Liuen Liang's research while affiliated with Macquarie University and other places

Applications of nanoparticles (NPs) in the life sciences require control over their properties in protein-rich biological fluids, as an NP quickly acquires a layer of proteins on the surface, forming the so-called “protein corona” (PC). Understanding the composition and kinetics of the PC at the molecular level is of considerable importance for controlling NP interaction with cells. Here, we present a systematic study of hard PC formation on the surface of upconversion nanoparticles (UCNPs) coated with positively-charged polyethyleneimine (PEI) and negatively-charged poly (acrylic acid) (PAA) polymers in serum-supplemented cell culture medium. The rationale behind the choice of UCNP is two-fold: UCNP represents a convenient model of NP with a size ranging from 5 nm to >200 nm, while the unique photoluminescent properties of UCNP enable direct observation of the PC formation, which may provide new insight into this complex process. The non-linear optical properties of UCNP were utilised for direct observation of PC formation by means of fluorescence correlation spectroscopy. Our findings indicated that the charge of the surface polymer coating was the key factor for the formation of PC on UCNPs, with an ensuing effect on the NP–cell interactions.

Zinc oxide nanoparticle (ZnO NP)-based sunscreens are generally considered safe because the ZnO NPs do not penetrate through the outermost layer of the skin, the stratum corneum (SC). However, cytotoxicity of zinc ions in the viable epidermis (VE) after dissolution from ZnO NP and penetration into the VE is ill-defined. We therefore quantified the relative concentrations of endogenous and exogenous Zn using a rare stable zinc-67 isotope (67Zn) ZnO NP sunscreen applied to excised human skin and the cytotoxicity of human keratinocytes (HaCaT) using multiphoton microscopy, zinc-selective fluorescent sensing, and a laser-ablation inductively coupled plasma–mass spectrometry (LA-ICP-MS) methodology. Multiphoton microscopy with second harmonic generation imaging showed that 67ZnO NPs were retained on the surface or within the superficial layers of the SC. Zn fluorescence sensing revealed higher levels of labile and intracellular zinc in both the SC and VE relative to untreated skin, confirming that dissolved zinc species permeated across the SC into the VE as ionic Zn and significantly not as ZnO NPs. Importantly, the LA-ICP-MS estimated exogenous 67Zn concentrations in the VE of 1.0 ± 0.3 μg/mL are much lower than that estimated for endogenous VE zinc of 4.3 ± 0.7 μg/mL. Furthermore, their combined total zinc concentrations in the VE are much lower than the exogenous zinc concentration of 21 to 31 μg/mL causing VE cytotoxicity, as defined by the half-maximal inhibitory concentration of exogenous 67Zn found in human keratinocytes (HaCaT). This speaks strongly for the safety of ZnO NP sunscreens applied to intact human skin and the associated recent US FDA guidance.

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. Here, 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 is built as three-dimensional (3D) tissue engineering constructs (TECs) combining human MDA-MB-231 cells and decellular-ized 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 com-partments 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 diag-nosis 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.

In the natural fluidic environment of a biological system, nanoparticles swiftly adsorb plasma proteins on their surface forming a “protein corona”, which profoundly and often adversely affects their residence in the systemic circulation in vivo and their interaction with cells in vitro. It has been recognized that preformation of a protein corona under controlled conditions ameliorates the protein corona effects, including colloidal stability in serum solutions. We report on the investigation of the stabilizing effects of a denatured bovine serum albumin (dBSA) protein corona formed on the surface of upconversion nanoparticles (UCNPs). UCNPs were chosen as a nanoparticle model due to their unique photoluminescent properties suitable for background-free biological imaging and sensing. UCNP surface was modified with nitrosonium tetrafluoroborate (NOBF4) to render it hydrophilic. UCNP-NOBF4 nanoparticles were incubated in dBSA solution to form a dBSA corona followed up by lyophilization. As produced dBSA-UCNP-NOBF4 demonstrated high photoluminescence brightness, sustained colloidal stability after long-term storage and the reduced level of serum protein surface adsorption. These results show promise of dBSA-based nanoparticle pretreatment to improve the amiability to biological environments towards theranostic applications.

Introduction and Objectives: Despite multiple resections and long-term chemo and immunotherapy, most non-muscle invasive bladder cancer patients suffer from recurrence or progression leading to cystectomy and a less favorable outcome. Possible reasons for that are incomplete resection and reimplantation of cancer cells, which could be prevented by improved resection and adjuvant therapy. Our objective was to develop a targeted drug for detection, fluorescence-guided resection, and deep-penetrating adjuvant photodynamic therapy of urothelial carcinoma (UC). The agent was based on upconversion nanoparticles (UCNP), which can carry a photosensitizer and can transform deep-penetrating near-infrared light into high-energy visible light, demanded for tumor visualization and for production of reactive oxygen species in the photosensitizer. At this stage, we aimed to select an antibody that could be attached to UCNPs to deliver them to UC cells. Methods: We produced silica-coated UCNP of the composition NaYF4:Yb,Er amenable for conjugation with biomolecules. An anti-Glypican-1 (GPC-1) monoclonal antibody MIL-38 (Glytherix Ltd., Sydney, Australia), was chosen for targeted delivery of the nanoparticles as it had previously demonstrated affinity towards bladder cancer. UCNPs were conjugated with MIL-38 by using a fusion protein Linker-Protein G (LPG). Finally, to investigate targeted binding and molecular specificity of these nanoconjugates, we incubated them with GPC-1 positive and GPC-1 negative cells. The role of MIL-38 in targeted delivery of nanoconjugates was also validated by incubation of GPC-1 positive T24 cells with nanoparticles coupled to an isotype control antibody and without an antibody. Results: Targeted upconversion nanoconjugates UCNP-LPG-MIL-38 labeled almost 90% of T24 cells with high expression of GPC-1 and only 23.2% of C3 cells with low expression of this antigen, demonstrating high molecular selectivity and specificity. Incubation of T24 cells with nanoconjugates linked with a control antibody and without antibody resulted in labeling of 19.8% and 26.2%, respectively, demonstrating the role of MIL-38 in targeted delivery of these nanoconjugates. As a result of the labeling, mean photoluminescence of cells in targeted group was from five to eight times stronger than in control groups, allowing for easy identification of positive cells with low background autofluorescence. Conclusions: These results highlight the potential of these nanoconjugates for the diagnosis and therapy of UC, as they can bind to Glypican-1-positive BC cells and cause their bright photoluminescence, which could be used for detection of tumors and activation of photosensitizers. It was also confirmed that monoclonal antibody MIL-38 has high potential to be applied in experimental diagnosis, drug delivery, and targeted therapy of UC, as it mediated targeted binding of upconversion photoluminescent nanoconjugates to Glypican-1-positive UC cells. Citation Format: Liuen Liang, Andrew Care, Anwar Sunna, Douglas Campbell, Bradley Walsh, Andrei Zvyagin, David Gillatt, Dmitry Polikarpov. Photoluminescent nanoconjugates for molecular imaging of bladder cancer [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2019 May 18-21; Denver, CO. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(15_Suppl):Abstract nr B27.

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).

Early stages of colonization of distant organs by metastatic cancer cells (micrometastasis) remain almost inaccessible to study due to lack of relevant experimental approaches. Here, we show the first 3D tissue engineered model of hepatic micrometastasis of triple negative breast cancer (TNBC). It reproduces characteristic histopathological features of the disease and reveals that metastatic TNBC cells colonize liver parenchymal and stromal extracellular matrix with different speed and by different strategies. These engineered tumors induce the angiogenic switch when grafted in vivo, confirming their metastatic-specific behaviour. Furthermore, we proved feasibility and biological relevance of our model for drug and nanoparticle testing and found a down-regulatory effect of the liver microenvironment of the sensitivity of TNBC cells to chemotherapeutic drug doxorubicin in free and nanoformulated forms. The convenient and affordable methodology established here can be translated to other types of metastatic tumors for basic cancer biology research and adapted for high-throughput assays.

Acellular organ-specific scaffolds (AOSS) obtained by various methods of decellularization (DCL) are successfully applied in regenerative medicine and biomedical research. They can be used in reconstructive surgery as materials for wound healing and tissue replacement, or as implantable systems for delivery of cells, drugs, growth factors, nanoparticles and other biologically active compounds. High quality of DCL allows to reduce foreign body reactions after implantation and creates conditions for the activation of endogenous regeneration. AOSS-based 3D engineered tissues are also excellent for biologically accurate and reliable testing of new treatment and diagnostic approaches. However, wide application of AOSS-based tissue engineering faces several challenges, including the efficiency and scalability of production of high quality AOSS materials and long processing time. Application of the materials derived from the evolutionary distant animal species for reconstruction of human tissues in vitro and in vivo also induces concerns. We accumulated significant experience in scalable and cost-efficient production and applications of AOSS and AOSS-derived biomaterials from farm industry by-products. In particular, our work involves DCL of the internal organs, muscle, bone, articular cartilage and skin obtained from farm industry (pig, sheep, cow, horse, rabbit, chicken and fish) as food-quality materials or biological waste. We applied these products for preparation of AOSS, organ-specific ECM solutions and powders, and various ECM-polymer composites. Here, we report on our methodological findings, characteristics of the AOSS and AOSS-derived biomaterials, their applications for basic biological and medical research, pre-clinical testing of drugs and nanoparticles, and for regenerative medicine. We will present our vision of the past, present and future of AOSS-based applied in vitro tissue modelling and clinical use of AOSS in the context of CNBP research goals. We also will discuss the grant funding and commercialization potential of our results in relation to the future work in the field of animal welfare.