Cells-in-Touch: 3D Printing in Reconstruction and Modelling of Microscopic Biological Geometries for Education and Future Research Applications.

Additive manufacturing (3D printing) and computer-aided design (CAD) still have limited uptake in biomedical and bioengineering research and education, despite the significant potential of these technologies. The utility of organ-scale 3D-printed models of living structures is widely appreciated, while the workflows for microscopy data translation into tactile accessible replicas are not well developed yet. Here, we demonstrate an accessible and reproducible CAD-based methodology for generating 3D-printed scalable models of human cells cultured in vitro and imaged using conventional scanning confocal microscopy with fused deposition modeling (FDM) 3D printing. We termed this technology CiTo-3DP (Cells-in-Touch for 3D Printing). As a proof-of-concept, we created dismountable CiTo-3DP models of human epithelial, mesenchymal, and neural cells by using selectively stained nuclei and cytoskeletal components. We also provide educational and research context for the presented cellular models. In the future, the CiTo-3DP approach can be adapted to different imaging and 3D printing modalities and comprehensively present various cell types, subcellular structures, and extracellular matrices. The resulting CAD and 3D printed models could be used for a broad spectrum of education and research applications.

Diagnostic and prognostic biomarkers for tubulointerstitial fibrosis.

Renal fibrosis is the final common pathophysiological pathway in chronic kidney disease (CKD) regardless of the underlying cause of kidney injury. Tubulointerstitial fibrosis (TIF) is considered to be the key pathological predictor of CKD progression. Currently, the gold-standard tool to identify TIF is kidney biopsy, an invasive method that carries risks. Non-invasive diagnostics rely on an estimation of glomerular filtration rate and albuminuria to assess kidney function, but these fail to diagnose early CKD accurately or to predict progressive decline in kidney function. In this review, we summarize the current and emerging molecular biomarkers that have been studied in various clinical settings and in animal models of kidney disease and that are correlated with the degree of TIF. We examine the potential of these biomarkers to diagnose TIF non-invasively and to predict disease progression. We also examine the potential of new technologies and non-invasive diagnostic approaches in assessing TIF. Limitations of current and potential biomarkers are discussed and knowledge gaps identified.

CSF neopterin, quinolinic acid and kynurenine/tryptophan ratio are biomarkers of active neuroinflammation.

BACKGROUND: Defining the presence of acute and chronic brain inflammation remains a challenge to clinicians due to the heterogeneity of clinical presentations and aetiologies. However, defining the presence of neuroinflammation, and monitoring the effects of therapy is important given its reversible and potentially damaging nature. We investigated the utility of CSF metabolites in the diagnosis of primary neuroinflammatory disorders such as encephalitis and explored the potential pathogenic role of inflammation in epilepsy. METHODS: Cerebrospinal fluid (CSF) collected from 341 paediatric patients (169 males, median age 5.8 years, range 0.1-17.1) were examined. The patients were separated into a primary inflammatory disorder group (n = 90) and epilepsy group (n = 80), who were compared with three control groups including neurogenetic and structural (n = 76), neurodevelopmental disorders, psychiatric and functional neurological disorders (n = 63), and headache (n = 32). FINDINGS: There were statistically significant increases of CSF neopterin, kynurenine, quinolinic acid and kynurenine/tryptophan ratio (KYN/TRP) in the inflammation group compared to all control groups (all p < 0.0003). As biomarkers, at thresholds with 95% specificity, CSF neopterin had the best sensitivity for defining neuroinflammation (82%, CI 73-89), then quinolinic acid (57%, CI 47-67), KYN/TRP ratio (47%, CI 36-56) and kynurenine (37%, CI 28-48). CSF pleocytosis had sensitivity of 53%, CI 42-64). The area under the receiver operating characteristic curve (ROC AUC) of CSF neopterin (94.4% CI 91.0-97.7%) was superior to that of CSF pleocytosis (84.9% CI 79.5-90.4%) (p = 0.005). CSF kynurenic acid/kynurenine ratio (KYNA/KYN) was statistically decreased in the epilepsy group compared to all control groups (all p ≤ 0.0003), which was evident in most epilepsy subgroups. INTERPRETATION: Here we show that CSF neopterin, kynurenine, quinolinic acid and KYN/TRP are useful diagnostic and monitoring biomarkers of neuroinflammation. These findings provide biological insights into the role of inflammatory metabolism in neurological disorders and provide diagnostic and therapeutic opportunities for improved management of neurological diseases. FUNDING: Financial support for the study was granted by Dale NHMRC Investigator grant APP1193648, University of Sydney, Petre Foundation, Cerebral Palsy Alliance and Department of Biochemistry at the Children's Hospital at Westmead. Prof Guillemin is funded by NHMRC Investigator grant APP 1176660 and Macquarie University.

Deciphering the Kidney Matrisome: Identification and Quantification of Renal Extracellular Matrix Proteins in Healthy Mice.

Precise characterization of a tissue's extracellular matrix (ECM) protein composition (matrisome) is essential for biomedicine. However, ECM protein extraction that requires organ-specific optimization is still a major limiting factor in matrisome studies. In particular, the matrisome of mouse kidneys is still understudied, despite mouse models being crucial for renal research. Here, we comprehensively characterized the matrisome of kidneys in healthy C57BL/6 mice using two ECM extraction methods in combination with liquid chromatography tandem mass spectrometry (LC-MS/MS), protein identification, and label-free quantification (LFQ) using MaxQuant. We identified 113 matrisome proteins, including 22 proteins that have not been previously listed in the Matrisome Database. Depending on the extraction approach, the core matrisome (structural proteins) comprised 45% or 73% of kidney ECM proteins, and was dominated by glycoproteins, followed by collagens and proteoglycans. Among matrisome-associated proteins, ECM regulators had the highest LFQ intensities, followed by ECM-affiliated proteins and secreted factors. The identified kidney ECM proteins were primarily involved in cellular, developmental and metabolic processes, as well as in molecular binding and regulation of catalytic and structural molecules' activity. We also performed in silico comparative analysis of the kidney matrisome composition in humans and mice based on publicly available data. These results contribute to the first reference database for the mouse renal matrisome.

Decreased cerebrospinal fluid kynurenic acid in epileptic spasms: A biomarker of response to corticosteroids.

BACKGROUND: Epileptic (previously infantile) spasms is the most common epileptic encephalopathy occurring during infancy and is frequently associated with abnormal neurodevelopmental outcomes. Epileptic spasms have a diverse range of known (genetic, structural) and unknown aetiologies. High dose corticosteroid treatment for 4 weeks often induces remission of spasms, although the mechanism of action of corticosteroid is unclear. Animal models of epileptic spasms have shown decreased brain kynurenic acid, which is increased after treatment with the ketogenic diet. We quantified kynurenine pathway metabolites in the cerebrospinal fluid (CSF) of infants with epileptic spasms and explored clinical correlations. METHODS: A panel of nine metabolites in the kynurenine pathway (tryptophan, kynurenine, kynurenic acid, 3-hydroxykynurenine, xanthurenic acid, anthranilic acid, 3-hydroxyanthranilic acid, quinolinic acid, and picolinic acid) were measured using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). CSF collected from paediatric patients less than 3 years of age with epileptic spasms (n=34, 19 males, mean age 0.85, median 0.6, range 0.3-3 yrs) were compared with other epilepsy syndromes (n=26, 9 males, mean age 1.44, median 1.45, range 0.3-3 yrs), other non-inflammatory neurological diseases (OND) (n=29, 18 males, mean age 1.47, median 1.6, range 0.1-2.9 yrs) and inflammatory neurological controls (n=12, 4 males, mean age 1.80, median 1.80, range 0.8-2.5 yrs). FINDINGS: There was a statistically significant decrease of CSF kynurenic acid in patients with epileptic spasms compared to OND (p<0.0001). In addition, the kynurenic acid/kynurenine (KYNA/KYN) ratio was lower in the epileptic spasms subgroup compared to OND (p<0.0001). Epileptic spasms patients who were steroid responders or partial steroid responders had lower KYNA/KYN ratio compared to patients who were refractory to steroids (p<0.005, p<0.05 respectively). INTERPRETATION: This study demonstrates decreased CSF kynurenic acid and KYNA/KYN in epileptic spasms, which may also represent a biomarker for steroid responsiveness. Given the anti-inflammatory and neuroprotective properties of kynurenic acid, further therapeutics able to increase kynurenic acid should be explored. FUNDING: Financial support for the study was granted by Dale NHMRC Investigator grant APP1193648, Petre Foundation, Cerebral Palsy Alliance and Department of Biochemistry at the Children's Hospital at Westmead. Prof Guillemin is funded by NHMRC Investigator grant APP1176660 and Macquarie University.

Engineered Microenvironments for 3D Cell Culture and Regenerative Medicine: Challenges, Advances, and Trends.

The overall goal of regenerative medicine is to restore the functional performance of the tissues and organs that have been severely damaged or lost due to traumas and diseases [...].

Implantable Drug Delivery Systems and Foreign Body Reaction: Traversing the Current Clinical Landscape.

Precise delivery of therapeutics to the target structures is essential for treatment efficiency and safety. Drug administration via conventional routes requires overcoming multiple transport barriers to achieve and maintain the local drug concentration and commonly results in unwanted off-target effects. Patients' compliance with the treatment schedule remains another challenge. Implantable drug delivery systems (IDDSs) provide a way to solve these problems. IDDSs are bioengineering devices surgically placed inside the patient's tissues to avoid first-pass metabolism and reduce the systemic toxicity of the drug by eluting the therapeutic payload in the vicinity of the target tissues. IDDSs present an impressive example of successful translation of the research and engineering findings to the patient's bedside. It is envisaged that the IDDS technologies will grow exponentially in the coming years. However, to pave the way for this progress, it is essential to learn lessons from the past and present of IDDSs clinical applications. The efficiency and safety of the drug-eluting implants depend on the interactions between the device and the hosting tissues. In this review, we address this need and analyze the clinical landscape of the FDA-approved IDDSs applications in the context of the foreign body reaction, a key aspect of implant-tissue integration.

Chick Embryo Experimental Platform for Micrometastases Research in a 3D Tissue Engineering Model: Cancer Biology, Drug Development, and Nanotechnology Applications.

Colonization of distant organs by tumor cells is a critical step of cancer progression. The initial avascular stage of this process (micrometastasis) remains almost inaccessible to study due to the lack of relevant experimental approaches. Herein, we introduce an in vitro/in vivo model of organ-specific micrometastases of triple-negative breast cancer (TNBC) that is fully implemented in a cost-efficient chick embryo (CE) experimental platform. The model was built as three-dimensional (3D) tissue engineering constructs (TECs) combining human MDA-MB-231 cells and decellularized CE organ-specific scaffolds. TNBC cells colonized CE organ-specific scaffolds in 2-3 weeks, forming tissue-like structures. The feasibility of this methodology for basic cancer research, drug development, and nanomedicine was demonstrated on a model of hepatic micrometastasis of TNBC. We revealed that MDA-MB-231 differentially colonize parenchymal and stromal compartments of the liver-specific extracellular matrix (LS-ECM) and become more resistant to the treatment with molecular doxorubicin (Dox) and Dox-loaded mesoporous silica nanoparticles than in monolayer cultures. When grafted on CE chorioallantoic membrane, LS-ECM-based TECs induced angiogenic switch. These findings may have important implications for the diagnosis and treatment of TNBC. The methodology established here is scalable and adaptable for pharmacological testing and cancer biology research of various metastatic and primary tumors.

Local Delivery of Pirfenidone by PLA Implants Modifies Foreign Body Reaction and Prevents Fibrosis.

Peri-implant fibrosis (PIF) increases the postsurgical risks after implantation and limits the efficacy of the implantable drug delivery systems (IDDS). Pirfenidone (PF) is an oral anti-fibrotic drug with a short (<3 h) circulation half-life and strong adverse side effects. In the current study, disk-shaped IDDS prototype combining polylactic acid (PLA) and PF, PLA@PF, with prolonged (~3 days) PF release (in vitro) was prepared. The effects of the PLA@PF implants on PIF were examined in the rabbit ear skin pocket model on postoperative days (POD) 30 and 60. Matching blank PLA implants (PLA0) and PLA0 with an equivalent single-dose PF injection performed on POD0 (PLA0+injPF) served as control. On POD30, the intergroup differences were observed in α-SMA, iNOS and arginase-1 expressions in PLA@PF and PLA0+injPF groups vs. PLA0. On POD60, PIF was significantly reduced in PLA@PF group. The peri-implant tissue thickness decreased (532 ± 98 µm vs. >1100 µm in control groups) approaching the intact derma thickness value (302 ± 15 µm). In PLA@PF group, the implant biodegradation developed faster, while arginase-1 expression was suppressed in comparison with other groups. This study proves the feasibility of the local control of fibrotic response on implants via modulation of foreign body reaction with slowly biodegradable PF-loaded IDDS.

Machine learning reveals mesenchymal breast carcinoma cell adaptation in response to matrix stiffness.

Epithelial-mesenchymal transition (EMT) and its reverse process, mesenchymal-epithelial transition (MET), are believed to play key roles in facilitating the metastatic cascade. Metastatic lesions often exhibit a similar epithelial-like state to that of the primary tumour, in particular, by forming carcinoma cell clusters via E-cadherin-mediated junctional complexes. However, the factors enabling mesenchymal-like micrometastatic cells to resume growth and reacquire an epithelial phenotype in the target organ microenvironment remain elusive. In this study, we developed a workflow using image-based cell profiling and machine learning to examine morphological, contextual and molecular states of individual breast carcinoma cells (MDA-MB-231). MDA-MB-231 heterogeneous response to the host organ microenvironment was modelled by substrates with controllable stiffness varying from 0.2kPa (soft tissues) to 64kPa (bone tissues). We identified 3 distinct morphological cell types (morphs) varying from compact round-shaped to flattened irregular-shaped cells with lamellipodia, predominantly populating 2-kPa and >16kPa substrates, respectively. These observations were accompanied by significant changes in E-cadherin and vimentin expression. Furthermore, we demonstrate that the bone-mimicking substrate (64kPa) induced multicellular cluster formation accompanied by E-cadherin cell surface localisation. MDA-MB-231 cells responded to different substrate stiffness by morphological adaptation, changes in proliferation rate and cytoskeleton markers, and cluster formation on bone-mimicking substrate. Our results suggest that the stiffest microenvironment can induce MET.

Recent advances in liposome formulations for breast cancer therapeutics.

Among many nanoparticle-based delivery platforms, liposomes have been particularly successful with many formulations passed into clinical applications. They are well-established and effective gene and/or drug delivery systems, widely used in cancer therapy including breast cancer. In this review we discuss liposome design with the targeting feature and triggering functions. We also summarise the recent progress (since 2014) in liposome-based therapeutics for breast cancer including chemotherapy and gene therapy. We finally identify some challenges on the liposome technology development for the future clinical translation.

Oxygen-Carrying Polymer Nanoconstructs for Radiodynamic Therapy of Deep Hypoxic Malignant Tumors.

Radiodynamic therapy (RDT) is an emerging non-invasive anti-cancer treatment based on the generation of the reactive oxygen species (ROS) at the lesion site following the interaction between X-rays and a photosensitizer drug (PS). The broader application of RDT is impeded by the tumor-associated hypoxia that results in low availability of oxygen for the generation of sufficient amounts of ROS. Herein, a novel nanoparticle drug formulation for RDT, which addresses the problem of low oxygen availability, is reported. It consists of poly (lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) co-loaded with a PS drug verteporfin (VP), and the clinically approved oxygen-carrying molecule, perfluorooctylbromide (PFOB). When triggered by X-rays (4 Gy), under both normoxic and hypoxic conditions, PLGA-VP-PFOB nanoconstructs (NCs) induced a significant increase of the ROS production compared with matching PLGA-VP nanoparticles. The RDT with NCs effectively killed ~60% of human pancreatic cancer cells in monolayer cultures, and almost completely suppressed the outgrowth of tumor cells in 2-weeks clonogenic assay. In a 3D engineered model of pancreatic cancer metastasis to the liver, RDT with NCs destroyed ~35% of tumor cells, demonstrating an exceptional efficiency at a tissue level. These results show that PLGA-VP-PFOB is a promising agent for RDT of deep-seated hypoxic tumors.

Modeling of Old Scars: Histopathological, Biochemical and Thermal Analysis of the Scar Tissue Maturation.

Mature hypertrophic scars (HSs) remain a challenging clinical problem, particularly due to the absence of biologically relevant experimental models as a standard rabbit ear HS model only reflects an early stage of scarring. The current study aims to adapt this animal model for simulation of mature HS by validating the time of the scar stabilization using qualitative and quantitative criteria. The full-thickness skin and perichondrium excision wounds were created on the ventral side of the rabbit ears. The tissue samples were studied on post-operation days (PODs) 30, 60, 90 and 120. The histopathological examination and morphometry were applied in parallel with biochemical analysis of protein and glycosaminoglycans (GAGs) content and amino acid composition. The supramolecular organization of collagen was explored by differential scanning calorimetry. Four stages of the rabbit ear HS maturation were delineated and attributed with the histolomorphometrical and physicochemical parameters of the tissue. The experimental scars formed in 30 days but stabilized structurally and biochemically only on POD 90-120. This evidence-based model can be used for the studies and testing of new treatments of the mature HSs.

Amianthoid transformation of costal cartilage matrix in children with pectus excavatum and pectus carinatum.

BACKGROUND: It is unclear if amianthoid transformation (AT) of costal cartilage extracellular matrix (ECM) has an impact on the development of pectus excavatum (PE) and pectus carinatum (PC). METHODS: AT foci were examined in intrasurgical biopsy specimens of costal cartilages of children (8-17 years old) with PE (n = 12) and PC (n = 12) and in age-matching autopsy control samples (n = 10) using histological and immunohistochemical staining, atomic force and nonlinear optical microscopy, transmission and scanning electron microscopy, morphometry and statistics. RESULTS: AT areas were identified in the costal cartilage ECM in children with normal chest, PE and PC. Each type of the AT areas ("canonical", "intertwined", "fine-fibred" and "intralacunary") had a unique morphological pattern of thickness and alignment of amianthoid fibers (AFs). AFs were formed via lateral aggregation of collagen type II fibrils in the intact ECM. Foci of the AT were observed significantly more frequently in the PE and PC groups. The AT areas had unique quantitative features in each study group. CONCLUSION: AT is a structurally diverse form of ECM alteration present in healthy and pathological costal cartilage. PE and PC are associated with specific AT disorders.

Mechanisms for Tuning Engineered Nanomaterials to Enhance Radiation Therapy of Cancer.

Engineered nanomaterials that produce reactive oxygen species on exposure to X- and gamma-rays used in radiation therapy offer promise of novel cancer treatment strategies. Similar to photodynamic therapy but suitable for large and deep tumors, this new approach where nanomaterials acting as sensitizing agents are combined with clinical radiation can be effective at well-tolerated low radiation doses. Suitably engineered nanomaterials can enhance cancer radiotherapy by increasing the tumor selectivity and decreasing side effects. Additionally, the nanomaterial platform offers therapeutically valuable functionalities, including molecular targeting, drug/gene delivery, and adaptive responses to trigger drug release. The potential of such nanomaterials to be combined with radiotherapy is widely recognized. In order for further breakthroughs to be made, and to facilitate clinical translation, the applicable principles and fundamentals should be articulated. This review focuses on mechanisms underpinning rational nanomaterial design to enhance radiation therapy, the understanding of which will enable novel ways to optimize its therapeutic efficacy. A roadmap for designing nanomaterials with optimized anticancer performance is also shown and the potential clinical significance and future translation are discussed.

Application of Mitochondrially Targeted Nanoconstructs to Neoadjuvant X-ray-Induced Photodynamic Therapy for Rectal Cancer.

In this work, we brought together two existing clinical techniques used in cancer treatment-X-ray radiation and photodynamic therapy (PDT), whose combination termed X-PDT uniquely allows PDT to be therapeutically effective in deep tissue. To this end, we developed mitochondrially targeted biodegradable polymer poly(lactic-co-glycolic acid) nanocarriers incorporating a photosensitizer verteporfin, ultrasmall (2-5 nm) gold nanoparticles as radiation enhancers, and triphenylphosphonium acting as the mitochondrial targeting moiety. The average size of the nanocarriers was about 160 nm. Upon X-ray radiation our nanocarriers generated cytotoxic amounts of singlet oxygen within the mitochondria, triggering the loss of membrane potential and mitochondria-related apoptosis of cancer cells. Our X-PDT strategy effectively controlled tumor growth with only a fraction of radiotherapy dose (4 Gy) and improved the survival rate of a mouse model bearing colorectal cancer cells. In vivo data indicate that our X-PDT treatment is cytoreductive, antiproliferative, and profibrotic. The nanocarriers induce radiosensitization effectively, which makes it possible to amplify the effects of radiation. A radiation dose of 4 Gy combined with our nanocarriers allows equivalent control of tumor growth as 12 Gy of radiation, but with greatly reduced radiation side effects (significant weight loss and resultant death).

Autofluorescence excitation-emission matrices as a quantitative tool for the assessment of meat quality.

Commercially produced meat is currently graded by a complex and partly subjective multiparameter methodology; a quantitative method of grading, using small samples would be desirable. Here, we investigate the correlation between commercial grades of beef and spectral signatures of native fluorophores in such small samples. Beef samples of different commercial grades were characterized by fluorescence spectroscopy complemented by biochemical and histological assessment. The excitation-emission matrices of the specimens reveal five prominent native autofluorescence signatures in the excitation range from 250 to 350 nm, derived mainly from tryptophan and intramuscular fat. We found that these signatures reflect meat grade and can be used for its determination.

Tracing upconversion nanoparticle penetration in human skin.

Due to their unique optical properties upconversion nanoparticles (UCNPs) provide exceptionally high contrast for imaging of true nanoparticle distribution in excised human skin. It makes possible to show penetration of solid nanoparticles in skin treated with chemical enhancers. We demonstrated tracing upconversion nanoparticles in excised human skin by means of optical microscopy at the discrete particle level sensitivity to obtain their penetration profiles, which was validated by laser-ablation inductively-coupled-plasma mass-spectrometry. To demonstrate utilities of our method, UCNPs were coated with polymers, formulated in water and chemical enhancers, and applied on excised human skin mounted on Franz cells, followed by imaging using a custom-built laser-scanning microscope. To evaluate the toxicity impact on skin by polymer-coated UCNPs, we introduced a tissue engineering model of viable epidermis made of decellularized chick embryo skin seeded with keratinocytes. UCNPs formulated in water stopped in stratum corneum, whereas UCNPs formulated in ethanol-water solution crossed stratum corneum and reached viable epidermis - hence, the enhancement effect for solid nanoparticles was detected by optical microscopy. All polymer-coated UCNPs were found nontoxic within the accepted safety levels. The keratinocyte resilience to polyethyleneimine-coated UCNPs was surprising considering cytotoxicity of polyethyleneimine to two-dimensional cell cultures.

Non-Invasive Monitoring of Functional State of Articular Cartilage Tissue with Label-Free Unsupervised Hyperspectral Imaging.

Damage and degradation of articular cartilage leads to severe pain and loss of mobility. The development of new therapies for cartilage regeneration for monitoring their effect requires further study of cartilage, ideally at a molecular level and in a minimally invasive way. Hyperspectral microscopy is a novel technology which utilises endogenous fluorophores to non-invasively assess the molecular composition of cells and tissue. In this study, we applied hyperspectral microscopy to healthy bovine articular cartilage and osteoarthritic human articular cartilage to investigate its capacity to generate informative molecular data and characterise disease state and treatment effects. We successfully demonstrated label-free fluorescence identification of collagen type I and II - isolated in cartilage here for the first time and the co-enzymes free NADH and FAD which together give the optical redox ratio that is an important measure of metabolic activity. The intracellular composition of chondrocytes was also examined. Differences were observed in the molecular ratios within the superficial and transitional zones of the articular cartilage which appeared to be influenced by disease state and treatment. These findings show that hyperspectral microscopy could be useful for investigating the molecular underpinnings of articular cartilage degradation and repair. As it is non-invasive and non-destructive, samples can be repeatedly assessed over time, enabling true time-course experiments with in-depth molecular data. Additionally, there is potential for the hyperspectral approach to be adapted for patient examination to allow the investigation of cartilage state. This could be of advantage for assessment and diagnosis as well as treatment monitoring.

Controlled gene and drug release from a liposomal delivery platform triggered by X-ray radiation.

Liposomes have been well established as an effective drug delivery system, due to simplicity of their preparation and unique characteristics. However conventional liposomes are unsuitable for the on-demand content release, which limits their therapeutic utility. Here we report X-ray-triggerable liposomes incorporating gold nanoparticles and photosensitizer verteporfin. The 6 MeV X-ray radiation induces verteporfin to produce singlet oxygen, which destabilises the liposomal membrane and causes the release of cargos from the liposomal cavity. This triggering strategy is demonstrated by the efficiency of gene silencing in vitro and increased effectiveness of chemotherapy in vivo. Our work indicates the feasibility of a combinatorial treatment and possible synergistic effects in the course of standard radiotherapy combined with chemotherapy delivered via X-ray-triggered liposomes. Importantly, our X-ray-mediated liposome release strategy offers prospects for deep tissue photodynamic therapy, by removing its depth limitation.