An integrated multimodal imaging workflow of cryogenic super-resolution fluorescence microscopy and soft X-ray tomography, Orbitrap secondary ion mass spectrometry, and inductively coupled plasma-mass spectrometry has revealed the unexpected targeting of a half-sandwich cyclopentadienyl Rh(III) phenylazopyridine anticancer complex to cellular lipid membranes and lipid droplets. The complex accumulates in plasma membranes with a surprisingly intense switch-on luminescence in living cancer cells, drives remodeling of lipid droplet architecture, and penetrates deeply into lipid-rich tissue environments. DFT modeling shows strong supramolecular interactions between the complex and glycerophosphorylcholine lipids.

Abstract Tuberculosis (TB), caused by Mycobacterium tuberculosis ( Mtb ), remains a significant global health challenge. Currently treatment of drug-sensitive TB, involves a six-month regimen consisting of a combination of four anti-TB drugs, with drug-resistant TB requiring over two years of treatment and additional drugs. As toxicity of anti-TB drugs often leads to poor compliance, disease relapse and the emergence of drug-resistant strains, new strategies to reduce drug toxicity and shorten treatment duration are critical. We report nanocarrier-based drug delivery systems targeting macrophages, which primarily support replication and survival of Mtb . We have developed mannose-functionalized nanoparticles that bind to mannose receptors on macrophages and feature a pH-sensitive core which releases an encapsulated drug in the acidic lysosomal environment of macrophages. Rifampicin (RIF), a main anti-TB drug currently in use clinically, was encapsulated within the nanoparticles. We demonstrate that antibiotic-containing nanocarriers efficiently accumulated in macrophages without causing toxicity. Encapsulated RIF showed enhanced efficacy against both BCG and Mtb in primary macrophages. Biodistribution studies in mice revealed that the nanoparticles have extended circulation time and do not induce toxicity. In addition, the encapsulated RIF showed better targeting of mycobacteria when compared to free RIF in a murine model of mycobacterial infection. Such an enhanced bacterial killing using mannose-functionalised nanocarriers loaded with the key anti-TB drug rifampicin offers excellent potential for TB therapy.

An endogenous biological clock, the circadian clock, coordinates life with the 24-hour day/night environmental cycle. In mammals, a central pacemaker in the suprachiasmatic nucleus of the hypothalamus coordinates timing between peripheral clocks and with the environment and, for example, modulates immune responses to infections. However, its role in controlling bacterial infections at a cellular level is not understood. Here, we investigate the role of the host cellular clock during infection by a highly drug-resistant human pathogen, Staphylococcus aureus. Our findings revealed that S. aureus invasion into epithelial cells was dependent on the host circadian phase. Interestingly, cells deficient in BMAL1, a transcriptional activator and an essential clock protein, demonstrated increased bacterial uptake compared to parental A549 cells. The BMAL1-knockdown (KD) cells showed significant induction of GP340, a receptor of the S. aureus adhesin, SraP. An S. aureus sraP mutant exhibited blunted rhythmic uptake into A549 cells or an increased uptake into BMAL1-KD compared to parental A549 cells. Of note, other bacterial adhesin mutants showed rhythmic and higher uptake in BMAL1-KD cells. Hence, we report that S. aureus epithelial cell invasion is clock-modulated and mediated through the S. aureus SraP-GP340 pathway, suggesting potential for host clock-directed therapy against this pathogen. The cellular circadian clock shapes Staphylococcus aureus invasion dynamics in epithelial cells through modulation of surface marker abundance.

An endogenous biological clock, the circadian clock, coordinates life with the 24-hour day/night cycle of the environment. The unit of this circadian clock is the cell but in multicellular organisms, such as mammals, a circadian timing system (CTS) with a central pacemaker orchestrates peripheral clocks and the overall finely tuned temporal order. For example, the CTS changes immune responses to infections depending on time-of-day, however, its role in controlling bacterial infections at a cellular level is not understood. In this study, we investigated the role of the host cellular clock during infection by Staphylococcus aureus, a highly drug-resistant, facultatively intracellular human pathogen. Our findings revealed that S. aureus invasion into epithelial cells is dependent on the cellular circadian phase. Interestingly, in BMAL1, an essential clock protein, deficient cells bacterial uptake was significantly higher compared to parental A549 cells. RNA sequencing of BMAL1 knockdown (KD) cells showed a significant upregulation of GP340 , coding for the receptor of SraP, an S. aureus adhesin. An S. aureus mutant lacking SraP did not exhibit a circadian rhythm of uptake into A549 cells nor an increased uptake into BMAL1 KD compared to the parental A549 cells. Of note, bacterial mutants for other adhesins continued to show a rhythmic and higher uptake in BMAL1 KD cells. Hence, we report that S. aureus invasion of epithelial cells is clock-modulated and mediated through S. aureus SraP, suggesting potential for host clock-directed therapy against this pathogen. ### Competing Interest Statement The authors have declared no competing interest. University of Warwick, https://ror.org/01a77tt86, Warwick Research Development Fund Monash Warwick Alliance, Monash Warwick Alliance Fellowship Anglo American (United Kingdom), https://ror.org/0231mbz86

Cyclic peptide-polymer conjugate nanotubes have been shown to be powerful drug delivery vectors, due to their propensity for dynamic self-assembly, high aspect ratio morphology and structural interchangeability. Building upon previous studies that demonstrate the shielding abilities of the polymeric corona of nanotubes to enhance pro-drug bond stabilities and modulate hydrolysis, here the concept of a hydrophobic core building block with multiple drug units to improve drug loading capacity and overall efficiency of the nanotube carriers is utilized. By leveraging the intermolecular features of the drug core to strengthen assembly, it is hypothesized that these nanotubes have the potential as a responsive supramolecular delivery system whereby upon full hydrolysis of the labile drug, these core forming interactions disappear, and nanotubes can fall apart and undergo clearance. Herein, the self-assembly, in vitro efficacy and in vivo pharmacokinetic and anti-tumor pharmacodynamics of these nanotubes in colorectal cancer models, comparing the potent topoisomerase inhibitor SN-38 with its clinically-used parent pro-drug irinotecan, is explored.

Antimicrobial polymers, which have emerged as a promising alternative to antibiotics in the fight against antimicrobial resistance, are based on the design of cationic host defense peptides (CHDPs). Being part of the mammalian innate immune system, CHDPs possess both antimicrobial and immunoregulatory effects to manage bacterial infections. However, the immunomodulatory effects of antimicrobial polymers remain largely unexplored. Within this work, a library of 15 copolymers was synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization and their abilities to modulate pro-inflammatory pathways in lipopolysaccharide (LPS)-activated murine and human macrophages were investigated. We found that two diblock copolymers with cationic units copolymerized with either apolar or hydrophilic comonomers appeared to have anti-inflammatory activity through suppression of the activation of the nuclear factor kappa-light-chain enhancer of the activated B cell (NF-κB) signaling pathway, scavenging of reactive oxygen species, and reduced production of the pro-inflammatory cytokine interleukin-6 (IL-6). Furthermore, the cationic-apolar copolymer exhibits significant antimicrobial activity against P. aeruginosa. Thus, this promising copolymer holds potential as a dual-action therapeutic, effectively combating bacterial infections while curbing prolonged inflammation and thereby preventing sepsis at the site of infection.

The circadian rhythm influences homeostatic functions such as sleep, physical activity and food intake as well as pharmacotherapy, namely pharmacokinetics. To investigate the impact of the circadian rhythm on the pharmacokinetics of paroxetine, in vitro synchronized permeability studies were carried out in a tri-culture blood-brain barrier model. Paroxetine demonstrated lower apparent permeability when the cells were incubated at 24 h post-synchronization than at 36 h. Additionally, in vivo chronopharmacokinetic studies were performed in CD-1 female mice administered with paroxetine (5 mg/kg) by intranasal route in the early morning or evening. Paroxetine exposure in the brain was higher when it was administered at the beginning of the active phase (ZT13) compared with the rest phase (ZT1) (p < 0.001), probably owing to the lower levels of P-glycoprotein expressed in the brain at the active phase (p < 0.05). Since melatonin production depends on serotonin, its plasma concentrations were also assessed in vivo. The results demonstrated that melatonin concentrations increased 12 h after paroxetine nasal instillation at ZT13 (p < 0.05), but remained unchanged at ZT1, suggesting that the drug effect is influenced by administration time. In conclusion, the circadian rhythm impacted the pharmacokinetics of paroxetine, especially its distribution into the brain, the target organ. This emphasizes the importance of the time of administration in antidepressant dosing, highlighting its relevance for future studies.

Emerging evidence highlights the critical role of time-of-day (ToD) in immunotherapy, with large-scale retrospective studies showing that administering immune checkpoint inhibitors (ICIs) earlier in the day is associated with significantly improved efficacy across all cancer types. This observation aligns with our growing understanding of the circadian system, our internal biological clock, which governs a range of physiological processes, including immune regulation, over a 24-hour scale. Each individual possesses a distinct pace in their circadian rhythm, known as their chronotype, which reflects their natural preference for morning or evening activity, performance and rest. Chronotype has already proven to be a valuable predictor of outcomes in other areas of healthcare, and its application in immuno-oncology holds promising potential. In this commentary, we propose leveraging chronotype as a low-cost and minimally invasive strategy to personalize the timing of ICI administration and enhance therapeutic effectiveness. Despite encouraging retrospective data on ToD effects, current treatment protocols remain largely time-agnostic, hindered by the scantiness of prospective, chronotype-informed clinical trials. We outline here the key steps required to validate and implement chronotype-based scheduling, including rigorous clinical-translational studies. Embracing this temporal dimension could represent a transformative shift toward more precise, personalized, and effective circadian-based cancer immunotherapy.

Antimicrobial polymers, which have emerged as a promising alternative to antibiotics in the fight against antimicrobial resistance, are based on the design of cationic host defence peptides (CHDPs). Being a part of the mammalian innate immune system, CHDPs possess both antimicrobial and immunoregulatory effects to manage bacterial infections. However, the immunomodulatory effects of antimicrobial polymers remain largely unexplored. Within this work, a library of 15 copolymers were synthesised by reversible addition-fragmentation chain transfer (RAFT) polymerisation and their abilities to modulate pro-inflammatory pathways in LPS-activated murine and human macrophages were investigated. We found that two diblock copolymers with cationic units copolymerised with either apolar or hydrophilic comonomers appeared to have anti-inflammatory activity through suppression of the activation of the NF-κB signalling pathway, scavenging of reactive oxygen species and reduced production of the pro-inflammatory cytokine IL-6. Furthermore, the cationic-apolar copolymer exhibits significant antimicrobial activity against P. aeruginosa . Thus, this promising copolymer holds potential as a dual-action therapeutic, effectively combating bacterial infections while curbing prolonged inflammation and thereby preventing sepsis at the site of infection. ### Competing Interest Statement The authors have declared no competing interest.

Crossing the blood–brain barrier (BBB) remains a major obstacle for central nervous system therapeutics. Short peptides have emerged as promising vectors, including cell-penetrating peptides (CPPs) and brain-penetrating peptides (BPPs). However, the structural and physicochemical features that distinguish CPPs from BPPs remain poorly understood, limiting rational design. Here, we compiled a curated dataset of 490 peptides, encompassing CPPs, BPPs, and non-BPP controls, and systematically analysed their amino acid composition, sequence distribution, and physicochemical descriptors. BPPs were found to exhibit a more balanced distribution of cationic, polar, and hydrophobic residues compared to CPPs, which were enriched in contiguous arginine and lysine blocks. Physicochemical analysis revealed that BPPs had lower charge density, greater stability, and reduced aromaticity relative to CPPs. Dimensionality reduction confirmed BPPs occupy an intermediate chemical space between CPPs and non-BPPs. Machine learning classification, particularly with Extra Trees models, achieved strong performance in discriminating peptide classes, with charge, instability index, and aromaticity identified as the most predictive features. These findings suggest that BBB penetration is not a simple extension of cell penetration but requires finely tuned physicochemical properties. This study provides mechanistic insights into BPP design and highlights machine learning as a valuable tool for engineering next-generation BBB-penetrating peptides and peptide-mimetic materials. ### Competing Interest Statement The authors have declared no competing interest. Medical Research Council, https://ror.org/03x94j517

We report the synthesis and characterization of ten novel half‐sandwich Rh(III) azopyridine complexes as potential anticancer agents, with the general formula [(η 5 ‐Cp x )Rh(4‐R 2 ‐phenylazopy‐5‐R 1 )Cl]PF 6 , where Cp x = Cp*, Cp xPh or Cp xPhPh , R 1 = H, Br, or CF 3 , and R 2 = H, OH or NMe 2 . X‐ray crystallographic data for complex 2 (R 1 = Br, R 2 = OH, Cp x = Cp xPh ) and complex 3 (R 1 = CF 3 , R 2 = OH, Cp x = Cp xPh ) confirm their typical half‐sandwich “piano‐stool” geometry. The substituents have a major influence on the cytotoxicity of these complexes toward human ovarian (A2780 and cisplatin‐resistant A2780cis), lung (A549) and prostate (PC‐3) cancer cells, and non‐cancerous human lung fibroblasts (MRC‐5). Potencies range from sub‐micromolar to inactive (>50 µM). They were non‐cross‐resistant with cisplatin, and complex 9 (R 1 = H, R 2 = NMe 2 , Cp x = Cp * ) showed some selectivity (>3x) for A549 cancer cells versus normal cells. The highly active, lipophilic complex 2 was strongly accumulated by cells and catalyzed the oxidation of NADH (reduced nicotinamide adenine dinucleotide) to NAD + , and GSH (glutathione) to GSSG. Notably, complex 2 is almost an order of magnitude less toxic toward zebrafish in vivo than cisplatin, despite being 10‐fold more active in A549 cells. These studies demonstrate how the chemical and biological activities of this series of half‐sandwich organorhodium(III) complexes can be finely tuned by the choice of substituents on the cyclopentadienyl and azopyridine ligands. The complexes appear to have an unusual mechanism of anticancer activity, associated not only with Rh(III) but also with the phenylazopyridine, cyclopentadienyl, and the chlorido ligands.

The mammalian suprachiasmatic nucleus (SCN), situated in the ventral hypothalamus, directs daily cellular and physiological rhythms across the body. The SCN clockwork is a self-sustaining transcriptional-translational feedback loop (TTFL) that in turn coordinates the expression of clock-controlled genes (CCGs) directing circadian programmes of SCN cellular activity. In the mouse, the transcription factor, ZFHX3 (zinc finger homeobox-3), is necessary for the development of the SCN and influences circadian behaviour in the adult. The molecular mechanisms by which ZFHX3 affects the SCN at transcriptomic and genomic levels are, however, poorly defined. Here, we used chromatin immunoprecipitation sequencing to map the genomic localization of ZFHX3-binding sites in SCN chromatin. To test for function, we then conducted comprehensive RNA sequencing at six distinct times-of-day to compare the SCN transcriptional profiles of control and ZFHX3-conditional null mutants. We show that the genome-wide occupancy of ZFHX3 occurs predominantly around gene transcription start sites, co-localizing with known histone modifications, and preferentially partnering with clock transcription factors (CLOCK, BMAL1) to regulate clock gene(s) transcription. Correspondingly, we show that the conditional loss of ZFHX3 in the adult has a dramatic effect on the SCN transcriptome, including changes in the levels of transcripts encoding elements of numerous neuropeptide neurotransmitter systems while attenuating the daily oscillation of the clock TF Bmal1. Furthermore, various TTFL genes and CCGs exhibited altered circadian expression profiles, consistent with an advanced in daily behavioural rhythms under 12 h light–12 h dark conditions. Together, these findings reveal the extensive genome-wide regulation mediated by ZFHX3 in the central clock that orchestrates daily timekeeping in mammals.

DFT calculations on a conjugate of ferrocene to an octahedral Pt( iv ) complex reveal Fe-to-Pt charge transfer, promoting longer-wavelength photoactivation, enhanced cellular accumulation, potent anticancer activity, and cell death via ferroptosis.

The discovery of circadian clock genes greatly amplified the study of diurnal variations impacting cancer therapy, transforming it into a rapidly growing field of research. Especially, use of chronomodulated treatment with 5-fluorouracil (5-FU) has gained significance. Studies indicate high interindividual variability (IIV) in diurnal variations in dihydropyrimidine dehydrogenase (DPD) activity - a key enzyme for 5-FU metabolism. However, the influence of individual DPD chronotypes on chronomodulated therapy remains unclear and warrants further investigation. To optimize precision dosing of chronomodulated 5-FU, this study aims to: (i) build physiologically-based pharmacokinetic (PBPK) models for 5-FU, uracil, and their metabolites, (ii) assess the impact of diurnal variation on DPD activity, (iii) estimate individual DPD chronotypes, and (iv) personalize chronomodulated 5-FU infusion rates based on a patient's DPD chronotype. Whole-body PBPK models were developed with PK-Sim(R) and MoBi(R). Sinusoidal functions were used to incorporate variations in enzyme activity and chronomodulated infusion rates as well as to estimate individual DPD chronotypes from DPYD mRNA expression or DPD enzymatic activity. Four whole-body PBPK models for 5-FU, uracil, and their metabolites were established utilizing data from 41 5-FU and 10 publicly available uracil studies. IIV in DPD chronotypes was assessed and personalized chronomodulated administrations were developed to achieve (i) comparable 5-FU peak plasma concentrations, (ii) comparable 5-FU exposure, and (iii) constant 5-FU plasma levels via "noise cancellation" chronomodulated infusion. The developed PBPK models capture the extent of diurnal variations in DPD activity and can help investigate individualized chronomodulated 5-FU therapy through testing alternative personalized dosing strategies.

Recent studies have established that the circadian clock influences onset, progression and therapeutic outcomes in a number of diseases including cancer and heart diseases. Therefore, there is a need for tools to measure the functional state of the molecular circadian clock and its downstream targets in patients. Moreover, the clock is a multi-dimensional stochastic oscillator and there are few tools for analysing it as a noisy multigene dynamical system. In this paper we consider the methodology behind TimeTeller, a machine learning tool that analyses the clock as a noisy multigene dynamical system and aims to estimate circadian clock function from a single transcriptome by modelling the multi-dimensional state of the clock. We demonstrate its potential for clock systems assessment by applying it to mouse, baboon and human microarray and RNA-seq data and show how to visualise and quantify the global structure of the clock, quantitatively stratify individual transcriptomic samples by clock dysfunction and globally compare clocks across individuals, conditions and tissues thus highlighting its potential relevance for advancing circadian medicine.

Immunoaffinity-based liquid biopsies of circulating tumor cells (CTCs) hold great promise for cancer management but typically suffer from low throughput, relative complexity, and postprocessing limitations. Here, we address these issues simultaneously by decoupling and independently optimizing the nano-, micro-, and macro-scales of an enrichment device that is simple to fabricate and operate. Unlike other affinity-based devices, our scalable mesh approach enables optimum capture conditions at any flow rate, as demonstrated with constant capture efficiencies, above 75% between 50 and 200 μL min–1. The device achieved 96% sensitivity and 100% specificity when used to detect CTCs in the blood of 79 cancer patients and 20 healthy controls. We demonstrate its postprocessing capacity with the identification of potential responders to immune checkpoint inhibition (ICI) therapy and the detection of HER2 positive breast cancer. The results compare well with other assays, including clinical standards. This suggests that our approach, which overcomes major limitations associated with affinity-based liquid biopsies, could help improve cancer management.

Aims/hypothesis The objective was to investigate if metformin pharmacokinetics is modulated by time-of-day in humans using empirical and mechanistic pharmacokinetic modelling techniques on a large clinical dataset. This study also aimed to generate and test hypotheses on the underlying mechanisms, including evidence for chronotype-dependent interindividual differences in metformin plasma and efficacy-related tissue concentrations. Methods A large clinical dataset consisting of individual metformin plasma and urine measurements was analysed using a newly developed empirical pharmacokinetic model. Causes of daily variation of metformin pharmacokinetics and interindividual variability were further investigated by a literature-informed mechanistic modelling analysis. Results A significant effect of time-of-day on metformin pharmacokinetics was found. Daily rhythms of gastrointestinal, hepatic and renal processes are described in the literature, possibly affecting drug pharmacokinetics. Observed metformin plasma levels were best described by a combination of a rhythm in GFR, renal plasma flow (RPF) and organic cation transporter (OCT) 2 activity. Furthermore, the large interindividual differences in measured metformin concentrations were best explained by individual chronotypes affecting metformin clearance, with impact on plasma and tissue concentrations that may have implications for metformin efficacy. Conclusions/interpretation Metformin’s pharmacology significantly depends on time-of-day in humans, determined with the help of empirical and mechanistic pharmacokinetic modelling, and rhythmic GFR, RPF and OCT2 were found to govern intraday variation. Interindividual variation was found to be partly dependent on individual chronotype, suggesting diurnal preference as an interesting, but so-far underappreciated, topic with regard to future personalised chronomodulated therapy in people with type 2 diabetes. Graphical abstract

Sleep behaviour is broadly regulated by two drives, the circadian (Process C), which is orchestrated by the suprachiasmatic nuclei (SCN), and controls sleep timing, and the homeostatic (Process S), which controls sleep amount and the response to sleep deprivation (Borbély et al ., 2016). However, the molecular pathways that mediate their independent effects, and their interactions remain unclear. Adenosine is an important integrator of both processes (Bjorness & Greene, 2009; Jagannath et al ., 2021, 2022), such that adenosine levels track and modulate wakefulness, whilst adenosine signalling inhibits the circadian response to light. Therefore, we studied the sleep/circadian behaviour, and cortical and SCN transcriptomic profiles of a mouse model overexpressing Adenosine Kinase (Adk-Tg) (Fedele et al ., 2005), (Palchykova et al ., 2010). We found that overall, the Adk-Tg mouse slept less and showed lower amplitude circadian rhythms with an altered sleep/wake distribution across the 24h day, which correlated with changes in transcription of synaptic signalling genes that would shift the excitatory/inhibitory balance. In addition, the Adk-Tg mouse showed a reduced level of ERK phosphorylation, and attenuation of DNA repair related pathways. After sleep deprivation, however, the Adk-Tg mouse significantly increased relative to wildtype, immediate early gene expression levels including of Arc , but paradoxically reduced ERK phosphorylation. Thus, baseline sleep levels and timing are regulated by ERK signalling, whereas the response to sleep loss is mediated by the alteration of the transcriptomic landscape independently of ERK.

The hypothalamic type 2 corticotropin releasing hormone receptor (CRH-R2) plays critical roles in homeostatic regulation, particularly in fine tuning stress recovery. During acute stress, the CRH-R2 ligands CRH and urocortins promote adaptive responses and feeding inhibition. However, in rodent models of chronic stress, over-exposure of hypothalamic CRH-R2 to its cognate agonists is associated with urocortin 2 (Ucn2) resistance; attenuated cAMP-response element binding protein (CREB) phosphorylation and increased food intake. The molecular mechanisms involved in these altered CRH-R2 signalling responses are not well described. In the present study, we used the adult mouse hypothalamus-derived cell line mHypoA-2/30 to investigate CRH-R2 signalling characteristics focusing on gene expression of molecules involved in feeding and circadian regulation given the role of clock genes in metabolic control. We identified functional CRH-R2 receptors expressed in mHypoA-2/30 cells that differentially regulate CREB and AMP-activated protein kinase (AMPK) phosphorylation and downstream expression of the appetite-regulatory genes proopiomelanocortin (Pomc) and neuropeptide Y (Npy) in accordance with an anorexigenic effect. We studied for the first time the effects of Ucn2 on clock genes in native and in a circadian bioluminescence reporter expressing mHypoA-2/30 cells, detecting enhancing effects of Ucn2 on mRNA levels and rhythm amplitude of the circadian regulator Aryl hydrocarbon receptor nuclear translocator-like protein 1 (Bmal1), which could facilitate anorexic responses in the activity circadian phase. These data uncover novel aspects of CRH-R2 hypothalamic signalling that might be important in regulation of circadian feeding during stress responses.

Abstract Introduction Adipose tissue transfer is used for correcting breast contour defects after cancer surgery. Graft survival can be as poor as 15% at four years follow-up. Hyaluronic acid (HA) hydrogels can be used to suspend adipocytes to aid survival. Methods A unique HA gel with cross-linked aldehyde and hydrazine groups allowing self-healing around cells after injection was engineered. The gel's rheology and degradation properties in vitro were characterised. 3T3-L1, murine and human adipocytes were encapsulated within HA hydrogel, with survival and gene expression interrogated. A murine animal model underwent subcutaneous injection of primary murine adipocytes mixed with HA gel, with survival and gene expression examined. Results The novel HA hydrogel can be degraded by tissue hyaluronidases in 4–5 days. Self-healing occurred in 30 minutes and the gel is robust enough to be injected through a needle. 3T3-L1, murine and human adipocytes were encapsulated within HA hydrogel and showed a <15% deduction in survival compared to control. Mature adipocyte gene expression markers compared to control showed no difference. Primary murine adipocytes injected in vivo showed no difference in survival at higher gel volumes compared to control. Conclusion The novel engineered HA hydrogel has suitable mechanical characteristics for injection and can support adipocyte survival in vitro and in vivo and is ready for a pilot human trial. Take-home message Our novel HA gel can support adipocytes in vivo and in vitro, and offers a potential method for augmentation of adipose tissue transfer.