In vitro and in vivo applications of a universal and synthetic thermo-responsive drug delivery hydrogel platform

thermo-responsive


Introduction
Sustained drug release has emerged as an important approach for drug delivery as it offers numerous advantages including maintenance of the drug level at a desired range, preventing overdosing, minimizing potential adverse side effects and drug toxicity.Moreover, sustained drug delivery can improve patient compliance by reducing the number of dosage administrations (Adepu and Ramakrishna, 2021).Hydrogels may have promising potentials as drug delivery vehicles due to their ability to encapsulate a wide range of therapeutics while preserving their stability (Ciolacu et al., 2020;Lin and Metters, 2006;Patel et al., 2019;Sosnik and Seremeta, 2017).Importantly, some hydrogel systems have shown potentials to release therapeutics at a predictable rate and significantly improve the drug bioavailability as well as overcoming the rapid elimination of certain delivered drug to achieve the intended therapeutic outcome (Alasvand et al., 2017).
Among numerous polymeric hydrogel-based drug delivery matrices, thermo-responsive hydrogel systems are recognized as a promising modality for sustained drug release due to their in situ gel-forming ability in response to the physiological temperature (Patel et al., 2019).This particular characteristic is especially advantageous from a clinical usability point of view as formulations can be easily handled in a clinical setting.Post-administration, these thermo-responsive formulations form a hydrogel network without the need for addition of other chemicals or reagents to induce the gelation process.Further, these hydrogel systems must be ideally adhesive to the target site to allow sustained and localized drug release (Jeong et al., 2002) and to enhance its bioavailability by prolonging the residence time of the drug (Ciolacu et al., 2020).While, a plethora of thermo-responsive polymers have been synthesized, such as chitosan, cellulose and pluronic F127, none has been widely adopted due to the lack of adhesivity and stability (Pollock and Healy, 2010), unpredictable biodegradation characteristics (Bayat et al., 2017), and severe inflammatory response to the carrier systems (Lian et al., 2012).
We hypothesized that these unmet challenges can be addressed by using poly(N-isopropylacrylamide)-co-(polylactide/2-hydroxy methacrylate)-co-(oligo (ethylene glycol)), denoted as PNPHO that has been clinically used for tissue regeneration and exhibited structural stability, tunable biodegradation characteristics, and favorable biocompatibility (Calder et al., 2022;Dehghani et al., 2017;Fathi et al., 2014).As such, a unique molecular structure of PNPHO copolymer is engineered to optimize the properties of the product for drug delivery applications.This research, for the first time, studies the potential of using PNPHO as a universal platform (denoted as TP) for sustained drug delivery.The cytotoxicity of TP as a suitable platform for drug delivery was evaluated by using in vitro models of human bronchial and nasal epithelia cells (Horváth et al., 2016).The platform nature of TP as a drug delivery vehicle was systematically investigated by determining encapsulation capabilities of a range of bioactive molecules with different molecular weights, chemical compositions, and bioactivities.The tested therapeutics include ciprofloxacin HCl (Cip; 0.37 kDa), tumor necrosis factoralpha (TNF-α; 17 kDa), transforming growth factor-beta 1 (TGF-β1; 25 kDa), and human bone morphogenetic protein 2 (BMP2; 26 kDa).The release profiles of these moieties from TP matrix were evaluated in vitro and their therapeutic efficiency after encapsulation within TP was assessed both in vitro and in vivo.

Preparation of the TP hydrogel platform
PNPHO polymer was supplied by Tetratherix TM (Sydney, Australia) in a solid, freeze-dried, powder form.The polymer's chemical composition is 81 mol% N-isopropylacrylamide, 7 mol% polylactide/2hydroxy methacrylate, with lactate length of 5, 7 mol% N-acryloxy succinimide ester and 5 mol% oligo (ethylene glycol).The synthesis and preparation of the polymer are reported elsewhere (Dehghani et al., 2017;Fathi et al., 2014).Briefly, reactant monomers and macromonomers with the specified feed ratios were dissolved in dimethyl formamide and reacted by using 4,4 ′ -Azobis(4-cyanovaleric acid) as an initiator at 70 • C for 24 h.The synthesised polymer was then purified in water and freeze dried.The freeze-dried PNPHO polymer was stored in a sealed container at (2-10 • C).TP140 carrier solution is formulated by dissolving 140 mg/ml of PNPHO polymer in PBS at 4 • C for 48 h.The carrier system is a single-phase transparent solution that is flowable through 18G needle via thumb pressure and transitions into a hydrogel phase at 37 • C.

Encapsulation of drugs within TP140 hydrogel
Different bioactive molecules were encapsulated within the TP polymeric matrix to evaluate its applicability for sustained drug delivery.To prepare TP140 incorporated with Cip as a model drug and TNF-α, TFG-β, and BMP2 cytokines, a solution of the drug or cytokines in cold PBS (4 • C) was first prepared.Then, an appropriate amount of the freeze-dried PNPHO polymer was dissolved in the solution at 140 mg/ mL concentration.In case of Cip solution in PBS, a stock solution in water was initially prepared followed by dilution in PBS.The encapsulated drug or cytokine concentrations in the final mixtures (TP140/Cip, TP140/TNF-α, TP140/TFG-β, and TP140/BMP2) were as follows: Cip, 20 mg/mL; TNF-α, 250 ng/mL; TFG-β, 250 ng/mL; and BMP2, 0.5 mg/ mL.These amounts were used based on the known solubility of these therapeutics in aqueous media, as well as the encapsulation loads reported in the literature for sustained drug delivery (Pollock and Healy, 2010;Re'em et al., 2012;Varanda et al., 2006).Each TP140/drug mixture was continuously stirred for 48 h at 4 • C to obtain a homogeneous clear solution.

Zeta potential and particles size measurement
The surface charge of the tested therapeutics was measured by using a zetasizer instrument (Zetasizer Nano ZS, Malvern Panalytical, UK).A solution of each drug and TP in PBS was prepared (TNF-α: 250 pg/mL, TGF-β1: 150 pg/mL, BMP2: 0.4 mg/mL, TP: 140 mg/mL).The prepared solutions of TNF-α TGF-β1, and BMP2 were diluted 10X and transferred to a polystyrene capillary zeta cell (DTS1070, Malvern Panalytical, UK).The measurements were performed at room temperature (25 • C) in triplicate and the average zeta potential is reported.The measurement for TP140 was performed at 15 • C to prevent thermal gelation.
The particles size was measured by dynamic light scattering for TP140, empty and loaded with the test drugs by using the dynamic light scattering instrument (Zetasizer Nano ZS, Malvern Panalytical, UK).To do so, the TP140 as blank and TP140 loaded with drugs were prepared according to section 2.3 and transferred (~1.5 mL) into a polystyrene cuvette (DTS0012, Malvern Panalytical, UK).The measurements were performed in triplicate at 15 • C to prevent the thermal gelation during the measurements and the average particles size is reported.

Thermal gelation time of TP140
The gelation time of TP140 as blank and loaded with the drugs was measured by using a vial tilting method.As such, 0.5 mL of the TP140, TP140/Cip, TP140/TNF-α, TP140/TGF-β1, or TP140/BMP2 solutions were transferred into Eppendorf tubes and the tubes were incubated in a water bath at 37 • C. The gel formation was monitored by inverting the tubes at every 20 s in addition to observing the colour change from a transparent solution to a translucent semi-solid gel.The gelation time was recorded when no flow was observed as the tubes were inverted, and a uniform colour change was observable throughout the corresponding solutions.

Cell culture
A healthy bronchial epithelial cell line (BEAS-2B; CRL-9609, ATCC, USA) was grown in DMEM supplemented with 10% v/v of FBS and 1% v/v of 2 mM L-glutamine solution.Nasal epithelial cell line (RPMI-2650; CCL-30, ATCC, USA) was maintained in MEM supplemented with 10% v/v of FBS and 1% v/v of 2 mM L-glutamine.The human lung fibroblast cell line (MRC-5; CCL-171, ATCC, USA) was maintained in MEM supplemented with 10% (v/v) FBS, 1% v/v of 2 mM L-glutamine, 1 mM sodium pyruvate, and 1.5 g/L sodium bicarbonate as recommended by the supplier.Cells were incubated at 37 • C, 5% CO 2 , and 95% humidity.The cell growth media was changed every 3 days and the confluent cells were passaged according to the manufacturer's recommendations.

Cytotoxicity assay
The safety of TP140 hydrogel was evaluated by assessing its potential toxic interactions with bronchial epithelial BEAS-2B and nasal epithelial RPMI-2650 cells using two MTS and live/dead cell staining assays.The cells were first exposed to TP140 leachables and then, the cellular metabolic activity was evaluated by MTS assay, as well as obtaining microscopic images from the stained live and dead cells.

MTS assay
BEAS-2B and RPMI-2650 cells were seeded in a 24-well plate at 4.2 × 10 4 and 1.26 × 10 5 cells/cm 2 densities, respectively and incubated for 24 h (37 • C and 5% CO 2 ).To treat the cells with the TP140, 100 μL of TP140 solution was transferred into the apical chamber of empty Transwell inserts and allowed for thermo-responsive gel formation for 30 min at 37 • C.During incubation, the Transwell inserts were placed on a flat surface to avoid any potential leakage through the Transwell membrane.The Transwell inserts with the gel formed in the apical chamber were placed in a 24-well cell culture plate, where the cells were cultured in the basolateral chambers.Fresh MEM media (100 μL) was added on top of the hydrogels in the apical chamber of the Transwell inserts and to the basolateral chamber (600 µL).The cells were incubated further for 24 and 48 h.Empty Transwell inserts with either fresh growth media or 20% v/v dimethyl sulfoxide (DMSO) were used as negative and positive controls, respectively.After the incubation time, the growth media on top of the cells was removed and the cells were rinsed with PBS.Then, the MTS reagent was added to the wells followed by 2 h incubation time.Afterwards, the colour intensity of the reagent in each well was measured by determining the absorbance at 490 nm using a microplate reader (Spectromax M2; Molecular Devices, USA).Cell viability was then calculated as the percentage of the measured colour intensity as compared to the untreated cells (negative control) for each time point.

Live/dead cell staining
Live/dead cell staining was performed to assess the potential cytotoxicity of the TP140 hydrogel on BEAS-2B and RPMI-2650 cell lines after 24 and 48 h treatment periods.The cells were treated with TP140 hydrogel following the procedure outlined in section 2.7.1 for the viability MTS assay.The cells were then treated with calcein-AM (4 µM, Invitrogen, Australia), Ethidium homodimer-1 (EthD, 4 µM, Invitrogen, Australia), and Hoechst (25 ng/mL, Sigma Aldrich, Australia) and incubated according to the manufacturer's instructions to stain the viable cells, dead cells, and the nuclei, respectively.Microscopic images were immediately taken after incubation using a time-lapse microscope (Nikon Eclipse Ti, Japan) equipped with a CoolSNAP ES2 highresolution digital camera (Photometrics, USA).

Inflammatory effects of TP140 hydrogel in vitro
To assess the suitability of TP140 as a drug delivery carrier, its potential anti-and pro-inflammatory effects were studied in vitro using BEAS-2B bronchial epithelial cells.

Pro-inflammatory effect of TP140 hydrogel
The pro-inflammatory effect of the TP140 hydrogel was evaluated by treating the BEAS-2B cells with the TP140 hydrogel using Transwell inserts similar to the procedure explained for the indirect cytotoxicity assay (section 2.7).After 24 and 48 h of treatment, the basolateral media was collected, centrifuged (1000 rfc, 10 min) and stored at − 24 ℃ until further analysis.Lipopolysaccharide (LPS) from E. coli at 2 μg/mL concentration and cell growth media were used as positive and negative controls, respectively.Samples were analysed by ELISA to quantify the secreted IL-6 and IL-8 inflammatory cytokines after the cells were treated with the TP140 and the control.

Anti-inflammatory effect of TP140 hydrogel
The anti-inflammatory effect of TP140 hydrogel was evaluated by treating the BEAS-2B cells that were pre-stimulated with TNF-α.BEAS-2B cells were seeded at a density of 4.2 × 10 4 cells/cm 2 in 24-well polystyrene culture plates and incubated at 5% CO 2 and 37 • C for h.Cells were stimulated for 24 h with TNF-α (2 ng/mL) prepared in 0.5% (v/v) bovine serum albumin (BSA).Afterwards, the hydrogels prepared in Transwells, as outlined in section 2.7, were transferred to the 24-well plates containing the pre-stimulated cells.Fresh cell growth media (100 μL) was then added to the apical chamber on top of the formed hydrogel and the basolateral chamber (600 µL), followed by incubation at 5% CO and 37 • C for 24 and 48 h.Empty Transwell inserts with cell growth media were also used as control.After incubation, samples were collected from the basolateral chamber, centrifuged (1000 rfc, 10 min), and stored at − 24 ℃ until further analysis.Samples were analysed to quantify the secretion of IL-6 and IL-8 using ELISA.

Burst and sustained release of therapeutics from TP140 hydrogel
The burst release was considered as the mass of bioactive molecule expelled from the TP140 during gel formation.To evaluate this released mass, TP140 was loaded with different bioactive molecules (Cip, TNF-α, TGF-β1, and BMP2).Then, 200 μL of TP140/therapeutic mixture was transferred to the apical chambers of Snapwell inserts and allowed for gel formation at 37 • C for 30 min.During this time, the Snapwell inserts were placed on a flat surface to avoid any potential leakage through the membrane of the Snapwells before gel formation.After gelation, the liquid expelled from the gel was gently collected from the top of the gel and analysed to quantify the mass of the released bioactive molecule.The quantified mass was considered as the amount of burst release (unencapsulated mass).This amount was then used to evaluate the encapsulation efficacy (EE) for each of the therapeutics.The EE was calculated based on the following formula (Newton and Kaur, 2019;Xu et al., 2012) after measuring the burst release in the sample collected from the supernatant liquid on top of the formed gel, where m L is the mass of the therapeutic loaded within the TP140 hydrogel and m R is the mass of the therapeutic released from the TP140 hydrogel via the burst release.
To evaluate the sustained drug release from the TP140 hydrogel, the Snapwell inserts with the formed hydrogel in the apical chamber were placed in a 6-well plate and 2 mL of pre-warmed PBS (37 • C) was added to the basolateral chambers.The plate was then incubated at 37 • C under constant 60 rpm orbital shaking.Samples of 200 μL were withdrawn from the basolateral media at different time points (i.e., every hour for the first 6 h followed by every 24 h thereafter up to 120 h).After each sample collection, an equal amount of fresh pre-warmed PBS was replaced in the basolateral chambers.Samples were then analysed to quantify the released mass of encapsulated therapeutic.

Quantification of therapeutics
Cip was quantified based on a validated High-Performance Liquid Chromatography (HPLC) method using a Shimadzu Prominence UFLC system equipped with a LC20AT pump, SIL20AHT autosampler and SPD-20A UV-VIS detector (Shimadzu, Sydney, NSW, Australia).The quantification was performed in isocratic mode by using a reverse-phase Luna C-18 Column (Phenomenex, Torrance, USA) with 150 × 4.6 mm dimensions and 3 µm particle size.The mobile phase was prepared as phosphate buffer (pH ~ 7.2) and acetonitrile mixed at 75: 25 volumetric ratio.The mobile phase flow rate was 0.7 mL/min with detection at 275 nm wavelength.An injection volume of 100 µL was applied and Cip eluted at 2.4 min.A standard curve was obtained by freshly prepared standards with 0.05 -100 µg/mL concentrations of Cip in a mixture of acetonitrile and Milli-Q water at 50: 50 volumetric ratio.The linearity was obtained with R 2 ≥ 0.999.TNF-α, TGF-β1, and BMP2 were quantified by ELISA.

Bioactivity of the released therapeutics from TP140 hydrogel
The bioactivity of the released drug or cytokines was assessed to determine the therapeutic efficacy of TP140 loaded with bioactive molecules.

Antibacterial activity of the Cip encapsulated within TP140 hydrogel
The antibacterial activity of TP140/Cip hydrogel against Staphylococcus aureus (S. aureus; ATCC 29213) was evaluated by broth microdilution assay according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (Wiegand et al., 2008;Wikler, 2006).Bacteria were streaked from glycerol-frozen stocks onto Luria-Bertani (LB) agar plates and incubated overnight at 37 • C. For each experiment, overnight liquid cultures of S. aureus were prepared in CaMHb at 37 • C and 200 rpm.Overnight liquid cultures were diluted (1:100) in fresh media and allowed to grow to mid-log phase (~2h) and then standardized to 1 × 10 6 CFU/mL.
Cip released from the TP140/Cip hydrogel into PBS (section 2.9) upon 48 h incubation was collected and filtered to remove the hydrogel debris, followed by quantifying the amount of released Cip by HPLC (section 2.10).Similarly, a control sample was prepared by using TP140.The control and treatment dilutions (Cip released from TP140/Cip, TP140 extract, and free Cip) were prepared in a 96-well plate by twofold serial dilutions in 100 µL of CaMHb.The 96-well plate with the dilutions was inoculated with 100 µL of the standardized bacterial suspension to make a final inoculum of 5 × 10 5 CFU/mL.The plates were then sealed with AeraSeal™ sterile film (Sigma Aldrich, Australia) and incubated for 20 h at 37 • C. The inoculum concentration was verified by plating the diluted standardized suspension on LB agar plates immediately after inoculation and counting the colonies on the following day.The bacterial growth was quantified by measuring the optical density (OD) at 600 nm.The minimum inhibitory concentration (MIC) was determined as the lowest concentration with clear wells and visible inhibited cells growth.The minimum bactericidal concentration (MBC) was determined by plating 10 µL of each of the clear wells with no bacterial growth on LB agar followed by incubation for 24 h at 37 • C. The MBC value was then determined as the lowest concentration where no bacterial growth was observed.

Pro-inflammatory activity of TNF-α and TGF-β1 encapsulated within TP140 hydrogel
The bioactivity of the released TNF-α and TGF-β1 from TP140 hydrogel was evaluated in terms of the pro-inflammatory effect on MRC-5 healthy lung fibroblast cells.MRC-5 cells were seeded at a density of 9.38 • × 10 4 • cells/cm 2 in a 24-well culture plate and incubated (5% CO 2 , 37 • C, and 95% RH) for 24 h.Cells were serum-starved (0.5% • FBS) for 24 h and exposed to the treatments (TP140/TNF-α and TP140/TGF-β1) and controls (free TNF-α and TGF-β1 solutions in the cell growth media, and empty TP140 loaded with cell growth media) for 24 h.The treatments (200 µL) were transferred into the apical chambers of the Transwell inserts and placed in the 24-well plate, where the MRC-5 cells were cultured in the basolateral chambers to treat the cells with TNF-α or TGF-β1 released from the hydrogels in the apical chambers.Treatments were performed for 24 h and afterwards, samples were collected from the basolateral chamber and stored at − 24 ℃ before quantification of the secreted IL-6 and IL-8 by ELISA.

Bone regeneration activity of BMP2 encapsulated within TP140 hydrogel
The bioactivity of TP140/BMP2 hydrogel was evaluated by determining the bone formation in a mouse ectopic osteogenesis model implant.
2.11.3.1.Animal model and surgical procedure.Experiments were conducted following the guidelines set out in the National Health and Medical Research Council, Australian Code of Practice for the Care and Use of Animals for Scientific Purposes, 8th edition, 2013 (Health et al., 2013).The study has been approved by Sydney Local Health District Animal ethic committee (ethical approval no.2017/026).
Twenty male Balb/c mice (10-week-old) were randomly divided into 2 groups of 10 animals.Each animal had two intramuscular implants with bilateral placement in the muscle pouches of right and left hind limbs.Two different formulations were tested: TP140/BMP2 (0.015 mg of BMP2) and control (0.045 mg of BMP2, in standard collagen film).TP140/BMP2 treated sites were administered via direct injection of the formulations to the pre-formed muscle pouches.+ Control group animals were implanted with collagen membranes, pre-soaked in BMP2 for 30 min prior to the surgeries.The mice were radiographed (X-ray) at week 0, as well as weeks 2, 4 and 6 post-implantation.Eventually, the implanted hydrogels from all animals were harvested from the implant sites and fixed by 4% paraformaldehyde (PFA) for imaging purposes and histological evaluations.

Specimen analysis.
Representative explants showing bone formation, based on radiography, were taken for micro-CT scanning.Xray images of ectopic bone formation were taken by the Faxitron (MX 20/DX 50, Wheeling, USA) and the density evaluated using Fiji software (ImageJ).Micro-CT scan was obtained using Skyscan 1172 scanner (SkyScan, Kontich, Belgium) and the reconstruction of raw micro-CT sections was performed by GPU Accelerated NRecon software (Sky-Scan, Kontich, Belgium).CTVol software (Skyscan, Kontich, Belgium) was used for 3D image reconstruction.
Histological preparation for microscopic analysis was performed using the Histostar® tissue processing (Thermo Fisher Scientific, Waltham, MA, USA) and the paraffin blocks were cut using a Leica RM 2125 RT microtome (Leica Microsystems GmbH, Wetzlar, Germany).Sectioned samples were stained with Hematoxyline and Eosin (H&E) and analysed using a BX60 F-3 microscope (Olympus Optical Co., Ltd., Japan).

Data analysis
Data are expressed as mean ± SD of triplicate experiments.Statistical analyses were performed using GraphPad Prism 5 software.Unpaired t-test and analysis of variance (ANOVA) were performed to compare the means of two and more than two data sets, respectively.The statistical significance was considered as p < 0.05.NS in figures represents no statistical difference and asterisk sign (*) represents statistically significant differences, corresponding to identified p values (*p ≤ 0.05, ** p ≤ 0.01, and ***p ≤ 0.001).

Surface charge of the tested therapeutics and TP140
Surface charge is a critical parameter in compound encapsulation and efficiency due to its role in formation of ionic bonds and the resulting particle size (Mackenzie et al., 2015).To this end, the compounds tested in combination with TP140 as model therapeutics cover a wide range of surface charges.The results from measuring the net charge of the tested therapeutics and TP140 are shown in Table 1.The results showed that TP140 is negatively charged, and cytokines (TNF-α, TGF-β1, and BMP2) possessed a wide range of negative surface charges (p < 0.0001) while Cip molecules are positively charged in the prepared solution with pH 4.6 (Kooti et al., 2018).The use of wide range of compounds with varying surface charges enables the assessment of TP's potential and suitability for the delivery of different drug molecules across different therapeutic areas, e.g.anti-bacterial and tissue regeneration.

Effect of drug encapsulation on TP140 characteristics
The gelation kinetics of TP carrier and its particle size are critical parameters for effective drug delivery applications.As such, the interaction of the loaded therapeutics with TP140 carrier and its impact on gelation and particle size are investigated in this section.

Thermo-responsive gelation time
In order to evaluate whether TP140 maintains its thermo-responsive properties after drug loading, the gelation time was measured prior to and after the addition of therapeutic compounds.All solutions, regardless of the loaded therapeutics, transitioned from transparent to an opaque solution within 3-4 s of exposure to 37 • C in an incubator and required ~ 40 s to form a homogeneous hydrogel.The recorded gelation times showed that no significant difference (0.1323 ≤p ≤ 0.8416) was observed between the gelation time of the TP140 (without any encapsulated therapeutics) and TP140 loaded with the different therapeutics.It is important to note that the heat transfer in this benchtop testing was by convection that could be different from the body fluid and consequently, the rate of hydrogel formation was slower to that expected in vivo.Nonetheless, the gelation time study confirmed that TP140 maintains its gelation characteristics and the addition of loaded compounds did not interfere with the gelation kinetics.

Particle size
The particle size of TP140 solutions, loaded with different therapeutics was characterised to further understand the potential interactions of the tested compounds with the polymer.As such, the particles size was measured before and after drug loading.No particle was detected for solutions of TP140, and TP140/Cip at 15 • C. While, for particle size of TP140/TNF-α, TP140/TGF-β1, and TP140/BMP2, particles were found to be 9.85 ± 3.06, 12.52 ± 6.14, and 164.23 ± 25.63 nm, respectively.The particles size detected for TP140/TNF-α and TP140/TGF-β1 were found statistically similar (p = 0.5364).However, the particles size detected for TP140/BMP2 was significantly larger than the sizes determined for both TP140/TNF-α and TP140/TGF-β1 (p = 0.0005 and p = 0.0006, respectively).This could be related to the large size, high molecular weight (26 kDa), and protein folding of BMP2, which could have resulted in observation of larger particles when it was loaded into TP140.Nonetheless, the noticed differences in the particle size of the solutions had no impact on the ability of the carrier system to be administered via injection.More importantly, in vivo injectability of TP140/BMP2 was assessed in the following section to challenge the carrier system for delivery and release kinetics.

Biological characterization of the TP140 hydrogel in vitro
Cytotoxicity and severe inflammatory responses to any drug carrier system are undesirable and deter the intended mechanism of action of the incorporated therapeutics.As such, the cytotoxicity of TP140 as a carrier system for drug delivery and its potential inflammatory properties were evaluated, using respiratory tract cells due to their sensitivity to cytotoxic and inflammatory properties of carrier systems and drugs.

Cytotoxicity of TP140
The cytotoxicity study was performed both by determining the metabolic activity of the cells with the MTS assay and Live/Dead™ staining of the cells treated with TP140.The results of MTS assay on the nasal and bronchial cells treated with TP140 for 24 and 48 h are presented in Fig. 1-A and -B, respectively.It was found that the viability of both nasal and bronchial cells treated with TP140 for 24 h was not significantly different (p = 0.4735 for nasal and p = 0.1493 for bronchial cells) to that measured in the − Control (DMEM media only).Similarly, after 48 h of treatment, the viability of nasal epithelial cells remained unchanged compared to the − Control, and the viability of the bronchial epithelial cells was only slightly decreased to 87.13 ± 6.55% (p = 0.0476).Accordingly, the results showed that TP140 did not have any appreciable toxic effect on either of the tested cell lines.
The microscopic images obtained from the live/dead cell staining are presented in Fig. 2A and -B for 24 h and 48 h of treatment, respectively.The live cells are stained green, whereas dead cells and nuclei are in red and blue, respectively.Based on the microscopic images, the population of the dead cells was increased in the images taken after 48 h of treatment for both nasal and bronchial epithelial cells compared to 24 h treatment.This result further endorses the findings from the MTS assay.Importantly, the visual comparison of images obtained from TP140 treatment and the − Control cells show similar viability of cells.Therefore, it can be concluded that TP140 is a safe carrier platform for drug delivery and does not exhibit toxic effect on the cells.

Inflammatory effects of TP140 hydrogel
Pro-inflammatory properties of a carrier system can potentially interfere with loaded therapeutics and their intended mechanism of action at the host site (Lian et al., 2012).As such, the pro-and antiinflammatory effects of the TP140 hydrogel on the bronchial epithelial cells were evaluated by determining the secretion of IL-6 and IL-8 inflammatory cytokines over 24 h and 48 h post-treatment with TP140.The results in Fig. 3-A and -B showed that cells treated with LPS (+Control) expressed significantly higher level of IL-6 and IL-8 levels (p ≤ 0.001 and p ≤ 0.046, respectively) compared to those in − Control

Table 1
The Zeta potential values measured for TP140 solution and the free solutions of the therapeutics used in this study to investigate the application of TP140 for drug encapsulation (n = 3, mean ± SD). (media only).These findings validated the effectiveness of this in vitro investigation to assess inflammatory properties of TP140.More importantly, the level of IL-6 (Fig. 3-A) and IL-8 (Fig. 3-B) in the treated cells with TP140 was similar (p ≥ 0.0790) to those in the − Control (media only) and thus confirmed that TP140 did not induce any proinflammatory response in bronchial epithelial cells.
To assess the anti-inflammatory properties of TP140, epithelial cells were stimulated with pro-inflammatory TNF-α and were subsequently treated with TP140.The level of IL-6 and IL-8 before and after treatment with TP140 were measured.The results in Fig. 4-A showed that the level of IL-6 secretion from stimulated bronchial cells was decreased by treating cells with TP140 after 24 h (p = 0.0253) and 48 h (p = 0.0295).Similarly, the level of IL-8 in stimulated cells was significantly decreased after 24 hr (p = 0.0311).However, no significant decrease was noticed after 48 h of treatment with TP140 (Fig. 4-B).The slight decrease in IL-6 and IL-8 levels, secreted from stimulated cells after treatment with TP140 might be due to the retention of ILs within the hydrogel matrix.Nonetheless, findings from anti-inflammatory studies of TP140 endorsed and confirmed that TP140 had no pro-inflammatory response, which is of great benefit as a platform drug delivery vehicle.

Encapsulation efficacy (EE) of therapeutics in TP140 hydrogel
The efficacy of TP140 to encapsulate therapeutics was evaluated by using a model antibiotic (Cip) and different cytokines (TNF-α, TGF-β1, and BMP2).Intentionally, high amount of these therapeutics was loaded to TP140 solution to challenge the carrier system and its encapsulation efficiency (EE).In addition, the solubility of therapeutics within TP140 solution was considered in determining the loaded mass and the corresponding EE of the carrier.For instance, for Cip encapsulation, the drug concentration within the TP hydrogel (20 mg/mL) was determined lower than the standard solubility of the drug in aqueous media at 25 • C, which is 36 mg/mL (Pollock and Healy, 2010;Varanda et al., 2006) to ensure the complete solubility of Cip in the TP140.
The amount of non-encapsulated therapeutics within TP140 hydrogel was quantified by measuring the secreted quantity of the compound released immediately post-gelation due to the hydrophilic to hydrophobic phase transition of the polymer system.The EE determined for the therapeutics loaded in TP140 solution after hydrogel formation at 37 • C is presented in Table 2.The lowest EE was achieved when TNF-α was loaded within TP140 solution, which may stem from weaker interactions between the TP polymer and TNF-α compared to other tested therapeutics.This could be explained by the weak electrostatic interactions between TNF-α and TP140 that have similar charges as outlined and discussed in the previous sections and shown in Table 1.The surface charges of TNF-α and TP140 result in a weak electrostatic interaction between the compound and the carrier and thus may explain low EE of TP140/TNF-α.However, the results in Table 2 showed that TP140 had an EE of more than 90% for encapsulation of Cip, TGF-β1, and BMP2.Accordingly, in the application of TP140 as a carrier system, the electrostatic charge of the target compound and the corresponding EE must be taken into the account.Nonetheless, high EE of TP140 to deliver a wide range of compounds suggest its potentials as a carrier vehicle to encapsulate and deliver therapeutics.

In vitro release profiles of the encapsulated therapeutics within TP140 hydrogel
The capability of TP140 hydrogel system to control the release of different therapeutics was investigated, using different in vitro models.The release of Cip from TP140/Cip hydrogel over time is shown in Fig. 5-A.A rapid initial release rate was observed during the first 4 h, where 21.63 ± 2.94% of the loaded dose of Cip was released.This release phase was followed by a sustained release rate until 24 h, where 27.28 ± 3.03% of the total Cip mass was released, which is equal to 1.09 ± 0.12 mg of the compound.Afterwards, a gradual increase in the mass of released Cip was observed from 24 h until the end of the one-week study period, where the amount of released Cip reached 1574.59 ± 110.87 µg (40.4 ± 2.77%).This sustained increase follows a three-phase profile, including a rapid 4-hour initial release of Cip from TP140 hydrogel, followed by a slower secondary phase until 24 h and a third phase of slower release until 144 h.The positive charge of Cip molecules in the TP140 solution and the resultant ionic bond with the negatively charged TP carrier are thought to provide the observed sustained release profile.
Fig. 5-B shows the TNF-α release profile, where the total released mass reaches 38.91 ± 2.7 pg after 4 days.This is equal to 0.08 ± 0.005% of the loaded TNF-α mass., TNF-α shows a continuous increase in the released mass in a two-phase profile.An initial rapid release rate within the first 2 h, where 16.10 ± 1.43 pg (0.03 ± 0.003%) was released followed by a slower release up to 96 h timepoint.The quantification of TNF-α in the initial burst release showed that 44.1 ± 10 ng (88.19 ± 17.11%) was expelled during gel formation, which correlates with the low EE determined for TNF-α (Table 2).Hence, the majority of the encapsulated TNF-α was released during the burst release and a lower amount remained within the gel resulting in the low released mass of TNF-α during the release study.
TGF-β1 and BMP2 release profiles are depicted in Fig. 5-C and D, respectively.The results indicate a plateaued profile, where the released mass was maintained at the same level throughout the assay for 7 days.TGF-β1 mass was determined as 5.12 ± 2.8 pg after 4 days in addition to 75.5 ± 12.8 pg quantified for the burst release.Hence, in total, 0.16 ± 0.02% of the loaded TGF-β1 mass in the TP140 hydrogel was released during the study.Similarly, only 1.78 ± 0.38 ng of BMP2 was detected after 5 days, with 49.59 ± 3.3 ng being expelled during gel formation (burst release).Thereby, in total, 51.38 ± 3.48 ng BMP2 was released from the TP140/BMP2 matrix, equates to 0.05 ± 0.003% of the total encapsulated BMP2 mass.
One potential reason for observing such low amounts of released TGF-β1 and BMP2 compared to Cip and TNF-α could be the stronger electrostatic interaction between these proteins and the polymeric matrix of TP140.Moreover, the molecular weights of TGF-β1 and BMP2 are similar (25 and 26 kDa), which are higher than the other tested therapeutics and this could have resulted in lower diffusion coefficients of TGF-β1 and BMP2 within the hydrogel matrix.Accordingly, the release mechanism of TGF-β1 and BMP2 from TP140 is purely driven by surface erosion of the carrier system and will likely be different in vivo.Therefore, the results achieved from these in vitro studies are indictive and confirmed that TP140 is capable of providing a sustained release profile for delivery of these compounds and further highlight the importance of the following biological activity studies.

Bioactivity of the released therapeutics from TP140 hydrogel
The therapeutic performance of Cip, TNF-α and TGF-β1, released from the TP140 matrix was assessed through a range of in vitro studies.To investigate the biological activity of BMP2 released form TP140, an in vivo standard animal study was used as in vitro results in the previous section showed very low amount of this compound was released in simulated physiological conditions.

Antibacterial activity of Cip released from TP140/Cip hydrogel
The bioactivity of the Cip released from the TP140/Cip hydrogel was assessed by determining its antibacterial efficacy using a susceptible strain of S. aureus.The growth inhibition curves for free Cip and released from TP140 were obtained (Fig. 6-A); results showed 99.5 ± 1.5% and 98.7 ± 2.1% decrease in the OD values were observed when free Cip solution and TP140/Cip hydrogel were used, respectively.The results showed that TP140/Cip was able to maintain its antibacterial efficacy while the carrier allows for a sustained release rate as reported in section 3.4.2.The results in Fig. 6-B showed that no significant differences were observed between MIC values for the released Cip from TP140 and free Cip solution (p = 0.2996).The determined MIC values are also similar to the target MIC value reported for Cip against S. aureus by the European Committee for Antimicrobial Susceptibility Testing (EUCAST), which is 0.25μ g/mL (EUCAST, 2003).Moreover, EUCAST guidelines state that MIC value must be within one two-fold dilution of the reference value (0.25μ g/mL) as a reference for quality control of the microdilution tests.This corroborates the bioactivity of Cip released from the TP140 matrix and that the application of TP140 carrier system did not impede the biological activity of the encapsulated antibiotic compound.
Comparison of the MBC values obtained for the released Cip from TP140 and free Cip treatments showed a significant difference (p = 0.0103); the free Cip treatment has an MBC value of 2.9 times lower than Cip from TP140 (Fig. 6-B).This indicates that the bactericidal effect of the encapsulated Cip is achieved at a higher concentration when compared to the free drug solution.This must be considered during the formulation optimization and dose estimation, where a higher mass of drug can be loaded within the matrix to achieve the desired therapy depending on the severity of the infection.Overall, the observations suggest that the encapsulation of antibacterial drugs such as Cip within TP140 matrix can result in an efficient controlled delivery of the drugs without reducing the antibacterial efficacy of the drug.

Pro-inflammatory activity of the released TNF-α from TP140/TNF-α hydrogel
The pro-inflammatory activity of the released TNF-α from the TP140 hydrogel was evaluated by determining its capability to induce IL-6 and IL-8 secretion by healthy lung fibroblast MRC-5 cells.The cells were treated with TP140/TNF-α, and the IL-6 and IL-8 secretion was compared with the cells treated with free TNF-α solution (2 ng/mL).It was found that the released TNF-α from TP140 significantly increased both IL-6 (Fig. 7-A) and IL-8 (Fig. 7-B) levels compared with the untreated cells (p = 0.0254 and 0.0078, respectively).Treatment of the cells with free TNF-α solution also resulted in a significant increase in the levels of the IL-6 and IL-8 (p = 0.0012 and 0.0015, respectively) compared to the control cells.Importantly, no statistical differences (p > 0.05) of IL-6 and IL-8 measurements were noticed between free TNF-α and the released compound from TP140/TNF-α.This suggests that proinflammatory effect of TNF-α on the cells were preserved after encapsulation within the TP140 hydrogel, further supporting its application as a suitable carrier for the efficient delivery of this cytokine.

Pro-Inflammatory activity of TGF-β1 released from TP140/TGF-β1 hydrogel
The bioactivity of released TGF-β1 from the TP140/TGF-β1 hydrogel was evaluated by its ability to induce IL-6 (Fig. 8-A) and IL-8 (Fig. 8-B) secretion in healthy lung fibroblast MRC-5 cells and comparing findings from that achieved with free TGF-β1 solution (2 ng/mL).Treatment of the cells with the free TGF-β1 solution significantly increased the level of IL-6 secretion by the cells (p = 0.0064).The treatment of cells with either encapsulated or free TGF-β1 did not cause any significant difference in the level of secreted IL-8 (p = 0.1739 and 0.1227, respectively) when compared to the untreated control cells (Fig. 8-B) and therefore showed a limitation in the study to induce IL-8 expression.Focusing on results from IL to 6 measurements, results showed that the cells treated with TP140/TGF-β1 (Fig. 8-A) presented a significant increase in the IL-6 secretion (p = 0.0043) compared with the untreated control.Importantly, there was no significant difference (p = 0.2299) in IL-6 levels between the cells treated with TGF-β1 released from TP140 or its free counterpart.Based on the TGF-β1 release profile (Fig. 5-C), 4.22 ± 2.7 pg of the TGF-β1 is released after 24 h incubation, which is equivalent to 7.03 ± 4.5 pg/mL concentration in the basolateral chamber.This concentration is ~ 250X lower than the concentration of the + Control (free TGF-β1) solution.Given that the lower mass of released TGF-β1 expressed statistically similar (p = 0.2299 for IL-6 and p = 0.2876 for IL-8) pro-inflammatory effect as the free TGF-β1 solution, it can be concluded that the bioactivity of TGF-β1 is maintained after being encapsulated within the TP140 matrix and that the carrier system can be used for the delivery of this pro-inflammatory cytokine.

Osteogenic activity of BMP2 released from TP140/BMP2 hydrogel in a mouse ectopic bone model
The ability of TP140 to deliver and control the release of therapeutics in vivo was assessed by using a model osteoinductive growth factor in a standard animal study.Use of an in vivo model to assess the biological activity of BMP2 was essential as the results in section 3.4 showed that small amount of BMP2 is released in vitro, highlighting the limitations associated with simulated physiological conditions in benchtop testing.As such, two treatments, TP140/BMP2 (with 0.015 mg of BMP2) and a + Control (standard collagen matrix with 0.045 mg of BMP2), were implanted into pre-formed muscle pouches in mice.The capability of these two treatments to induce ectopic bone formation was assessed at weeks 2, 4, and 6 post intra-muscular pouch implantation of hydrogels.This in vivo study was performed to investigate the extent of ectopic bone formation over a prolonged period of time, i.e. 6 weeks, to confirm that TP140 hydrogel is able to control the release of large proteins and maintain their functional activity overtime.
Quantification of bony tissue from CT-scan results (in Fig. 9-A) at different time points were used to investigate the osteoinductivity of released BMP2 from the + Control and from TP140.In addition, the quantification of femur bone density in Fig. 9-B validated the accuracy of measurements as no significant difference at different time points was noticed among the femur bone density.More importantly, results in Fig. 9-C showed that despite the low dose incorporated within TP140 (0.015 mg), BMP2 released from the carrier promoted ectopic bone Fig. 7.The therapeutic effect of the TP140/TNF-α on the MRC-5 healthy lung fibroblast cells expressed as the secretion of the inflammatory cytokines (IL-6 and IL-8).
The MRC-5 cells were treated with the TNF-α released after 24 h from TP140/TNF-α hydrogel in the apical chamber of Transwell inserts into the basolateral chamber, where the cells were cultured.The observed inflammatory response is compared with the free TNF-α solution and empty TP140 matrix as the controls (n = 3, mean ± SD, ** p ≤ 0.01).
Fig. 8.The therapeutic effect of the TP140/TGF-β1used for treating the MRC-5 healthy lung fibroblast cells in terms of the inflammatory cytokines (IL-6 and IL-8) secretion.The cells, cultured in the basolateral chamber of the Traswell inserts were treated with the released TGF-β1 from TP140/TGF-β1hydrogel formed in the apical chamber after 24 h.The inflammatory response of the cells is compared with free TGF-β1 solution and empty TP140 matrix as the control (n = 3, mean ± SD, **p ≤ 0.01).formation 2 weeks post-implantation surgery.However, the extent of ectopic bone regeneration in the + Control group was greater than that in TP140/BMP2 at early stages post-surgery which might be attributed to 3-fold higher dose of BMP2 used in this group.The density of ectopic bone in the + Control group decreased at weeks 4 and 6 postimplantation due to the high osteoclast activity at the implantation site and the ectopic nature of the regenerated bone.This result confirmed that there has been a burst release of BMP2 from the control group at an early stage, which leads to high osteoclast activity at subsequent time points.In TP140/BMP2 group, however, the sustained release of BMP2 allowed gradual formation of ectopic bone at weeks 4 and 6 post-implantation.As such, the density of ectopic bone at week 6 for TP140/BMP2 group was significantly (p < 0.001) higher than in the control group.The long-term and sustained release of BMP2 from the TP vehicle in this in vivo investigation confirmed that the developed carrier hydrogel addresses the limitation associated with current thermoresponsive hydrogel systems, including their low structural stability (Pollock and Healy, 2010)and lack of adhesion at the target site (Bayat et al., 2017).
The hematoxylin and eosin (H&E) staining of the TP140 implants, shown in Fig. 10, is consistent with the findings from x-ray imaging.Histological evaluation of the implants at weeks 2, 4, and 6 post-surgery showed that TP140/BMP2 implants retained at the implantation site and confirmed the potential of TP140/BMP2 to support ectopic bone formation.Results showed that at 2 weeks post-implantation, the sites are filled with the newly formed bone mainly woven bone, which then progressed to more mature lamellar bone at weeks 4 and 6.No sign of inflammatory response or severe foreign body reaction to the product was noticed.This result was in agreement with our previous findings from different in vivo application of the background technology (Calder et al., 2022;Dehghani et al., 2017;Fathi et al., 2014).The majority of the ectopic bone was formed at the surface of TP140/BMP2 implants with some degree of bone ingrowth within the matrix.Despite the lower dose of BMP2 used in TP140/BMP2 compared to + Control, there was no   distinct difference in the osteogenesis patterns between two treatment groups.These results further endorse high potential of the TP140 carrier system as a delivery vehicle for therapeutics.

Conclusions
The findings from this study collectively show that TP possesses all critical characteristics of a universal carrier for sustained delivery of therapeutics.Specifically, the vehicle system did not induce cytotoxic or inflammatory effects in vitro.Loading of a model antibiotic drug and multiple cytokines of varying molecular weights and surface charges did not impede the flowability and the thermo-responsive characteristics of the carrier system.Further, the vehicle system provided a relatively high encapsulation efficiency, mitigated the burst release and thus provided a sustained release profile for a wide range of therapeutics.Importantly, a range of in vitro and in vivo studies confirmed that the carrier system and its gelation process were benign and did not detrimentally impact the biological activity of the loaded compounds.Therefore, this study confirmed the potential applications of the TP hydrogel system as a drug delivery platform, whereas future in vivo and clinical studies are required to provide more insights on the clinical translatability this class of carriers.

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.

Fig. 1 .
Fig. 1.The percentage of the viability of RPMI-2650 (A) and BEAS-2B (B) cells, exposed to TP140 leachables as the TP140 hydrogel was formed in the apical chamber and the cells were cultured in the basolateral chamber of Transwells after 24 and 48 h incubation assessed by the MTS assay (n = 3, mean ± SD, *p ≤ 0.05 and *** ≤ 0.001).

Fig. 2 .
Fig. 2. Microscopic images obtained from RPMI-2650 and BEAS-2B cells treated with TP140 leachables while the TP140 hydrogel was formed in the apical chamber and the cells were cultured in the basolateral chamber of Transwells after 24 h (A) and 48 h (B) incubation.Live cells, dead cells, and the nuclei are indicated by Calcein-AM (green), EthD (red), and Hoechst (blue).The images are taken by a 10x air objective (APO Fluor, Nikon, Japan).The scale bars show 100 µm.

Fig. 3 .
Fig. 3. Inflammatory response of the BEAS-2B cells to TP140 treatment in terms of IL-6 (A) and IL-8 (B) secretion assessed in Transwell inserts setup after 24 and 48 h treatment, where the cells were cultured in the basolateral chamber and the TP140 hydrogel was formed in the apical chamber (n = 3, mean ± SD, *p ≤ 0.05).

Fig. 4 .
Fig. 4. The anti-inflammatory effect of TP140 treatment on the pre-stimulated BEAS-2B cells in terms of the IL-6 (A) and IL-8 (B) secretion after 24 and 48 h treatment in the Transwell inserts, where the TP140 hydrogel was formed in the apical chamber and the cells were cultured in the basolateral chamber (n = 3, mean ± SD, *p ≤ 0.05).

Fig. 5 .
Fig. 5.In vitro release profiles of (A) Cip, (B) TNF-α, (C) TGF-β1, and (D) BMP2 encapsulated within TP140 hydrogel matrix over time (n = 3, mean ± SD) assessed in Snapwell inserts, where the drug release from the drug loaded TP140 hydrogel in the apical chamber into the basolateral chamber was determined.

Table 2
Encapsulation efficacy (EE) values determined for encapsulation of different therapeutics within the TP140 hydrogel (n = 3, mean ± SD).