv) – 750 µg/ml & B (v) – 1000 µg/ml) & PG-Mg-Gal (C(ii) – 250 µg/ml, C (iii) – 500 µg/ml, C (iv) – 750 µg/ml & C (v) – 1000 µg/ml); (Scale bar − 10 μm); (D) – Live/Dead cells staining images of D-551 cells incubated with Mg-Gal, PG & PG-Mg-Gal at 24 hrs (Scale bar − 10 μm); (E) – Graphical representation of the cell viability indicating the biocompatibility of Mg-Gal, PG & PG-Mg-Gal; (F) – Graphical representation of percentage of dead cells quantified using ImageJ. The results in E & F parts of the figure were expressed as mean ± SD (n = 3) and statistically analyzed by One-Way ANOVA with Tukey’s multiple comparison post test. The comparisons for cell viability graph were made between results at 1000 µg/ml concentration and their respective control results. The statistical significance was denoted as p < 0.001 (*); p < 0.01 (). The comparison for Live/Dead cells assay was made between control and all the experimental groups. The statistical significance was denoted as Not significant (ns).
HIFU triggered gallic acid release
3.6
Influence of external device (HIFU) on trigger or manipulation of the gallic acid release was reported in this study. HIFU renders highly localized mechanical or thermal effect on the target samples which is governed by the ultrasound parameters such as frequency, duty cycle, and exposure time. In our study, we propose the mechano-physical damage of the hydrogel caused by the HIFU leveraging on high acoustic power. Acoustic radiation force is one of the possible physical outcomes of high acoustic power that transfers the momentum of the propagating acoustic field to the target samples. In case of soft material formulations such as hydrogels their network architecture depends on extent of crosslinking and hence, the absorbed acoustic energy can cause significant deformation and structural collapse at the microlevel [2], [63], [64], [65]. Mg-Gal that act as crosslinker for PG-Mg-Gal hydrogel in our study, can release gallic acid upon exposure to HIFU mediated mechanical disruption of the network architecture with minimal to negligible influence of HIFU-induced thermal effect. All experiments were performed using the same HIFU system under identical operating conditions and the only experimental parameter intentionally varied was the HIFU intensity in order to observe the change in release pattern and extent of hydrogel disruption. The acoustic power is one of the key parameters we could obtain from the HIFU instrument with varying intensities (Fig. 7A & 7B). The change in acoustic power corresponds directly to changes in acoustic intensity and results in variation in radiation force [66], [67]. Hence, we selected two different acoustic powers (250 mWatts and 750 mWatts) in our study and we did not explore beyond 750 mWatts in our study, due to the chance of possible increase in temperature and pertaining effects on the hydrogel. However, influence of ultrasound parameters such as central frequency, duty cycle, pulse repetition frequency (PRF) and focal dimensions which were not reported in the current study, will be extensively explored to demonstrate the optimal parameters required for HIFU mediated burst/on-demand drug release from a hydrogel platform. Another important influencing factor in our experiment with HIFU is thermal effect and our study complied with FDA diagnostic ultrasound safety criteria (Mechanical Index < 1.9, Thermal Index < 6.0) [19], [21], [35]. Further, to establish that temperature has negligible impact and mechano-physical damage is the predominant mechanism involved in the drug release process, we performed temperature monitoring of the HIFU irradiated gels and found that there is very minimal difference in temperature between untreated and treated samples (Fig. 7C & 7D). The mechanical damage caused in the hydrogel was also demonstrated with SEM images of the gels exposed to HIFU (Fig. 7E – 7H). We have exploited the mechanical destruction of hydrogel utilizing the HIFU to trigger the gallic acid release from PG-Mg-Gal and our observation clearly mentioned that HIFU had enhanced the gallic acid release (Fig. 8 A-F). The experiments were conducted at intensities 2 & 4 (250 mW & 750 mW) as per the user manual provided by the manufacturer and the scheme of the experiment was illustrated in Fig. 8A – 8C. There was an increase in gallic acid release at 750 mW compared to 250 mW. Amount of gallic acid release at both the intensities were showing drastic increase until 40 mins and attained saturation until 90 mins. The amount of gallic acid released at 750 mW HIFU irradiation was 2 folds higher than 250 mW throughout the study period. In the first 10 mins HIFU with 750 mW showed 19.4% gallic acid release, which was significantly higher than 250mW (11.6%). The same pattern was observed in 40 mins where the gallic acid release was 46.09% with 750mW 4 and ∼26.6% 250 mW (Fig. 8D).Fig. 7. Characterization of HIFU parameter and HIFU induced temperature monitoring. (A) – Acoustic power output as a function of intensity denoted as per manufacturer; (B) – Linearity of acoustic power with increase in intensity; (C) − Infrared thermal images (FLIR) showing temperature of the hydrogel; (D) – Change in temperature of the hydrogel samples over time after HIFU exposure compared to control. The results were expressed as mean ± SD (n = 3) and statistically analyzed by One-Way ANOVA with Tukey’s multiple comparison post test. The comparison was made between Control & HIFU (250 mWatts); Control & HIFU (750 mWatts) and HIFU (250 mWatts) & HIFU (750 mWatts). The statistical significance was denoted as p < 0.05 () & Not significant (ns). (E) – SEM image of untreated hydrogel; (F) – SEM image of hydrogel soaked in PBS for 10 mins; (G & H) – SEM images of hydrogel exposed to 250 mWatts (G) & 750 mWatts (H) HIFU for 10 mins.Fig. 8HIFU triggered gallic acid release from PG-Mg-Gal. (A − C) – Illustration of experimental set up (A), steps involved in the experimental process (B) and overall experimental outcome in the study (C); (D) – Cumulative release of gallic acid after HIFU irradiation at acoustic powers of 250 & 750 mWatts; (E) – On-demand release of gallic acid after each cycle of HIFU irradiation; (F) – Comparative analysis of gallic acid release with and without HIFU irradiation. The results were expressed as mean ± SD (n = 3) and statistically analyzed by One-Way ANOVA with Tukey’s multiple comparison post test. The comparison was made between HIFU (250 mWatts) & HIFU (750 mWatts) at 90 mins (D); HIFU (250 mWatts) & HIFU (750 mWatts) 30 mins after each HIFU cycle (E); Untreated and HIFU treated hydrogels at 90 mins. The statistical significance was denoted as p < 0.0001 (****); p < 0.01 ().
Acoustic power directly correlates to radiation force exerted by the ultrasound wave motion on the medium, which is assumed to be one of the key mechanisms for drug release in our study which causes disruption of the interaction between Mg, gallic acid and side chains of the polymers (gelatin & PVP) [68]. Similar studies were conducted on hydrogel with different polymeric compositions like hyaluronic acid, xanthan gum, sodium alginate, etc and they reported rapid drug release with influence of ultrasound which supports our observation in the current study [69], [70], [71]. Further, we wanted to examine the suitability of PG-Mg-Gal for HIFU triggered on-demand drug release pattern and we observed that major amount of gallic acid was released in the first cycle of HIFU irradiation which comprised of 30 s of US irradiation followed by monitoring gallic acid release for 30 mins (Fig. 8E). There is a gradual decrease release of gallic acid in the subsequent cycles of HIFU, which clearly showed the complete disorientation Mg-Gal crosslinked hydrogel network and almost 90% release of gallic acid by the end of 3rd cycle of HIFU. These results indicate that unlimited on-demand release cannot be claimed from the current study, but a pulsatile release pattern until complete depletion of gallic acid from the hydrogel.
Polymeric materials used in the hydrogel plays a key role in exhibiting the on-demand drug delivery property and in our study the responsiveness of hydrogel to ultrasound was obviously demonstrated with amount of gallic acid release. Alginate based hydrogel, liposomal and microbubble formulations were reported to show on-demand drug release patterns that could help in personalized therapeutic strategy for conditions like, diabetes, wound healing, pain management and etc [72]. Fig. 8F depicts the comparative observation of drug release pattern between HIFU irradiated and untreated PG-Mg-Gal, where the gallic acid release from the untreated gel was 17.7% after 90 mins, while the hydrogel under HIFU irradiation showed enhanced drug release at an acoustic power of 750 mW (53.7%). Among the different acoustic powers utilized in our study, 250mW at intensity 2 showed significantly lower drug release (27.4%) than intensity 4. However, the release was statistically higher than the untreated gel. These results suggests that by utilizing the mechanical effect of HIFU, we can improve the drug release from PG-Mg-Gal. Further, we wanted to demonstrate the accelarated bioactivity of Mg-Gal from the hydrogel through acoustic burst of gallic acid triggered by HIFU.
HIFU mediated cytotoxicity of PG-Mg-Gal
3.7
Enhanced bioactivity of PG-Mg-Gal with and with out HIFU irradiation was depicted in Fig. 9. Quantification of viable and dead cells after incubation with PG-Mg-Gal without HIFU irradiation and PG-Mg-Gal exposed to HIFU was performed using Live/Dead cells staining assay. According to Fig. 8D, maximum release of gallic acid was achieved by HIFU in 90 mins and hence, we wanted to study the percentage of dead cells after 4 hrs for demonstrating the effect of HIFU in enhancing the bioactivity of hydrogel in a shorter period. Fig. 9A shows that at the end of 4 hrs the number of dead cells (red staining) were significantly higher in the group where hydrogel was treated with HIFU compared to untreated hydrogel. This indicated the rapid release of gallic acid from HIFU treated hydrogel resulting in enhanced and early cell death with shorter incubation time (4 hrs). Conversely, the after 24 hrs, the number of viable cells were reduced significantly in both the HIFU treated and untreated hydrogel. This shows there is a saturated release of gallic acid rendering consistent bioactivity when the hydrogels were incubated for longer time. Gallic acid being a potent anti-cancer agent was reported to upregulate the pro-apoptotic pathways and the initiation of cytotoxicity should have achieved with disrupting the membrane permeability leading imbalance in cellular processes [53], [73]. Increased cell death in HIFU treated PG-Mg-Gal at 4 hrs denotes the increase in gallic acid release than untreated hydrogel leading to accelerated upregulation of cytotoxic pathways in the cells than the untreated hydrogel with lesser cell death at 4 hrs due to reduced gallic acid release. Percentage of dead cells in 24 hrs was the highest in both HIFU treated and untreated hydrogel which is when a consistent release of gallic acid was achieved.Fig. 9HIFU induced cytotoxicity of PG-Mg-Gal using live/dead cells staining. (A) – Live/Dead cells staining images of different experimental groups observed at 4 hrs & 24 hrs (Scale bar − 10 μm). (B) – Graphical representation of percentage of dead cells quantified using ImageJ. The results were expressed as mean ± SD (n = 3) and statistically analyzed by One-Way ANOVA with Tukey’s multiple comparison post test. The comparison was made between all the experimental groups at 4hrs & 24 hrs. The statistical significance was denoted as p < 0.0001 (****); p < 0.01 (**); Not significant (ns).
ImageJ software was utilized to count the number of dead cells observed in each group and Fig. 9B depicts the percentage of dead cells among the total number of cells. The graph shows that there is no significant number of dead cells in control, negative control (only HIFU irradiation) and untreated hydrogel groups showing 0.3%, 2.7% & 0.6% respectively, at the end of 4 hrs. Hydrogel exposed to HIFU treatment showed ∼14.1% of dead cells which is significantly higher than the other groups. Cells stained after 24 hrs showed there is no major difference between hydrogel treated and untreated groups (84.3% & 80.3%, respectively), and they are significantly higher than the control and negative control groups (0.7% & 1.1%, respectively). These results showed at 4 hrs HIFU treated gel resulted in enhanced anti-cancer activity compared to untreated gel due to burst release of gallic acid. While at 24 hrs due to consistent release of gallic acid in both HIFU treated and untreated hydrogels, the bioactivity was almost comparable.
Conclusion
4
Our study summarizes, the ability of Mg-Gal to stabilize the polymeric network (gelatin & PVP) by chemical cross-linkages at the functional groups to form a hybrid polymeric hydrogel. The physico-chemical characterization of Mg-Gal & PG-Mg-Gal revealed a highly crosslinked network of the hydrogel with Mg-Gal. Mechanical characterization of PG-Mg-Gal showed better compression strength with appreciable elasticity. Water absorption and retention studies clearly indicated the porous nature of the hydrogel that can ably accommodate water molecules and there was a significant responsiveness of hydrogel observed under varied pH & temperature conditions. Mg-Gal showed relatively better cytotoxic effect on 4T1 breast cancer cells than PG-Mg-Gal, while increase in the concentration of the hydrogel with increase in Mg-Gal content, in turn improved the cytotoxic and anti-migration effect. Both the hydrogel formulation and its components showed excellent biocompatibility at the stipulated concentration where anti-cancer properties were reported. HIFU triggered burst release pattern was studied and the results indicated maximum release rate was observed around 2 hrs, with minimal/no apparent increase in temperature. Our observation indicated depletion of drug concentration from the hydrogel across cycles and hence on-demand release pattern demonstrated in this study we cannot claim an unlimited release of gallic acid. Although current study demonstrates the role of mechano-physical effect in drug release process, the influence of cavitation and microstreaming cannot be ruled out among the mechanisms of gallic acid release, which will be explored with our future experiments. The cytotoxic potential of hydrogel irradiated with HIFU showed better results compared to untreated hydrogel demonstrating the accelerated cytotoxic effect of gallic acid at a shorter incubation time. In conclusion, the acoustic degradation of PG-Mg-Gal has resulted in burst release of gallic acid and this enhanced the biological property of the drug. The dual property of the material to be a crosslinker and the bioactive materials shows the uniqueness of the hydrogel. Hence, when the crosslinking interaction was disrupted by external force, the release of drug happens rapidly with degradation of crosslinking network in the hydrogel. Further, optimization of polymer and crosslinker compositions are required for in vivo applications of the hydrogel for management of multiple ailments including cancer.
Funding information
This work was supported, in part, by National Research Foundation of Korea (NRF) grants funded by the Ministry of Science and ICT, Republic of Korea (2022R1A5A8023404, and RS-2024-00338853) and, in part, by the Samsung Research Funding Center of Samsung Electronics (No. SRFC-IT2202-02).
Data availability
The original contributions are included in the article. The data have not been uploaded to a public repository. However, the authors are willing to share them upon request.
CRediT authorship contribution statement
Badrinathan Sridharan: Writing – review & editing, Writing – original draft, Visualization, Validation, Software, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Cho Eun Lee: Writing – review & editing, Writing – original draft, Visualization, Validation, Software, Methodology, Investigation, Formal analysis, Data curation. Daehun Kim: Writing – review & editing, Writing – original draft, Validation, Software, Methodology, Investigation, Formal analysis, Data curation. Jin Hyeong Park: Writing – review & editing, Writing – original draft, Software, Methodology, Investigation, Formal analysis, Data curation. Wooram Um: Writing – review & editing, Writing – original draft, Validation, Supervision, Resources, Project administration, Funding acquisition, Conceptualization. Seung Yun Nam: Writing – review & editing, Writing – original draft, Validation, Supervision, Resources, Project administration, Funding acquisition, Conceptualization. Juhyun Kang: Writing – review & editing, Writing – original draft, Visualization, Validation, Software, Methodology, Investigation, Data curation. Hae Gyun Lim: Writing – review & editing, Writing – original draft, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Conceptualization.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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