Immune checkpoint inhibitors (ICIs) have fundamentally changed the treatment landscape of various cancers. While ICI treatments result in improved survival, quality of life and are cost-effective, the majority of patients experience at least one immune-related adverse event (irAE). Many of these side effects cause little discomfort or are asymptomatic; however, irAEs can affect any organ and are potentially life-threatening. Consequently, early diagnosis and appropriate treatment of irAEs are critical for optimizing long-term outcomes and quality of life in affected patients. Some irAEs are diagnosed according to typical symptoms, others by abnormal findings from diagnostic tests. While there are various guidelines addressing the management of irAEs, recommendations for the early recognition of irAEs as well as the optimal extent and frequency of laboratory tests are mostly lacking. In clinical practice, blood sampling is usually performed before each ICI administration , often for several months, representing a burden for patients as well as health care systems. In this report, we propose essential laboratory and functional tests to improve the early detection and management of irAEs and in cancer patients treated with ICIs. These multidisciplinary expert recommendations regarding essential laboratory and functional tests can be used to identify possible irAEs at an early time point, initiate appropriate interventions to improve patient outcomes, and reduce the burden of blood sampling during ICI treatment. Since the approval of ipilimumab in 2011, immune checkpoint inhibitors (ICIs) are increasingly used in the standard-of-care for multiple cancer types. ICIs have changed the treatment paradigm in many cancer types and have improved survival in a subset of patients with advanced or metastatic cancers . HoweverReported hospitalization rates of irAEs ranged from 8.5 to 25%, with an increased risk in older individuals > 65 years) and those undergoing combination therapy  5 years a–9, with Most irAEs occur during the first 3–4 months of treatment initiation, although delayed toxicity (defined as irAEs arising after one year of treatment) is also possible , 16. In Given these findings, the early recognition and appropriate management of irAEs are essential to optimize long-term outcomes and quality of life. The diagnosis and treatment of irAEs typically require consultation by a specialist and specific laboratory tests, imaging, and functional tests. The interpretation of test results for suspected irAEs can be challenging if no baseline values are available, particularly in patients with comorbidities that limit specific organ function. In addition, the assessment of irAE occurrence, grading, and timing is highly variable among clinicians . The thoWe provide practical recommendations for essential baseline laboratory and functional tests compiled by a multidisciplinary team of experts for the systematic assessment of various organ systems before starting ICIs and at regular intervals during ICI treatment. If patients present with any abnormal baseline values, the frequency and extent of the blood and functional tests should be adapted on an individual basis.A multidisciplinary panel composed of experts from medical oncology, cardiology, endocrinology, gastroenterology, hepatology, nephrology, neurology, pneumology, rheumatology, and public health who convened to develop the clinical recommendations. The experts met via teleconference and corresponded through e-mail. After agreement on the scope and objectives of the consensus manuscript, key topics were discussed in an iterative process until full agreement was reached. Typically authors had the last authority on discussion points pertaining to their specific area of expertise.A scoping literature search was performed using the PubMed database to obtain key literature on ICI-related toxicity and laboratory tests published using ICI–specific terms combined with “irAEs, laboratory tests, functional tests, dataset, recommendations”. Consensus discussions were repeated after gathering new evidence, and specific articles to include in the manuscript were selected by the experts based on their methodological rigor and importance for the key objectives.Clinically apparent liver toxicity due to ICI treatment is frequent. The risk for hepatotoxicity is higher with ICI combination treatment (13%) than with anti-PD1/PDL1 monotherapy (5%) and is dose-dependent –24. HepaWe recommend assessing baseline liver tests in two stages. Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyl transferase (GGT), alkaline phosphatase (ALP), and total bilirubin should be measured in all patients Table . In caseConsidering the high incidence of thyroid disorders (approximately 20%)  and hypoAlthough the long-term consequences of ICIs on gonadal tissues are currently not well known , orchitin = 8) of the patients with hypophysitis manifesting with ACTH deficiency were diagnosed with serial cortisol measurements [Although the incidence of autoimmune adrenalitis under ICIs is low (0.9%) , ICI-indurements . As suchICIs can induce insulin deficiency in about 2% of cases, presenting as new onset type 1 diabetes or worsening of preexisting type 2 diabetes . Given tICI-induced cardiotoxicity may involve all parts of the heart and is attributed to both inflammatory and non-inflammatory mechanisms. Inflammatory cardiotoxicity may manifest as myocarditis, peri-myocarditis, pericarditis, left ventricular dysfunction without myocarditis or coronary vasculitis , 39. TheA baseline transthoracic echocardiography should also be discussed in all patients. In resource-limited settings, this test should be discussed in patients at high risk of developing ICI-induced myocarditis: patients treated with combined ICI therapy or with a combination of ICI therapy with another cardiotoxic therapy, patients with ICI-related non-cardiovascular events, prior cancer therapy-induced cardiac condition  , or cardThe diagnosis of ICI-induced myocarditis is often challenging because of the wide range of potential clinical presentations. Having a baseline cardiological assessment facilitates the process of establishing the imputability of ICI when facing abnormal EKG, troponin dosage or echocardiography results during ICI therapy. In case of abnormal results at baseline, such as baseline troponin elevation, further explorations are required in collaboration with the cardio-oncology team in order to unveil an underlying cardiac condition. Although troponin is an organ-specific biomarker, it is not specific of myocarditis and several diagnoses should be considered: coronary artery disease, heart failure, arrhythmias, pulmonary embolism, cardiomyopathy, amyloidosis, hypertension, pulmonary hypertension, or arterial thromboembolism . Thus, aThe reported incidence of rheumatic irAEs varies between 1.5 and 22% , 52. Becex juvantibus treatment with corticosteroids would not be beneficial but actually be harmful. Additionally, concomitant treatment with non-steroidal anti-inflammatory drugs (NSAIDs) and/or proton pump inhibitors as well as ICI combination treatments have been shown to be risk factors for the development of AIN. We recommend assessing creatinine and urea levels prior to ICI treatment and before every administration during the frist 3 months and every 4 to 6 weeks thereafter to estimate renal function (Table The incidence of acute kidney injury (AKI) from ICIs, defined as an increase in serum creatinine of at least 1.5 times the baseline, was estimated to be 17% in a retrospective cohort study . While aIn a meta-analysis of phase II and phase III randomized controlled trials, ICI treatment was associated with an increased risk of hyponatremia  and hypokalemia  in patients with non-small cell lung cancer (NSCLC) . Hyperna3 status should be assessed before ICI treatment and patients with deficiency should initiate oral supplementation.Corticosteroids are applied in about 30–48% of patients with irAEs –60. LongHematological irAEs are rare  but potentially life-threatening . In partIt is important to identify patients with latent tuberculosis or viral infections. A latent infection not only influences the choice of immunosuppression for possible irAEs, but might require anti-infectious treatment depending on the level of immunosuppression. In agreement with other international guidelines , we recoIn a retrospective cohort study, 12% of lung cancer patients treated with ICIs developed diseases associated with disorders of the coagulation and fibrinolysis system . It was Similar to other irAEs, the incidence of colitis is higher among patients receiving ICI combination treatment (32%) compared to those treated with anti-PD1/PDL1 antibodies alone (6%) . It was Although pulmonary toxicity is a rare ICI-associated complication, it has one of the highest death rates among all irAES . An estiICIs have been associated with several neurological irAEs, including paraneoplastic neurological syndromes (PNS) which can affect the central nervous system , the peripheral nervous system , the neuromuscular junction  and muscles  . ApproxiThe majority of cancer patients are elderly and neurological examinations in this age group frequently reveal abnormal results independently of a cancer diagnosis and treatment . It can be challenging to differentiate preexisting neurological conditions from PNS or irAEs that require different treatment that are critical for the patient’s outcome. We recommend a baseline clinical neurological examination including motoric and sensory function and cranial nerve status Table . In patiConsensus guidelines on the use of essential laboratory and functional tests prior to ICI initiation and at regular intervals during ICI treatment are lacking. Given the considerable variation in clinical practice in different countries, international interdisciplinary consensus guidelines are needed that follow a delphi process to reach true consensus with a sound methodological approach across subject experts. Our recommendations were developed for optimal early detection and management of irAEs with the minimal burden for patients and health care systems. In addition to different organ specialists, the inclusion of pharmacists and pharmacologists into the expert panel can facilitate the interpretation of abnormal test results which might be caused by ICI as well as other drugs co-administered during ICI treatment.In addition to improving long-term outcomes and quality of life, this approach might even be cost-effective considering that the costs of laboratory tests are minimal compared to the costs of ICIs and hospitalization for the management of irAEs .The systematic collection of a uniform data set in large patient cohorts might also enable future research and validation of diagnostic and prognostic biomarkers which ultimately facilitate early diagnosis and management of irAEs that contribute to a more favorable quality of life and survival in patients living with cancer .


Progress in treatment for glioblastoma is hindered by the blood-brain barrier (BBB). In genetic mouse models recapitulating brain invasion and abnormal angiogenesis of human glioblastoma, Cai and colleagues demonstrate that optical modulation of the BBB with nanoparticles boosts intratumoural chemotherapy concentration, prolonging survival. We discuss prospects for clinical translation of exemplary innovative techniques. The standard of care remains maximal safe surgical resection, followed by radiotherapy and temozolomide chemotherapy2. One of the most important hurdles to overcome to improve patient outcomes in glioblastoma is to enable therapies to pass through the blood–brain barrier (BBB) and reach the tumour directly for cancer cell killing.There are several challenges facing the treatment of brain cancer. Gliomas are the most common primary intrinsic brain tumours. Glioblastoma, or a high-grade glioma, is a type of brain cancer that has a devastating prognosis, and for which oncogene-targeted and immune-targeted therapies have yet to make a significant impact in the clinic3. Furthermore, delivery to brain cancer specifically has nuanced issues: glioblastomas are heterogeneous cancers from a molecular and histopathological perspective, such that some areas of a tumour have BBB breakdown and increased permeability, whereas others do not. Additionally, the invasive margins of glioblastoma are composed of normal brain tissue infiltrated by cancer cells. In this region, the BBB remains intact, restricting drug delivery to invasive margins that have a unique immune microenvironment and where tumour recurrences are most likely to occur after treatment4.The BBB is made up of microvascular endothelial cells separating blood from brain interstitial fluid, while communicating with brain cells such as astrocytes and pericytes. The BBB is strengthened by tight and adherens junctions between neighbouring endothelial cells, creating a small pore diameter (1.4–1.8 nm) through which only limited particles of a small size can pass. Indeed, around 98% of small molecules and almost all biological macromolecular agents  are blocked from passing through the BBB, severely hampering drug delivery to the brain5 a recent advancement in this method is MRI acoustic emission-controlled focused ultrasound6. However, novel technologies for BBB opening are critical to ensure maximal drug penetration and minimal toxicity in brain cancer treatment.A plethora of techniques is being developed to improve BBB penetration in glioblastoma Fig. . These t7. While in vitro systems offer some advantages such as being easier to implement, faster and more cost-effective, in vivo cancer models are essential for recapitulating the three-dimensional organisation of tumours and their interactions within the intricate tumour microenvironment and host response. In this context, glioblastoma mouse models provide a more physiologically relevant setting to study cancer behaviour and drug responses. Several types of brain cancer mouse models exist, including syngeneic transplantation of mutated neural or cancer cells, genetically engineered mouse models (GEMMs), and patient-derived xenografts8. Different models have their own strengths and weaknesses, but a common issue is the inability to fully replicate cancer invasion into the surrounding brain and the aberrant microvasculature of glioblastoma.While new methods are being explored to improve drug delivery across the BBB, mouse models remain essential tools for preclinical testing of potential chemotherapies for glioblastoma before advancing to clinical trialsNature Communications, the authors developed two transplantable glioblastoma mouse models that reflect significant histopathological features observed in patient cancers9. They subsequently demonstrate that optical modulation of the BBB using tight junction-targeted gold nanoparticles can significantly enhance the delivery of the chemotherapeutic drug Taxol into brain cancers. Importantly, this intervention resulted in improved survival rates in mice.Now, Cai et al. introduce an alternative technique to enhance drug delivery into brain tumours, employing optical nanoparticle-based modulation of the BBB. In their study, published in BrafV600E, Ink4ab/Arf-/-, Pten-/- mutations and the other featuring a different set of mutations . With the exception of the relatively infrequent oncogene Braf mutation observed in human high-grade gliomas, the loss of the other tumour suppressor genes is a frequent occurrence in patients’ tumours10. These mouse models accurately portray key clinicopathological aspects of human glioblastomas: one model displays diffuse single-cell infiltration of cancer cells throughout the brain parenchyma, while the second model exhibits highly aberrant tumour vasculature yet limited brain invasion. Together, these two models effectively capture important features seen in human glioblastomas.While numerous mouse models of glioblastoma are employed in preclinical research, many fail to fully replicate the genomic or clinicopathological characteristics of human glioblastoma. Cai et al. genetically engineered two mouse brain cancer cell lines and implanted them into the mouse cortex Fig. . These cIn the subsequent phase, the authors assessed the technique of optical blood–brain-tumour barrier (optoBBTB) modulation utilising gold nanoparticles Fig. . They ch11. This weakening facilitated the delivery of Taxol into the tumour. The BBB modulation demonstrated several attributes: (1) it was region-specific, enhancing drug delivery exclusively to the tumour and not the rest of the brain; (2) it was reversible, as the BBB restored within a single day; and 3) it was repeatable, allowing for three rounds of chemotherapy with BBB modulation and drug delivery to the tumour on each occasion. Notably, this approach not only increased the concentration of Taxol within the tumours, but it also significantly prolonged the survival of mice in both the infiltrative and angiogenic glioblastoma models. This extension in survival time correlated with decreased tumour cell proliferation and increased cell death in the tumours of mice treated with opto-BBTB.Gold nanoparticles with a diameter of 50 nm, designed to target the tight junction protein JAM-A, were intravenously administered to mice harbouring glioblastoma. Subsequently, a transcranial 532 nm picosecond laser was applied to the tumour region to activate the gold nanoparticles and induce temporary weakening of the BBB, likely through increased paracellular permeability related to calcium influx12. Therefore, it remains to be determined whether optoBBTB modulation can effectively facilitate drug delivery to tumours located at greater depths within the brain. Moreover, it is important to note that mice possess relatively thin skulls compared to humans. As such, parameters for transcranial laser stimulation through thicker human cranial bone will require optimisation for practical application in humans. Proposed strategies for addressing these challenges include the use of near-infrared light-absorbing nanoparticles (as light at this wavelength has greater tissue penetration), or the placement of optical fibres into the cancer surgical cavity, allowing the delivery of side-emitting light to and beyond the tumour margins in patients. Another recent experimental tool is the use of “See-Shells”, digitally designed, transparent polymer skulls that offer long-term optical access to the brain. Although these “See-Shells” have the potential for optical BBB modulation, their application has only been tested in mice so far13.One limitation of this model pertains to the superficial location of the glioblastomas, situated in the cerebral cortex directly adjacent to the skull’s surface. While certain brain tumours in human patients share a similar location, a significant number are positioned deeper within the brain tissueIt is important to recognise the substantial size disparity between glioblastomas commonly observed in patients and those in mice. The translation process to the clinic must also account for the requirement of significantly more repeated chemotherapy doses in human patients. Furthermore, adaptations must consider the alterations induced in tumours due to extended radiation and chemotherapy regimens. Additionally, careful consideration should be directed towards the risk of immunological responses associated with long-term or repetitive treatment of the nanoparticles. Addressing these factors comprehensively will be imperative before successfully transitioning this approach into clinical practice.EGFR amplification, PDGFRA amplification, or an IDH1 mutation associated with a distinct glioma subtype15. Moreover, incorporating immunocompetent mice into the study would provide a more realistic scenario, as both the tumour behaviour as well as drug delivery and therapeutic efficacy could be influenced by intact immune responses.To further validate the approach, diverse mouse models like GEMMs and patient-derived xenografts could be explored. While the study employed clinically relevant glioblastoma mouse models to evaluate optical BBB modulation, it remains essential to investigate this technique in glioblastoma models that harbour other common oncogenic drivers with clinical relevance. Examples of such drivers include 16. By exploring the potential of optoBBTB modulation, researchers can gain valuable insights into enhancing drug delivery to the brain and improving the effectiveness of various treatments for glioblastoma and other brain cancers.Finally, while Taxol is a relevant drug for glioblastoma whose efficacy is limited due to the BBB, it would be similarly interesting to test other chemotherapeutic drugs and antibodies with similar limitations using this methodology. In particular, antibodies targeting immune checkpoints PD-1 and CTLA-4 (such as pembrolizumab and ipilimumab) have not shown major clinical benefits for glioblastoma and brain metastases, despite their remarkable success in peripheral tumours. This disparity may be attributed to BBB challenges, which could potentially be overcome by exploring methods like optoBBTBBypassing the BBB to enhance drug concentrations within tumours has long been a formidable challenge in the field of brain cancer therapy. Alongside the development of innovative methods to facilitate drug penetration into the brain, it remains crucial to advance the preclinical modelling of this disease. Such advancements are essential to ensure that emerging tools and therapies can be effectively evaluated, optimising their clinical potential.17 BBB-penetrating bacteria loaded with photosensitive nanoparticles that destroy themselves and adjacent tumour cells upon laser irradiation (‘Trojan bacteria’)18; and nanomotors targeting brain endothelial cells, relying on glioblastoma microenvironment chemoattractants to infiltrate the tumour and directly deliver chemotherapy19. While promising, future endeavours must address challenges associated with the clinical application of optoBBTB and similar technologies, particularly the anatomical disparities between humans and mice. Nevertheless, this study, alongside recent advancements in BBB modulation techniques, holds great promise for enabling a new generation of targeted therapies, including oncogene-targeted and immune-based drugs, to effectively penetrate brain cancers.In essence, the study conducted by Cai et al. not only introduces mouse models that replicate the invasive and angiogenic characteristics of human glioblastoma but also convincingly demonstrates the effectiveness of optical BBB modulation using nanoparticles. This approach enhances the infiltration of chemotherapy agents into brain tumours, ultimately extending overall survival. Other promising nanoscale technologies for BBB penetration are emerging, such as synthetic protein nanoparticles that leverage the BBB-penetrating capacity of natural proteins;


Substance abuse is common among patients with schizophrenia, is related toworse course and outcome of illness. Unfortunately, little is known about how substanceabuse affects the cognitive function of schizophrenia patients, whose cognitive function isoften already comprised. Neurocognitive functioning includes inhibition control and decision-making, and both schizophrenia and substance use disorder are related to impairmentsof inhibition control. However, the influence of substance abuse on inhibition capacitiesamong schizophrenia patients is unclearThis study measured the influence of substance use disorder on inhibition capacities and risky decision-making in a group of 39 schizophrenia patients that were evaluated using a socio-demographic questionnaire and clinical assessment using the Positive and Negative Syndromes Scale for Schizophrenia. To assess inhibition control we utilized the Matching Familiar Figure Test (MFFT) and the Stroop task, and to evaluate decision-making we used the Iowa Gambling Task (IGT) and self-report questionnaire, the Barratt Impulsiveness ScaleUnivariate analysis found significant differences between the groups with regard to criminal history , smoking status , and total BIS score . Our model did not find a significant effect of substance abuse on the first response time and number of errors on the MFFT or in the total interference index of Stroop performance and net score on risky decision-making in the IGT.The two groups did not differ significantly either in first response time or in number of errors on the MFFT , nor did they differ in the total interference index of the Stroop task (F(1)¼0.49, p¼.49, d¼0.25, 95% CI ).The analyses did not detect any statistically significant effect of substance abuse on inhibition control or risky decision-making processes in outpatients diagnosed with schizophrenia, despite increased impulsivity, criminal history and smoking status. These results neither support nor disprove previous findings.None Declared


Nature and Cell, Weatherbee et al.1, Pedroza et al.2 and Liu et al.3 have demonstrated how human pluripotent stem cells can be coaxed to self-organize into compartmentalized structures that mirror post-implantation embryos. Building on the successful establishment of stem cell-derived mouse embryo models ex utero, these models now shed light on human embryo development during the period between implantation and gastrulation, which has so far been challenging to investigate.In three recent articles published in 4Following the first few cell divisions after fertilization, the resulting blastomeres become fate-restricted and form a blastocyst, consisting of the inner cell mass, which gives rise to the embryo, and the surrounding trophectoderm, an extraembryonic tissue that gives rise to the placenta Fig. . The inn1 approached this challenge by first inducing extraembryonic lineages separately through transient overexpression of transgenes  to induce intermediate pluripotency states , which show potency for both embryonic and extraembryonic tissues (Fig. 5 reported self-assembly of an embryo model grown in shaking culture that includes trophoblast-like cells derived from non-transgenic naïve hESCs. This did indeed support not only authentic morphological organization and orchestrated temporal progression but also a nearly full complement of developmental lineages, including most extra-embryonic compartments, albeit with a relatively low efficiency. Nonetheless, incomplete models that cannot develop into viable embryos may be better able to assuage ethical concerns around this kind of research while the regulatory frameworks catch up with these new developments.In the third study, Liu et al.ues Fig. . The modCollectively, these sophisticated methodologies for establishing models of human post-implantation embryos have already illuminated the interplay between embryonic and extraembryonic compartments and now allow in-depth study of human development to advance reproductive and regenerative medicine. Notably, they share several technical and mechanistic concepts with sophisticated co-culture models based on adult stem cells that allow investigations into intercellular communication and self-organization in mature tissues. Much may be gained from synergizing the different strengths of these fields.


Precision control via molecular structure over adaptiveconjugatedpolymer properties in aqueous environments is critical for realizingtheir biomedical applications. Here, we unravel the dependence ofamphiphilic peptide-polydiacetylene (PDA) conjugate properties onthe characteristic steric and hydrophobic contributions within peptidesegments that serve as a biomimetic template for diacetylene polymerizationin water. We investigated the functional impacts of molecular volumeand polarity changes brought by dipeptide substitution domains onthe following peptide-PDA material properties at multiple length scales:supramolecular assembly behavior, chain conformation-dependent photophysicalproperties, cell-material interfacing, and for the first time, bulkelectrical properties of their films processed in water. A libraryof peptide-PDAs with systematically varied sequences show that thecontributions of steric effects predominantly influence the electronicstructure and resulting trends in photophysical properties, whilethe interplay between size and hydrophobicity of individual residuesbecomes more significant for higher-order assemblies affecting bulkproperties. This work demonstrates sequence-tunable molecular volumeand polarity as synthetic handles to rationally modulate PDA materialproperties across length scales, providing insights into the programmabilityof biomimetic conjugated polymers with adaptive functionalities. Moreover,the progression of the impact of individual residue substitution tobulk property changes for PDAs remains elusive. Considering the widespreaduse of PDA-based materials in various biomedical applications nowadays,38 there is a critical need for more studies that shed light on howto control material function based on the molecular design of biologicallyrelevant templating groups for PDAs.44To date, self-assembled biomolecules such as peptidesare amongthe DA templating groups shown to successfully drive and influencethe photopolymerization and chromogenic transitions of PDAs.In this study, we contribute toward addressing the knowledgegapin predicting the structural influence of the templating groups onPDA properties across length scales by molecularly controlling theproperties of peptide moieties through steric effects and hydrophobicinteractions 1. In par47 Here, peptidestructure control was utilized to modulate supramolecular interactionsbetween monomers and the conformation that they adapt after polymerizationunder aqueous environments. To do so, we prepared a library of amphiphilicDA monomers appended to a hexapeptide segment (with a dipeptide substitutiondomain) with comparable length as the aliphatic segments of PCDA:DDGDXX- and DXXDGD- . The dipeptide substitution domain  is implemented at two differentpositions within the hexapeptide: (i) closer to the aliphatic chainor (ii) closer to the periphery (C-terminus) of the peptide region.We varied the position of the dipeptide substitution domains to assessthe positional dependence of the perturbations in sterics and polarityafforded by each amino acid variation. Three aspartic acid (D) unitsper hexapeptide segment, with glycine (G) as a spacer between the2nd and 3rd D positions and at least one D in the C-terminus, arealso included as ionizable residues to improve aqueous solubilityand to generate a pH-responsive template that can easily induce PDAconformational changes due to electrostatic repulsion between thecarboxylates of D units when the pH > pKa of the monomer. When pH < apparent pKa, protonated aspartic acids support hydrogen bonding along with othersupramolecular interactions that guide the topochemical polymerizationof PDAs. Hence, all peptide-PDAs here are assembled at pH 2, afterwhich these aqueous suspensions were irradiated with 254 nm UV irradiationto initiate the polymerization process. One aspartic acid unit atthe C-terminus of all monomers is kept constant throughoutthe library as the unaltered position at the polar end of the amphiphilicregion. The synthesis and characterization data for these peptide-DAmonomers and peptide-PDA polymers can be found in the Supporting Information.The sequence composition of oligopeptideswith fixed lengths is systematically varied herein to understand howsteric effects and individual residue hydrophobicity impact PDA propertiesacross length scales. We chose 10,12-pentacosadiynoic acid (PCDA)as a commercially available PDA precursor that has been previouslyreported for use in sensing or tissue engineering applications.Through molecular variations in the peptidic template region of theamphiphiles, we sought to understand the distinct contributions ofhydrophobicity and steric effects on conformations adapted by theDA assemblies and PDA nanostructures, as well as their resulting properties.For example, hexapeptides with divaline (VV) vs dithreonine (TT) domainshave similar molecular volume, but the molecular polarity of VV-containingmoiety is lower than the TT-containing analog. The same rationalegoes for comparing diphenylalanine (FF) and dityrosine (YY) substitutions.As a point of reference, we used a diglycine (GG) substitution togenerate a sequence with the lowest molecular volume but high molecularpolarity value within the series. We can also consider that the molecularvolume of TT and YY domains are larger than that of GG-, but all threeare inherently more polar and less hydrophobic than the monomers withVV and FF domains. Since peptide-induced torsional impacts on thePDA backbone can result in measurable chromatic transitions, we utilizethe photophysical properties of peptide-PDAs as a probe for monitoringthe conformational changes imparted by the amino acid substitutionacross the peptide-polymer library studied herein.50 for a single amino acid, dipeptides, hexapeptidesegments, and peptide-DA monomers . Unlike one of theconventional methods for quantifying hydrophobicity differences calledgrand average of hydropathy, which is based on the individual residuehydropathy and disregards the order of amino acids within a sequence,51 the quantitative analysis used in this studywas performed using geometries optimized with density functional theory.Here, we focus on comparing the differences between the molecularvolume and MPI values for energy-minimized peptide segments to emphasize the characteristic differences that the peptide segmentsimpart on DA-bearing monomers. We hypothesize that the systematicvariation within the substitution domains will allow us to probe theinfluence of the following supramolecular interactions between DAassemblies/PDA chains pre- and post-polymerization: (i) steric repulsionof bulky residues that could impact the distance of DA monomers and(ii) changes in hydrophobic interactions, intermolecular hydrogenbonding, and solvent–peptide segment interactions due to varyingMPI values among the monomers.To quantitativelycompare the size and polarity differences ofpeptide segments, we calculated the molecular volume and molecularpolarity index (MPI)segments 2b,c rathXX-PDA and DXXDGD-PDA  over a course of 40 min. The increase in thedegree of polymerization for peptide-PDAs becomes insignificant after∼30 min as shown in the absorption spectra . We observed a progressive predominanceof peak signatures for red phase PDAs (λmax ∼540nm) as the UV exposure increases, which is consistent with the observationfor other reports on alkyl PDAs.53 The resultsof this absorption-based kinetic monitoring of PDA formation suggestthat the degree of polymerization of our peptide-PDAs at pH 2 canbe saturated within 40 min of exposure to 254 nm irradiation. In addition,the SEC characterization confirms that all samples were polymerizedwithin the range of ∼105 to ∼106 Da ,which is consistent with literature reports.55 A concentration-driven partial aggregation of peptide-PDAs at pH10 condition and sample-column interaction may still exist, as indicatedby the relatively short retention time and broad peak. With this,all subsequent experiments use polymer solutions resulting from peptide-DAsthat are UV-irradiated with 254 nm for 40 min. From these samples,we visualized the resulting nanostructure morphologies formed by theresulting peptide-PDAs using transmission electron microscopy (TEM).All unpolymerized assemblies under acidic conditions, as well as thepeptide-polymer conjugates in acidic and basic environments, formed1-D nanostructures with fiber-like morphologies (∼5 to 16 nm)similar to other peptide amphiphiles .56 Although no apparent trend changesin nanoscale morphology were observed across the sequence libraryor even between acidic and basic conditions, other than varying degreesof apparent twists, polydispersity, and aspect ratios, this does noteliminate the influence of peptide sequence in chain conformationand how the sequence impacts PDA properties at other length scales.To maximizethe degree of polymerization that can be afforded by each peptidicmonomer and standardize the length of UV irradiation time needed byall monomers within our library, we monitored the evolution of absorptionpeaks for 1 mM acidic solution of DDGD spectra S46–S48. 57 Here, we utilize the distinct PDA chromaticphases, particularly the blue and red phases, to spectroscopicallyinvestigate the torsion experienced by PDAs along the π-conjugatedchain as a consequence of the peptide templating groups  absorption with respect to blue phase DDGDGG-PDA, significant hypsochromic shifts of λmax (up to 100 nm) due to dominance in the absorption spectraof red phase peaks are observed. This trend shows that substitutingglycines with larger side chains, when they are nearest to the alkylDA region, leads to pronounced differences in the PDA chain conformation are observed when comparingthe λmax for DDGDVV-PDA vs DDGDTT-PDA, and DDGDFF-PDA vs DDGDYY-PDA absorption, whose molecular volume of peptide segmentsare relatively similar while having a considerable difference in polarity. To better quantify the torsional impact of the peptidevariation on the PDA backbone, we took the intensity ratio of peak1  and peak 2 . Despite DDGDGG-PDA having the highest P1/P2 ratio, we found that the ratioof the less planar red phase increases along with increasing peptideMPI when the molecular volumes of hexapeptides are larger than 600Å3 ,and circular dichroism (CD) spectroscopy on dilute aqueous dispersionsof peptide-PDA nanostructures. It is well established that the photophysicalproperties of PDA are directly correlated to the monomer assemblybehavior and ultimately, the conformation of the polymer backbone.g groups 1. Comparormation1. To betn 600Å3 4e. Thus,XXDGD-PDA; X = G, V, T, F, and Y), less impact of steric effectscan be expected on polymer conformation. All peptide-PDAs within thisseries had λmax of absorption ∼530 to 550nm  compared to DGGDGD-PDA. These results suggest that size differences in DXXDGD-PDA, from G- to the large F-/Y-,do not endow as much difference in conformation compared to DDGDXX-PDA; which is consistent with ourexpectation. This is also supported by the small 2–10 nm hypsochromicshift recorded for the λmax of T-, F-, and Y-substitutedpeptides-PDA absorption with respect to DGGDDG-PDA absorption. However, 3. These spectral results suggestthat although the impact of steric repulsion on the absorption maximais not as drastic as in DDGDXX-PDA, the size of residues in the substitution domain for DXXDGD-PDA still affects the distributionof blue- or red-phase chains within the ensemble polymers that existin the acidic aqueous solutions. The P1/P2 values are also comparablewhen comparing DTTDDG-PDA to DVVDDG-PDA and DFFDDG-PDA to DYYDDG-PDA,indicating that the polarity has a subtle impact on PDA conformationwhen the substitution domain is more accessible to the solvent. Forthe DXXDGD-PDA series, the absorptiondata demonstrate that the differential interaction of a peripheralXX domain with the solvent does not significantly perturb the DA assemblyand the conformation of the resulting peptide.62 This is also consistent with the smaller MPI differencebetween DTTDGD vs DVVDGD than that of DDGDTT vs DDGDVV, as well aswhen comparing the MPI difference between the FF- vs YY-substitutionof DDGDXX and DXXDGD peptides. For the sequences containing aromaticside chains, regardless of the position of the substitution, the additionalπ interactions did not favor the blue planar conformation asall spectra for the 4 peptide-PDAs with aromatic group have low P1/P2.This trend further highlights the contribution of steric effects tothe resulting PDA conformation. The collective absorption profilesof PDAs from DDGDXX- and DXXDGD-PDA show that the torsional impactof peptides on PDA conformation is significantly different when thesubstitution domain is closer to the solvent or to the DA group evenif the two substitution domain positions being compared here are justseparated by one amino acid position. Moreover, while hydrophobicityand steric effects both contribute to the final chain conformationof the PDA, our results show that steric repulsion presented to bethe more predominant factor in driving the preference toward a planar,blue or twisted, red PDA phase.When the substitution domain is further away fromthe conjugatedbackbone, but more accessible to water .63For the aqueous dispersionsof both DDGDished P1 1. More se series 4g, indicXX-PDAs exhibitedmore apparent spectral differences across the series than the DXXDGD-PDAs . The trends in the shifts between λem at pH 10 do not exactly follow the molecular volume or MPI trends,illustrating the dominance of repulsive electrostatic effects oversteric and hydrophobic effects on PDA conformation. We note that thePL spectra of acidic samples are not reported due to their very lowsignal-to-noise ratio that can be attributed to the existence of amore planar, blue phase with ultrafast relaxation of the excited state.65 Taken together, the absorption and PL data suggest that when theelectrostatic repulsion between D residues is in full effect at highpH, the impacts of size and hydrophobicity on PDA conformation becomeless significant than when the carboxylates of the peptide segmentsare fully protonated. Yet, steric and hydrophobic contributions stillshow more effect when the XX domain is nearer to the DA units thanwhen they are more accessible to the solvent, the peptide C-termini.When we examined the PL spectra of the polymer samplesunder basicconditions, DDGD7 However,when comparing the blue and red phases, several experimental and computationaldemonstrations argued that a decrease in order and a correspondingshortening of the conjugation length is not appropriate for describingthe red phase of PDA as an extended 1-D electronic system.67 We further explore the sequence-based structural modulation forthe peptide-PDAs here by performing CD spectroscopy to evaluate anycorrelation between the observed photophysical properties and thesequence-defined order via the secondary structure adapted by peptidicmoieties . The characteristic β-sheet-like band wasstill observed for all samples after polymerization under pH 2, indicatingthat polymerization did not completely disrupt the initial assemblyand that the sequence-specific peptide interactions promoted DA geometricarrangements favorable toward the topochemical formation of PDA  to that signature observed for the polymeric equivalents . Theseresults suggest that the steric contribution of bulky residues hindersthe formation of secondary structures that are appropriate for theformation of a planar conjugated backbone.69 Hence, the subtle steric and hydrophobic residue differences withinour monomer library demonstrate distinct effects on the molecular-and chain-level order adapted by the peptide-polymer conjugates asshown by the spectroscopic differences. The molecular simulationsreported here (S53–S57) further support themolecular ordering behavior of peptide-polymer conjugates as wellas the spectroscopic differences brought by differences in chain conformation.Comparing the acidic and basic peptide-PDA solutions, CD spectra showthat increasing the electrostatic repulsion in the peptide regionresulted in a structural shift from β-sheet-like to random coilssequences (VV- and FF-substituted) show characteristic negative excitonicchirality with cross-over wavelengths that clearly coincide with theλmax of red phase PDAs absorption. Meanwhile, DXXDGD-PDAs with bulky and hydrophobicresidues at the substitution domain have CD spectra with low intensityand asymmetric signals, suggesting that the interaction of residueswith the solvent is more likely to affect the secondary structure.Upon switching to basic conditions, the structural reorganizationof PDAs to a more disordered state is demonstrated by the quenchedbisignate signature between 450 and 700 nm, alongside the transitionfrom β-sheet-like structure to random coil observed for thepeptide segment. Based on the photophysical properties of the peptide-PDAsstudied here, both steric effects and hydrophobic interactions havemore synergistic contributions to the PDA order at the secondary structurelevel than the conformation at the chain level. Steric repulsion inthe peptide moiety has a larger impact on the chain conformation andelectronic structure, and thus can be used to modulate the chromaticphase of the resulting PDA. On the other hand, residue hydrophobicityand general peptide polarity, via peptide segment-solvent interactions,become more relevant when considering order at the secondary structurelevel and excitonic chirality of peptide-PDAs. When triggering electrostaticrepulsion by switching to basic conditions, regardless of the positionor nature of the residue at the substitution domain, all samples adapteda disordered PDA assembly, peptide moieties in random coil, and dominanceof the red PDA phase.Within the region of PDA absorption (450–700nm), the CDspectra of all peptide-PDAs at pH 2 exhibited a bisignate profileindicative of the Cotton effect 5c,f, andS53–S55). All carboxyl groups were keptin the protonated state to simulate polymer structures under acidicconditions. In particular, we conducted these simulations on a decamerpeptide-PDA model to shed light on the synergistic role of hydrogenbonding with the other supramolecular factors such as steric and hydrophobiceffects investigated here. To do so, weak and moderate intermolecularhydrogen bonds70 formed between peptideunits and peptide-water interactions were quantified based on thestructural snapshots from molecular dynamics simulations . Regardless of the substitutionposition, the average number of hydrogen bonds formed between peptideunits is highest when the residues at the XX domain have the bulkiest,aromatic amino acids (FF and YY). This result is consistent with thespectroscopic data, where we concluded that steric effect plays asignificant role in templating the conformation of PDA , there are more H-bonding interactionswhen these polar residues are closer to the nonpolar core of the assembly.These simulation results can also partially explain why the intensitiesof CD signals for DXXDGD-PDA around220 nm are relatively weaker compared to those for DDGDXX-PDA at pH 2. The observed solvation effectson the model may hinder the hydrophobic interactions necessary toform an ordered secondary structure when XX is further from the PDAbackbone, which is also indicated when there are fewer D-units accessibleto water.To better explain thespectroscopic differences within our sample library, force-field-basedmolecular dynamics simulations were performed to obtain atomic-levelapproximations of the designed peptide-PDA structures in aqueous environments. All peptide-PDAfilms made from pH 2 and pH 10 solutions showed measurable conductivityranging from 10–7 to 10–3 S cm–1 , indicatingthe relatively more ordered domains in peptide-PDA films with significantlyreduced intrachain electrostatic repulsion. These results are consistentwith the expectation that global ionization of peptides results inbulk disorder due to electrostatic repulsion under basic conditions,hence decreasing conductivity.PDAs have been previously used to dope and conjugatewith other organic compounds for improved charge transport.segments 7. Althouectrodes 7c. The f3 S cm–1 7a,b. TheXX-PDA andDXXDGD-PDA filmsmade under pH 2 conditions, specifically resulting in increased conductivityalong with the decrease of the peptidic molecular volume . Samplefilms for this analysis were made by drop-casting 1 mM acidic andbasic solutions directly to clean glass slides and then dried overnightunder ambient conditions. Throughout the peptide-PDA library, thefilms exhibited an overall bathochromic shift in spectra comparedto the absorption profiles recorded from the aqueous solutions , which could be attributed to furtheraggregation brought by the drying process. It is also noticeable thatthe substitution nearest to the PDA region resulted in films withspectra that are more sensitive to the residue variation comparedto when the substitution is at the periphery. After spectra collectionand mapping multiple regions of interest per film, we performed spectralangle mapping (SAM) and assessed the homogeneity of the chromaticphase adapted across the films. For each film sample, we identifieda spectral profile per film that was most represented within a definedregion and calculated the percentage of spectral matching per area. The percentageof spectral matching, which is indicative of both spectral homogeneityand surface roughness throughout the films, generally decreases withthe increase of residue size for both DDGDXX-PDA at pH 2 and pH 10 solutions  but not for DXXDGD-PDA . Collectively,considering the results of the conductivity measurements and hyperspectralmicroscopy, we show that the subtle residue variations in the peptidetemplate region are sufficient to modulate the bulk optical and electricalproperties of peptide-PDA films. These measurements also strengthenthe argument that the trends in conformation-dependent chromatic phasesin solution, blue or red PDAs, are not strictly correlated to bulkfilm properties. More specifically, the chain conformation-dependentchromatic phases are influenced by molecular design in a distinctmanner from the impacts in higher-level structural order, which furthersupports the need of studies that look into molecular design programmabilityof material properties at multiple length scales.We further investigated the optical properties of the sequence-variedpeptide-PDA films and their homogeneity using hyperspectral microscopywith dark-field imaging . Throughthese experiments, we assessed how varying the peptide sequence templatesimpact the feasibility of these PDAs to be utilized for applicationssuch as cutaneous tissue engineering or sensing at the interface ofdermal tissues.82 To assess how sequence variation differentially impacts cell viability,acute exposure of peptide-DAs was performed by incubating HDFs with1 mM monomer dispersions in an electrolytic, buffered medium for 6h . Except for theHDFs exposed to DDGDGG-DA, allother peptide-DAs show ≥90% viability, demonstrating the biocompatibilityof peptide-DAs under physiologically relevant conditions (GG-) had the lowest cell viability within our peptide-PDA library.In addition, at higher concentrations, the polymerized assembly canform a film serving as a substrate for HDF growth in vitro. Here, we prepared 5 mM peptide-DA solutions that were assembledwith the aid of the slow hydrolysis of glucono-δ-lactone (10mg/mL) to make uniform films. All polymers from our library successfullyformed polymerized films via UV irradiation, but only those resultingfrom DDGDVV-, DDGDTT-, and DDGDYY-PDA resulted in films that are stable in cell culture media forat least 5 days. These are the sequences with substitution domainsnearest to the PDA region, bearing mid-sized residues (V and T) anda bulky, but −OH-containing Y residue. This observation showsthe importance of peptidic design, particularly the balance betweenthe attractive and repulsive interactions within the peptide moieties,when considering building scaffolds that rely on hierarchical 1-Dassembly for polymer chain formation and chain entanglements to formstable films (or even hydrogels for future applications). After culturingHDFs on the VV-, TT-, and YY-substituted peptide-PDA films for 5 days,HDF monolayers formed on peptide-PDA films similar to those seededon control glass coverslips (S70). However,the cellular morphologies and apparent density of actin stress fibersqualitatively observed for DDGDYY-PDA, which has the largest monomer volume, and poorest surface homogeneity within the DDGDXX series, were different from the control andthe other peptide-PDA films. While a more deterministic set of designrules to predict this biointerfacing behavior would require a largerlibrary of peptides to screen, our results herein set the foundationfor programming cellular viability and cellular phenotype via peptidedesign for bioscaffolds built from peptide-PDAs materials, which willbe investigated in detail in the future.Since peptide-PDAs have been demonstrated to have potential usefor different biomedical applications,nditions 8a. The sIn this study, we demonstrate the abilityof systematic peptidesequence templating of amphiphilic DAs to rationally tune the propertiesof the resulting PDAs as a chromatically adaptive, π-conjugatedpolymer that can be utilized under physiologically relevant conditions.We built a library of hexameric peptide-DA monomers with dipeptidesubstitution domains that help delineate the contributions of stericsand residue polarity to the multiscale structure and behavior of peptide-PDAmaterials. Our results show that steric repulsion, especially nearthe PDA region, has more torsional impact on the conformation of thePDA chains than the inherent hydrophobicity of peptides. These findingsdemonstrated significant differences in properties based on the positionalisomers of peptide-polymer PDAs studied herein. Photophysical measurementsshow that substitutions with bulky residues generally lead to thetwisted, red phase PDA. We also show that changes in the secondarystructure of the peptide moieties translate the changes in the degreeof excitonic chirality experienced by the PDA chains. A transitionfrom β-sheet-like structures to random coils upon changing fromacidic to basic conditions guided the PDAs to adapt a more twistedchain along with disordered structures due to electrostatic repulsion.Molecular simulations showed that the impact of peptide variationis more pronounced on intramolecular hydrogen bonding within the peptideregion rather than on the polymer–solvent hydrogen bonding.The subtle systematic substitutions on the peptide segments resultedin measurable changes in bulk properties such as electrical conductivity,which is known to be dependent on the order of polymer ensembles infilm and was established to have distinct trends from how the chainconformation-dependent chromatic phase and electronic structure wasaffected by sequence design in solution state. Moreover, the systematicsequence variation on the peptide template was sufficient to observechanges in the interaction of peptide-DA monomer suspensions and peptide-PDAfilms with human dermal fibroblasts. In conclusion, the ability ofpeptide sequences to template the aqueous assembly of diacetylenemonomers toward a specific chromatic phase can be leveraged to modulatethe secondary structure, as well as bulk order of these peptide-polymerbiomaterials—demonstrating an approach to finely and distinctlytune properties at multiple length scales. These experimental findingson controllable material properties across length scales offer insightsinto the molecular programmability of the behavior of these peptide-polymerconjugates as functional biomaterials in physiologically relevantaqueous environments.