Improving the therapeutic ratio of radiotherapy against radioresistant cancers: Leveraging on novel artificial intelligence-based approaches for drug combination discovery.

Despite numerous advances in cancer radiotherapy, tumor radioresistance remain one of the major challenges limiting treatment efficacy of radiotherapy. Conventional strategies to overcome radioresistance involve understanding the underpinning molecular mechanisms, and subsequently using combinatorial treatment strategies involving radiation and targeted drug combinations against these radioresistant tumors. These strategies exploit and target the molecular fingerprint and vulnerability of the radioresistant clones to achieve improved efficacy in tumor eradication. However, conventional drug-screening approaches for the discovery of new drug combinations have been proven to be inefficient, limited and laborious. With the increasing availability of computational resources in recent years, novel approaches such as Quadratic Phenotypic Optimization Platform (QPOP), CURATE.AI and Drug Combination and Prediction and Testing (DCPT) platform have emerged to aid in drug combination discovery and the longitudinally optimized modulation of combination therapy dosing. These platforms could overcome the limitations of conventional screening approaches, thereby facilitating the discovery of more optimal drug combinations to improve the therapeutic ratio of combinatorial treatment. The use of better and more accurate models and methods with rapid turnover can thus facilitate a rapid translation in the clinic, hence, resulting in a better patient outcome. Here, we reviewed the clinical observations, molecular mechanisms and proposed treatment strategies for tumor radioresistance and discussed how novel approaches may be applied to enhance drug combination discovery, with the aim to further improve the therapeutic ratio and treatment efficacy of radiotherapy against radioresistant cancers.

Stealthiness and Hematocompatibility of Gold Nanoparticles with Pre-Formed Protein Corona.

Studies have established that a serum protein corona pre-formed around gold nanorods (NRs) could be exploited for loading photosensitizers and chemotherapeutics to result in efficient cell kill in vitro with an extremely low dose. In this study, we further demonstrated that pre-forming a serum protein corona (PC) around citrate-capped NRs (NR-Cit) to form NR-PC conferred them stealth property and high hematocompatibility similar to the common strategy of PEGylating NRs, which would otherwise not be able to evade the immune system. Specifically, the NR-PC caused minimal complement activation with significantly lower formation of the terminal complement complex SC5b-9 measured in human serum containing NR-PC, and this resulted in low uptake by phagocytic U937 monocytes of 5.9% of the initial gold dose compared to 55.8% of NR-Cit. In addition, NR-PC exhibited very low hemolytic activity of less than 0.2% hemolysis with no observable effect on RBC morphology as opposed to 0.6% for NR-Cit at the same concentration of 1 nM NRs. Furthermore, we showed that the high hematocompatibility and stealth property of NR-PC were maintained even after the loading of small molecules, photosensitizer Chlorine e6 (Ce6), into the protein corona, thus further establishing the potential clinical relevance of exploiting the inevitably formed serum protein corona on nanoparticles as an effective delivery vector for small molecular therapeutics.

Dosimetric uncertainties impact on cell survival curve with low energy proton.

There is an increasing number of radiobiological experiments being conducted with low energy protons (less than 5 MeV) for radiobiological studies due to availability of sub-millimetre focused beam. However, low energy proton has broad microdosimetric spectra which can introduce dosimetric uncertainty. In this work, we quantify the impact of this dosimetric uncertainties on the cell survival curve and how it affects the estimation of the alpha and beta parameters in the LQ formalism. Monte Carlo simulation is used to generate the microdosimetric spectra in a micrometer-sized water sphere under proton irradiation. This is modelled using radiobiological experiment set-up at the Centre of Ion Beam Application (CIBA) in National University of Singapore. Our results show that the microdosimetric spectra can introduce both systematic and random shifts in dose and cell survival; this effect is most pronounced with low energy protons. The alpha and beta uncertainties can be up to 10% and above 30%, respectively for low energy protons passing through thin cell target (about 10 microns). These uncertainties are non-negligible and show that care must be taken in using the cell survival curve and its derived parameters for radiobiological models.

Mannitol-induced gold nanoparticle aggregation for the ligand-free detection of viral particles.

Traditional virus detection methods require ligands that bind to either viral capsid proteins or viral nucleic acids. Ligands are typically antibodies or oligonucleotides and they are expensive, have limited chemical stability, and can only detect one specific type of virus at a time. Here, the biochemical surface properties of viruses are exploited for ligand-free, nonspecific virus detection. It has been found that the osmolyte mannitol can preferentially aggregate virus, while leaving proteins in solution. This led to the development of a ligand-free detection of virus using gold nanoparticle (AuNP) aggregation. Porcine parvovirus (PPV) was incubated with AuNPs and aggregation of the PPV-AuNP complex with mannitol was detected by dynamic light scattering (DLS). The lowest detectable concentration of PPV was estimated to be 106 MTT50 per mL, which is lower than standard antibody assays. PPV was also detected when swabbed from a dry surface and in the presence of a protein solution matrix. The enveloped bovine viral diarrhea virus (BVDV) was also detected using mannitol-induced aggregation of BVDV-coated AuNPs. The lowest detectable concentration of BVDV was estimated to be 104 MTT50 per mL. This demonstrates that gold nanoparticle aggregation can detect virus without the use of specific ligands.

Intra-patient and inter-patient comparisons of DNA damage response biomarkers in Nasopharynx Cancer (NPC): analysis of NCC0901 randomised controlled trial of induction chemotherapy in locally advanced NPC.

BACKGROUND: Inter-patient heterogeneity in radiation-induced DNA damage responses is proposed to reflect intrinsic variations in tumour and normal tissue radiation sensitivity, but the prediction of phenotype by a molecular biomarker is influenced by clinical confounders and assay reproducibility. Here, we characterised the intrapatient and inter-patient heterogeneity in biomarkers of DNA damage and repair and radiation-induced apoptosis. METHODS: We enrolled 85 of 172 patients with locally advanced nasopharynx cancer from a randomised controlled phase II/III trial of induction chemotherapy added to chemo-radiotherapy. G0 blood lymphocytes were harvested from these patients, and irradiated with 1, 4, and 8 Gy ex vivo. DNA damage induction (1 Gy 0.5 h) and repair (4 Gy 24 h) were assessed by duplicate γH2AX foci assays in 50-100 cells. Duplicate FLICA assays performed at 48 h post-8 Gy were employed as surrogate of radiation-induced apoptosis; %FLICA-positive cells were quantified by flow cytometry. RESULTS: We observed limited intrapatient variation in γH2AX foci and %FLICA readouts; median difference of duplicate foci scores was - 0.37 (IQR = - 1.256-0.800) for 1 Gy 0.5 h and 0.09 (IQR = - 0.685-0.792) for 4 Gy 24 h; ICC of ≥0.80 was observed for duplicate %FLICA0Gy and %FLICA8Gy assays of CD4+ and CD8+ T lymphocytes. As expected, we observed wide inter-patient heterogeneity in both assays that was independent of intrapatient variation and clinical covariates, with the exception of age, which was inversely correlated with %FLICAbackground-corrected (Spearman R = - 0.406, P < 0.001 [CD4+]; R = - 0.220, P = 0.04 [CD8+]). Lastly, an exploratory case-control analysis indicates increased levels of γH2AX foci at 4 Gy 24 h in patients with severe late radiotherapy-induced xerostomia (P = 0.05). CONCLUSION: Here, we confirmed the technical reproducibility of DNA damage response assays for clinical implementation as biomarkers of clinical radiosensitivity in nasopharynx cancer patients.

Polydopamine Nanoparticles Enhance Drug Release for Combined Photodynamic and Photothermal Therapy.

Our study shows a facile two-step method which does not require the use of core templates to load a hydrophobic photosensitizer drug chlorin e6 (Ce6) within polydopamine (PDA) nanoparticles (NPs) while maintaining the intrinsic surface properties of PDA NPs. This structure is significantly different from hollow nanocapsules which are less stiff as they do not possess a core. To our knowledge, there exist no similar studies in the literature on drug loading within the polymer matrix of PDA NPs. We characterized the drug loading and release behavior of the photosensitizer Ce6 and demonstrated the therapeutic efficacy of the combined photodynamic (PDT) and photothermal therapy (PTT) from Ce6 and PDA, respectively, under a single wavelength of 665 nm irradiation on bladder cancer cells. We obtained a saturated loading amount of 14.2 ± 0.85 µM Ce6 in 1 nM PDA NPs by incubating 1 mg/mL dopamine solution with 140 µM of Ce6 for 20 h. The PDA NPs maintained colloidal stability in biological media, whereas the pi-pi (π-π) interaction between PDA and Ce6 enabled a release profile of the photosensitizer until day 5. Interestingly, loading of Ce6 in the polymer matrix of PDA NPs significantly enhanced the cell uptake because of endocytosis. An increased cell kill was observed with the combined PDT + PTT from 1 nM PDA-Ce6 compared to that with PTT alone with 1 nM PDA and PDT alone with 15 µM equivalent concentration of free Ce6. PDA-Ce6 NPs could be a promising PDT/PTT therapeutic agent for cancer therapy.

Protein corona in drug delivery for multimodal cancer therapy in vivo.

The protein corona is inevitably formed on nanoparticles (NPs) when they are introduced in vivo and has been associated with a reduction in targeting yield, immune recognition and rapid blood clearance, leading to poor tumor accumulation. We have recently shown that it is possible to exploit the protein corona for drug delivery by exploiting it for loading and triggering the release of a photosensitizer Chlorin e6 (Ce6) for simultaneous photodynamic (PDT) and photothermal therapy (PTT) in vitro. Here, we extended our previous in vitro studies to evaluate its effectiveness in vivo. Specifically, we pre-formed the protein corona from mouse serum (MS) around gold nanorods (NRs) and loaded it with Ce6 to form NR-MS-Ce6. The intravenous delivery of NR-MS-Ce6 at a dose of 10 mg kg-1 Au loaded with 9.63 µg kg-1 Ce6 into tumor-bearing NCr nude mice resulted in their tumor accumulation reaching a peak concentration of 560.3 µg Au per kg tissue (0.0752% dose) within 6 h post-injection. Subsequent localized laser irradiation of the xenograft tumor resulted in a significant tumor temperature increase of 16.85 °C within 20 min. Combined with the simultaneous reactive oxygen species (ROS) production by Ce6 for PDT, complete tumor regression was achieved within 19 days with no tumor regrowth up to 31 days. Similar to other NPs, significant gold accumulation was observed in the major reticuloendothelial system (RES) organs, particularly the liver and spleen, although no acute toxicity was observed histologically 31 days post-treatment. Our results demonstrated for the first time an in vivo application of the protein corona around NPs in the loading and delivery of drugs in small animals. The ease of drug loading and the biocompatibility of the endogenous serum-based protein corona could make it useful for drug delivery and therapeutic applications instead of merely being considered as a biological artefact to be eliminated.

Exploiting the Protein Corona from Cell Lysate on DNA Functionalized Gold Nanoparticles for Enhanced mRNA Translation.

This study describes the use of DNA functionalized gold nanoparticles (AuNPs) to enhance the synthesis of proteins in cell lysate and examines the mechanisms behind the enhanced mRNA translation. With an appropriate DNA oligomer sequence that hybridizes to the 3'-untranslated region of two mRNA of interest, insulin and green fluorescent protein (GFP), we found that these DNA conjugated AuNPs (AuNP-DNA) introduced into HeLa cell lysate enhanced the synthesis of insulin and GFP by up to 2.18 and 1.80-fold, respectively, over baseline production with just the mRNA present. The insulin synthesis was markedly reduced with non-DNA citrate-capped AuNP (1.25-fold) and AuNP-DNA with a nonspecific poly(T) sequence (1.25-fold). We showed that both nonspecific adsorption of ribosomes and translation factors to form a lysate protein corona on AuNP-DNA and weak hybridization between DNA oligomers and mRNA of interest were important factors that brought translation factors, ribosomes, and mRNA into close proximity of each other. This could reduce the recycling time of ribosomes during mRNA translation, thereby increasing the efficiency of protein synthesis. The outcome of this work shows that with rational DNA design, it could be possible to modulate intracellular biological processes with AuNP-DNA and increase their production of proteins for various biomedical applications.

Exploiting the protein corona around gold nanorods for low-dose combined photothermal and photodynamic therapy.

A nanodevice comprising human serum (HS) protein corona coated gold nanorods (NRs) has been developed to perform both photothermal therapy (PTT) and photodynamic therapy (PDT) simultaneously at a very low dose under irradiation by a single laser. Here, we exploit the protein corona to load a photosensitizer, chlorin e6 (Ce6), to form NR-HS-Ce6, whose excitation wavelength matches with the longitudinal surface plasmon resonance (LSPR) of NRs. When excited by a single laser, the NRs caused photothermal ablation of cancer cells while Ce6 simultaneously produced reactive oxygen species (ROS) to kill cancer cells through oxidative stress in PDT. We found that the protein corona did not affect the photothermal heating of NRs and observed more than 5-fold increase in ROS generation when Ce6 was loaded on NR-HS compared to free HS-Ce6 dissolved in HS. The uptake of Ce6 by Cal 27 oral squamous cell carcinoma (OSCC) cells also increased 57-fold when loaded on NR-HS compared to free HS-Ce6. While both PDT and PTT have established modest success in reducing cancer cell viability on their own, we have shown that the combined therapy can achieve near complete eradication (95.2% cell kill) of cancer cells even at an extremely low dose of 50 pM of NR-HS-Ce6 containing an equivalent of 7.67 µg mL-1 Au and 4.83 nM Ce6. This near complete cell kill at such a low dose has not been reported previously. The advantages of this nanoscale delivery system showcase the application of protein corona in cancer treatment instead of considering it as an undesirable biological artefact.

Protein Corona around Gold Nanorods as a Drug Carrier for Multimodal Cancer Therapy.

A single nanodevice based on gold nanorods (NRs) coloaded with a photosensitizer, Chlorin e6 (Ce6), and a chemotherapeutic, Doxorubicin (Dox), on its endogenously formed human serum (HS) protein corona, i.e., NR-HS-Ce6-Dox was developed with the aim of performing multimodal cancer therapy: photodynamic (PDT), photothermal (PTT) and chemotherapy (CTX) simultaneously upon irradiation with a single 665 nm laser. Here, the excitation of NRs and Ce6 resulted in photothermal ablation (PTT), and production of reactive oxygen species (ROS) to kill Cal 27 oral squamous cell carcinoma (OSCC) cells by oxidative stress (PDT) respectively, while the laser-triggered release of Dox intercalated into the DNA of cancer cells to result in DNA damage and cell death (CTX). High laser-triggered Dox release efficiency of 71.5% and strong plasmonic enhancement of ROS production by Ce6 (4.8-fold increase compared to free Ce6) was observed. Uptake of both Ce6 and Dox by Cal 27 cells was greatly enhanced, with 3.3 and 52 times higher intracellular Dox and Ce6 fluorescence observed, respectively, 6 h after dosing with NR-HS-Ce6-Dox compared to free drugs. The simultaneous trimodal therapy achieved a near complete eradication of cancer cells (98.7% cell death) with an extremely low dose of 15 pM NR-HS-Ce6-Dox loaded with just 1.26 nM Ce6 and 12.5 nM Dox due to strong synergistic enhancement in cancer cell kill compared to individual therapies performed separately. No dark toxicities were observed. These drug concentrations were far lower than any previously reported in vitro, thus eliminating any potential systemic toxicity of these agents.

An instantaneous colorimetric protein assay based on spontaneous formation of a protein corona on gold nanoparticles.

Commercial protein assays used ubiquitously in laboratories typically require long incubation times due to the inherently slow protein-reagent reactions. In this study, we report a novel facile technique for the instantaneous measurement of total protein concentration by exploiting the rapid aggregation dynamics of gold nanoparticles (NPs). By adsorbing different amounts of proteins on their surface to form a protein corona, these NPs can be sterically stabilized to different degrees by aggregation, thus exhibiting a spectrum of color change which can be quantitatively characterized by UV-Vis absorption spectroscopy. We evaluated this technique on four model proteins with different structures: bovine serum albumin (BSA), normal mouse immunoglobulin G (IgG), fibrinogen (FBG) and apolipoprotein A-I (Apo-A1) using two approaches, sequential and simultaneous. We obtained an approach-dependent linear concentration range up to 80 µg mL(-1) and 400 µg mL(-1) for sequential and simultaneous approaches, respectively. This linear working range was wider than that of the commercial Bradford assay and comparable to the Micro BCA assay. The simultaneous approach was also able to produce a linear working range of 200 to 1000 µg mL(-1) (R(2) = 0.995) in human urine, while the sequential approach was non-functional in urine. Similar to Micro BCA, the NP-based protein assay was able to elicit a linear response (R(2) > 0.87) for all four proteins with different structures. However, unlike Micro BCA which requires up to 120 min of incubation, we were able to obtain the read-out almost instantaneously without the need for incubation. The NP-based technique using the simultaneous approach can thus be exploited as a novel assay for instantaneous protein quantification to increase the productivity of laboratory processes.

Dark-field circular depolarization optical coherence microscopy.

Optical coherence microscopy (OCM) is a widely used structural imaging modality. To extend its application in molecular imaging, gold nanorods are widely used as contrast agents for OCM. However, they very often offer limited sensitivity as a result of poor signal to background ratio. Here we experimentally demonstrate that a novel OCM implementation based on dark-field circular depolarization detection can efficiently detect circularly depolarized signal from gold nanorods and at the same time efficiently suppress the background signals. This results into a significant improvement in signal to background ratio.

Nanoparticle interface to biology: applications in probing and modulating biological processes.

Nanomaterials can be considered as "pseudo" subcellular entities that are similar to endogenous biomolecules because of their size and ability to interact with other biomolecules. The interaction between nanoparticles and biomolecules gives rise to the nano-bio interface between a nanoparticle and its biological environment. This is often defined in terms of the biomolecules that are present on the surface of the nanoparticles. The nano-bio interface alters the surface characteristics and is what the biological system sees and interacts with. The nanoparticle can thus be viewed as a "scaffold" to which molecules are attached. Intelligent design of this nano-bio interface is therefore crucial to the functionality of nanoscale systems in biology. In this review, we discuss the most common nano-bio interfaces formed from molecules including DNA, polymers, proteins, and antibodies, and discuss their applications in probing and modulating biological processes. We focus our discussion on the nano-bio interface formed on gold nanoparticles as our nanoparticle "scaffold" of interest in part because of our research interest as well as their unique physicochemical properties. While not exhaustive, this review provides a good overview of the latest advances in the use of gold nanomaterial interface to probe and modulate biological processes.