Volume 5 Number 11 Nanoscale November 2020 Pages 1433–1548 Horizons The home for rapid reports of exceptional significance in nanoscience and nanotechnology rsc.li/nanoscale-horizons ISSN 2055-6756 COMMUNICATION Kostas Kostarelos, Marilena Hadjidemetriou et al. The biomolecule corona of lipid nanoparticles contains circulating cell-free DNA Nanoscale Horizons This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online COMMUNICATION View Journal | View Issue The biomolecule corona of lipid nanoparticles contains circulating cell-free DNA† Open Access Article. Published on 27 August 2020. Downloaded on 2/11/2021 12:49:42 PM. Cite this: Nanoscale Horiz., 2020, 5, 1476 Lois Gardner, ab Jessica Warrington, a Jane Rogan,c Dominic G. Rothwell, b Received 2nd June 2020, Ged Brady,b Caroline Dive,b Kostas Kostarelos *ade and Accepted 11th August 2020 Marilena Hadjidemetriou *a DOI: 10.1039/d0nh00333f rsc.li/nanoscale-horizons The spontaneous adsorption of biomolecules onto the surface of nanoparticles (NPs) in complex physiological biofluids has been widely New concepts investigated over the last decade. Characterisation of the protein The aim of this study was to interrogate experimentally the fundamental composition of the ‘biomolecule corona’ has dominated research question whether circulating cell-free DNA (cfDNA) exists in the bio- molecule corona that spontaneously forms around nanoparticles upon efforts, whereas other classes of biomolecules, such as nucleic acids, incubation with complex biological fluids. Despite recent advances in the have received no interest. Scarce, speculative statements exist in the characterisation of the protein content of the biomolecule corona, to date literature about the presence of nucleic acids in the biomolecule corona, there has been no experimental evidence of the spontaneous interaction with no previous studies attempting to describe the contribution of between cfDNA and plasma-incubated nanoparticles. To test this hypo- genomic content to the blood-derived NP corona. Herein, we provide thesis and analyse such genomic content in the nanoparticle biomolecule corona, we incubated clinically-used liposomes with human plasma the first experimental evidence of the interaction of circulating cell-free samples, retrieved their coronas and subsequently quantified the total DNA (cfDNA) with lipid-based NPs upon their incubation with human corona cfDNA content using two different real-time quantitative PCR plasma samples, obtained from healthy volunteers and ovarian carci- (qPCR) assays. Our data revealed the presence of cfDNA in the liposomal noma patients. Our results also demonstrate an increased amount of biomolecule corona upon their incubation with human plasma samples obtained from healthy donors and ovarian carcinoma patients. More detectable cfDNA in patients with cancer. Proteomic analysis of the interestingly, an increased amount of corona cfDNA was detected in same biomolecule coronas revealed the presence of histone proteins, cancer patients. Finally, proteomic analysis of the same biomolecule suggesting an indirect, nucleosome-mediated NP–cfDNA interaction. coronas revealed the presence of histone proteins, suggesting an The finding of cfDNA as part of the NP corona, offers a previously indirect, nucleosome-mediated NP–cfDNA interaction. The revelation of unreported new scope regarding the chemical composition of the cfDNA as a component of the nanoparticle corona, opens up new ‘biomolecule corona’ and opens up new possibilities for the potential possibilities for the potential exploitation of the biomolecule corona for the enrichment and analysis of blood-circulating nucleic acids and the exploitation of the biomolecule corona for the enrichment and analysis genomic information they carry. of blood-circulating nucleic acids. Introduction of biomolecules onto their surface upon incubation with Over the last decade, biomedical applications of nanoparticles complex biofluids, known as the ‘protein’ or ‘biomolecule (NPs) have been challenged due to the spontaneous adsorption corona’.1,2 The bio-nanotechnology field has since invested considerable resources investigating the corona composition a Nanomedicine Lab, Faculty of Biology, Medicine and Health, in an attempt to prevent NP–protein interactions and con- The University of Manchester, AV Hill Building, Manchester, UK. sequently limit opsonisation-mediated clearance from blood E-mail: marilena.hadjidemetriou@manchester.ac.uk, and ‘masking’ of surface ligands.3–6 Protein corona formation kostas.kostarelos@manchester.ac.uk b Cancer Research UK Manchester Institute Cancer Biomarker Centre, is now a widely accepted phenomenon and has been documented The University of Manchester, Manchester, UK for a wide range of NPs, including lipid-, metal-, polymer- and c Manchester Cancer Research Centre Biobank, The Christie NHS Foundation Trust, carbon-based nanomaterials, with their composition and surface CRUK Manchester Institute, Manchester, UK chemistry altering the specific classes of proteins adsorbed.7 d Manchester Cancer Research Centre, The University of Manchester, More recently, attempts have been made to purposefully 555 Wilmslow Road, Manchester M20 4GJ, UK e Catalan Institute of Nanoscience and Nanotechnology (ICN2), UAB Campus, manipulate the NP corona to modify the function of NPs4,8 and Bellaterra, Barcelona, Spain this approach has since been exploited as an application in its † Electronic supplementary information (ESI) available. See DOI: 10.1039/d0nh00333f own right.9–12 For example, altering the surface properties of 1476 | Nanoscale Horiz., 2020, 5, 1476--1486 This journal is © The Royal Society of Chemistry 2020 View Article Online Communication Nanoscale Horizons NPs not only alters the corona composition but has also been of cfDNA from plasma samples prior to the incubation with shown to control the loading and release of drugs and inter- NPs.43,44 action with target molecules.13,14 Manipulating the corona by The aim of the present study was to offer experimental engineering the surface chemistry of NPs can therefore enhance evidence on the fundamental question as to whether cfDNA This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. their biological efficacy.4,6 In an alternative proposition, our exists in the biomolecule corona formed around NPs in human laboratory has illustrated the potential exploitation of protein plasma. In order to investigate this, we incubated clinically- corona as a proteomic biomarker discovery platform that enables used types of liposomes with plasma samples, retrieved the a higher-definition, in-depth analysis of the blood proteome and corona-coated liposomes, and subsequently quantified the total the enrichment of low abundant disease-specific molecules.9–12 corona cfDNA content using two different real-time quantitative Despite the comprehensive characterisation of the blood- PCR (qPCR) assays. Our data confirmed the presence of cfDNA Open Access Article. Published on 27 August 2020. Downloaded on 2/11/2021 12:49:42 PM. derived NP protein corona in mice and humans (both ex vivo molecules in the biomolecule corona. In addition, analysis of and in vivo),10,13,15–19 little attention has been placed on other the liposome corona formed in plasma samples obtained from types of biomolecules that may constitute the biomolecule ovarian carcinoma patients revealed higher total cfDNA content corona. Growing evidence suggests that lipids and metabolites compared to healthy controls, suggesting a disease-specific also interact with the surface of NPs.20–29 For example, a biomolecule corona. Finally, correlation with the proteomic comprehensive metabolomic analysis at the nano-bio interface analysis of the same liposomal coronas revealed nucleosome- between mineralo-organic NPs and human biofluids (serum, derived histone proteins, which suggested an indirect, protein- plasma, saliva, and urine) revealed a range of metabolites mediated cfDNA interaction with NPs. including amino acids, sugars, amides, fatty acids and glycero- phospholipids.24 More recently, the formation of a ‘metabolite’ corona has been shown using different NPs including, copper Results oxide, titanium dioxide, zinc oxide, zirconium dioxide, carbon25 and amino-functionalised polystyrene NPs.26 The lipid and meta- Plasma incubation and biomolecule corona formation bolite composition of the liposomal corona has also been To evaluate the cfDNA content of the biomolecule corona, characterised in human plasma, revealing a complex lipid and human plasma samples obtained from healthy volunteers were metabolite profile.27 Notably, a recent study investigating the incubated (37 1C, 10 minutes, 250 rpm) with PEGylated lipo- metabolite ‘small molecule’ corona formed around a range of somes (HSPC:Chol:DSPE-PEG2000), a formulation which con- nanomaterials revealed the presence of charged metabolites stitutes the basis of the anti-cancer agent Doxils (Fig. S1A and B, including amino acids and nucleosides.22 These studies highlight ESI†). Liposomes were employed in this study due to their the diverse and complex composition of the biomolecule corona. extensive protein corona characterisation, their use in nucleic Scarce speculative statements exist in the literature referencing acid-based biotechnology applications and more recently due the presence of nucleic acids in the biomolecule corona.30–32 to their promise as a proteomic enrichment tool.10,38,39,45 Our However, the genomic content of the blood-derived NP corona previous ex vivo and in vivo investigations on protein corona has never been investigated and there is no experimental evidence revealed that even though PEGylation reduces protein adsorption, to support that the NP corona contains cell-free DNA (cfDNA). it does not completely eliminate binding. PEGylated liposomes This could be partially due to the experimental challenges in were found to reproducibly interact with low molecular weight identifying minute levels of blood-circulating nucleic acids on the (MW) plasma proteins of low abundance.9–12,15 NPs surfaces. The very few studies that have tried to investigate In order to assess the potential interaction of cfDNA with any role that nucleic acids may play in the biomolecule corona, PEGylated liposomal surfaces, plasma-incubated liposomes focused on miRNAs using rat urine samples,33 or attempted to were purified by size exclusion chromatography (SEC), shown understand the interaction of NPs with added synthetic DNA in Fig. 1A, as described previously.15 Plasma control samples fragments in solution.31,34 NPs have also been extensively used (without prior incubation with liposomes) were subjected to as biosensors or as nano-carriers to deliver nucleic acids, with the exact same purification process. SEC column-eluted cfDNA positively charged lipid-, polymeric-, gold- and carbon-based was extracted from chromatographic fractions 1–15, using a (among many other) NPs used to deliver electrostatically- QIAamps circulating nucleic acid extraction kit (QIAGEN) and complexed nucleic acids for therapeutic applications.34–42 subsequently quantified using robust and highly sensitive In an alternative approach, Liang et al., intravenously adminis- LINE-1 real-time qPCR assay (Fig. 2A). Stewart assay was also tered cationic polymeric NPs to complex cfDNA in vivo to performed in order to quantify the amount of liposomes eluted. alleviate the symptoms of inflammation in rheumatoid arthritis As illustrated in Fig. 2A and in agreement with our previous (RA).53 Despite these approaches of electrostatic complexation studies,15 corona-coated liposomes were eluted in chromato- of nucleic acids with cationic NPs, the spontaneous association graphic fractions 5 and 6, while no detectable lipid content was of blood-derived cfDNA molecules with NPs has largely been found in the fractionated plasma control. Distribution of cfDNA ignored, presumably because it was thought that other blood across chromatographic fractions 1–15 revealed significant components competitively self-assemble into layers around differences between plasma-incubated liposomes and the NPs. It is also worth mentioning that the majority of matched plasma control. In the case of the plasma-incubated NP-based colorimetric cfDNA biosensors require the purification liposome sample the majority of cfDNA (45.8%) was eluted in This journal is © The Royal Society of Chemistry 2020 Nanoscale Horiz., 2020, 5, 1476--1486 | 1477 View Article Online Nanoscale Horizons Communication lower limit of detection for absorbance and fluorescence-based DNA quantification methods. Initially, a standardised TaqMans RNase P detection real-time qPCR assay (Applied Biosystemss) was used to quantify the cfDNA content of the biomolecule corona in This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. healthy plasma samples. As illustrated in Fig. 2B, the concentration of cfDNA measured in the corona samples was significantly higher in comparison to plasma control samples that underwent the full purification process (adjusted p-value o 0.0001). A small amount of cfDNA was identified in purified plasma controls, suggesting a co-elution of a small population of cfDNA molecules complexed Open Access Article. Published on 27 August 2020. Downloaded on 2/11/2021 12:49:42 PM. with large proteins or within extracellular vesicles (Fig. 2B). These data suggested that most of the cfDNA quantified in corona samples is associated (directly or indirectly) with the surface of liposomes and was not passively co-eluted in a size-dependent manner. In order to investigate whether the presence of proteins and/ or other molecules in the biomolecule corona affects the direct quantification of cfDNA, we compared the amount of cfDNA with and without prior extraction (QIAGEN’s QIAamps circu- lating nucleic acid extraction kit). Comparable amounts of cfDNA were detected using the TaqMans RNase P assay both in corona-coated liposome samples and in cfDNA subsequently purified from the same corona samples (Fig. 2B). These data indicated that the real-time qPCR assay was not significantly Fig. 1 Schematic representation of sample processing and cfDNA quantifi- inhibited by other molecules present in the corona, allowing cation method pipelines. (A) Schematic overview of human plasma and direct cfDNA measurements in the presence of lipid-based NPs liposomal nanoparticle (NP) incubation and subsequent size-exclusion and complex biofluid contaminants. To further investigate purification methodology. (B) Method analysis pipeline for plasma processing qPCR inhibition in NP-corona samples, a 2-fold dilution was (including cfDNA purification) and subsequent q-PCR quantification of performed prior to real-time qPCR quantification (Fig. S2A and cfDNA in NP corona samples and plasma control samples. B, ESI†). The cfDNA quantity of the 1 : 2 diluted corona sample was approximately half that of the original measurement (48%), providing further evidence to support the lack of RNase P qPCR chromatographic fraction 5, which also contained the largest inhibition in these direct real-time PCR measurements. The population of liposome NPs (66.7%), while liposome-free frac- concentration of cfDNA in the NP-corona samples and plasma tions 7–15 contained relatively small quantities of cfDNA controls (with no NPs) was confirmed with a robust and (o6%). In contrast, a normal distribution of cfDNA was evident sensitive LINE-1 qPCR assay (Fig. 2C). Both assays produced in the fractionated plasma control, with the highest amount of similar values, with RNase P and LINE-1 quantification cfDNA detected in fraction 10 (18.8%). Notably, in the absence methods consistently detecting significantly more cfDNA in of NPs, only 2.6% of the cfDNA content was detected in corona samples when compared to plasma controls, as shown fraction 5. The striking difference in cfDNA distribution in Fig. 2C. between corona-coated liposomes and the fractionated plasma In terms of reproducibility, the percentage of cfDNA recov- control suggests that a significant proportion of cfDNA eluted ered with liposomal NPs was consistent across healthy plasma in fraction 5 could be associated with the eluted liposomes. and liposome batches (Fig. S3A, ESI†). In addition, plasma linearity experiments revealed a significant reduction in total Quantitative detection of cfDNA in the liposome corona cfDNA content when plasma input volume was lowered, while To further purify corona-coated liposomes from any remaining the plasma : NP ratio was maintained (adjusted p-values o protein complexes and/or unbound cfDNA, chromatographic 0.01 for both 410 mL & 205 mL of plasma when compared to fractions 5 and 6 were pooled, concentrated and subsequently 810 mL) (Fig. S3B, ESI†). In contrast to the linear relationship washed three times using a membrane ultrafiltration column observed between plasma volume and cfDNA concentration, (Vivaspins, 1 million MWCO). This protocol has been previously altering the concentration of liposome NPs did not signifi- developed by us and also used successfully by others to purify cantly affect the amount cfDNA recovered (Fig. S2C, ESI†). corona-coated NPs from unbound plasma proteins.9,10,13 Combined, these data suggested that at the NP concentrations To determine the total cfDNA content of the liposomal investigated, liposomes interacted reproducibly with a sub- corona two different real-time qPCR assays were utilised, as population of plasma cfDNA molecules and that a NP : plasma outlined in Fig. 1B. A real-time qPCR approach was chosen as [mL : mL] ratio of 0.2 was found optimal to recover this fraction the concentration of cfDNA in blood commonly falls below the of cfDNA. 1478 | Nanoscale Horiz., 2020, 5, 1476--1486 This journal is © The Royal Society of Chemistry 2020 View Article Online Communication Nanoscale Horizons This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Open Access Article. Published on 27 August 2020. Downloaded on 2/11/2021 12:49:42 PM. Fig. 2 Characterisation of cfDNA content in the healthy ex vivo biomolecule corona. (A) cfDNA and liposomal lipid quantification across 15 chromatographic fractions. The purified cfDNA from a single healthy pooled plasma sample incubated with and without liposomal nanoparticles (NPs) was quantified by a highly-sensitive LINE-1 real-time PCR assay. NPs and cfDNA are expressed as percentage (%) of total recovered across chromatographic fractions. (B) RNase P real-time cfDNA quantification of pooled ex vivo NP(+) corona samples and NP( ) controls (size-purified plasma). cfDNA was measured directly and in samples with additional cfDNA purification step. (C) cfDNA concentrations in NP(+) corona samples and NP( ) controls were confirmed using the LINE-1 real-time PCR assay. For graphs B and C cfDNA is expressed as percentage recovery (%) relative to QIAGEN’s QIAamps Circulating Nucleic Acid extraction kit (average of three replicates). All error bars represent mean and standard deviation. Groups were compared using a Student t-test (p values o 0.05 were considered significant). Detection of cfDNA in ovarian carcinoma liposomal corona These samples were quantified directly using a robust high samples sensitivity LINE-1 qPCR assay and compared to corona samples To establish whether cfDNA could also be detected on the from 11 healthy aged matched females (Fig. 3). When normal- surface of liposomes incubated ex vivo with plasma obtained ised to post-purification liposome concentration, cfDNA was from cancer patients, corona-coated liposomes were prepared significantly higher in ovarian cancer samples (all stages, early upon incubation and purification from plasma samples stage (I and II) and late-stage (III and IV)) compared to healthy obtained from 43 patients with ovarian cancer (18 patients controls (p values = o0.001, o0.01 and o0.0001, respectively) with FIGO stage I, 8 with stage II, 12 with stage III and 5 with (Fig. 3). In addition, average cfDNA content increased from stage IV) (Table S1, ESI†). Patients with ovarian cancer classi- early (FIGO stage I and II) to late stage (FIGO stage III and IV), fied across all stages of the disease were included in the study although this was not statistically significant (Fig. 3B). These to determine whether cfDNA could be detected in NP corona data are consistent with previous studies that have proposed samples both at early stages and as the disease progressed. quantification cfDNA as a diagnostic and prognostic biomarker This journal is © The Royal Society of Chemistry 2020 Nanoscale Horiz., 2020, 5, 1476--1486 | 1479 View Article Online Nanoscale Horizons Communication This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Open Access Article. Published on 27 August 2020. Downloaded on 2/11/2021 12:49:42 PM. Fig. 3 Cell-free DNA (cfDNA) detection in the ex vivo ovarian cancer biomolecule corona. (A) Normalised cfDNA concentration (ng mM 1 lipid) in corona-coated liposomes (ovarian cancer samples and age- and sex-matched healthy controls), measured using a highly-sensitive LINE-1 real-time PCR assay and robust inhibitor-resistant polyermase. (B) The same data with ovarian cancer patients separated into early stage (1 & 2) and late-stage (3 & 4) cancers. All error bars represent mean and standard deviation. Three groups or more were compared using a one-way analyses of variance (ANOVA) test followed by the Tukey’s multiple comparison test. For comparisons of two groups a Student t-test was performed (adjusted p values o 0.05 were considered significant). for ovarian cancer, with increased cfDNA levels detected with nucleosome-interacting proteins were identified in these samples, disease progression.46,47 namely histone-lysine N-methyltransferase 2D and histone To determine whether direct cfDNA quantification in ovarian PARylation factor 1 (Fig. S4B, ESI†)48 Combined, these data cancer corona samples would be inaccurate and skewed, with confirmed the presence of cfDNA in the biomolecule corona of real-time qPCR inhibition increasing disproportionately with liposomes and suggested an indirect interaction which is poten- cancer stage, we compared cfDNA concentration in purified tially mediated via the nucleosome complex. and unpurified samples for eight late-stage (stage III n = 6, stage IV n = 2) high-grade serous ovarian cancer samples (details provided in Fig. S2E, ESI†). Similar cfDNA concentra- Discussion tions were measured for both unpurified ovarian cancer corona samples and their respective purified cfDNA samples (Fig. S2C, Understanding the composition of the biomolecule corona, ESI†). This suggests that real-time qPCR was not significantly spontaneously adsorbed onto the surface of nanoparticles inhibited in these biomolecule corona qPCR reactions and that (NPs), remains challenging due to the diverse physiochemical no significant cfDNA loss occurred during cfDNA extraction characteristics of NPs, as well as the complex multi-molecular using QIAGEN’s QIAamps circulating nucleic acid extraction composition of biological fluids.1 To date, the focus has been to kit. We were also able to measure the cfDNA content directly in elucidate the blood protein corona composition of various NPs ovarian cancer plasma samples (diluted 1 : 40), which again with different surface chemistry and physiochemical properties.49 showed no significant difference from the respective purified However, limited attention has been placed in profiling other bio- plasma cfDNA samples (Fig. S2D, ESI†). molecules present in the NP corona. Recent studies have high- Mass spectrometry (LC-MS/MS) proteomic analysis was then lighted the interaction of NPs with lipids and metabolites,21–23 performed on the 43 samples from ovarian cancer patients and yet the contribution of blood-circulating cell-free DNA (cfDNA) the 11 samples from healthy controls to investigate whether to the formation of the NP corona remains almost completely proteins known to associate with cfDNA could be detected in unexplored. the biomolecule corona (Fig. S4, ESI†). Histone proteins, H2A, Fragmented cell-free DNA can be actively secreted into the H2B and H4, which are found within the core nucleosome bloodstream and is also passively released into circulation complex, were detected in the biomolecule corona and were during cell death (i.e. apoptosis, necrosis).50 In healthy indivi- identified at significantly higher levels in ovarian cancer samples duals, cfDNA levels are usually extremely low, with elevated relative to healthy controls (Fig. S4A, ESI†). Two additional concentrations of cfDNA commonly triggered by pathological 1480 | Nanoscale Horiz., 2020, 5, 1476--1486 This journal is © The Royal Society of Chemistry 2020 View Article Online Communication Nanoscale Horizons disease states, such a tumorigenesis, inflammation, ischemia, successfully purified from lipid NPs using a standard cfDNA trauma and sepsis.50 Cell-free DNA is widely protected from extraction kit, highlighting the compatibility of lipid-based NPs nuclease digestion by its complexation with a core of histone with downstream purification and quantification methods. proteins, known as nucleosomes.51 Genomic analysis of the Consistent cfDNA recovery across NP batches (Fig. S3A, ESI†) This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. nucleic acid content in blood is of growing interest for diag- suggested its reproducible and stable interaction with the nostic and disease monitoring applications, particularly in the liposomal surface as part of the biomolecule corona. context of liquid biopsies for cancer.52 The PEGylated liposomes used in this study have a negative Despite recent advances in analysing the blood-circulating surface charge (Fig. S1A, ESI†), therefore it was considered genome, very little attention has been placed on the utilisation unlikely that DNA molecules would be bound directly onto of the spontaneous interaction of NPs with nucleic acids upon the liposome surface via electrostatic interactions. Considering Open Access Article. Published on 27 August 2020. Downloaded on 2/11/2021 12:49:42 PM. incubation with biological fluids. Recently, Qian and colleagues that cfDNA is protected within nucleosome complexes in the investigated the interaction of negatively-charged, carboxylated blood,51 we hypothesised that cfDNA may not be directly bound magnetic NPs with miRNAs in rat urine samples and suggested onto the liposome surface, but through the adsorption of an RNA-induced silencing complex (RISC)-mediated adsorption DNA–protein complexes (illustrated in Fig. 4). This indirect of miRNA.33 In a different study, Yun et al. developed a planar- mechanism of adsorption was further supported by the identifi- substrate based protocol in order to investigate the interaction cation of positively charged nucleosome core proteins, including of metal–phenolic network (MPN)-based nanomaterials with histone proteins H2A, H2B and H4, in the biomolecule corona by multiple biomolecules including synthetic single stranded 40 bp LC-MS/MS analysis (Fig. S4, ESI†). Our group has previously DNA fragments.31 It is important to note however, that these DNA detected histone proteins in human and mouse liposomal corona, molecules do not reflect the size and structure of endogenous both ex vivo and in vivo.9,11,12 Moreover, human histone proteins plasma cfDNA, nor the molecular complexity of blood.31 (H2B and H4) have also been found to interact with colloidal gold Despite the above-mentioned preliminary indications of the NPs upon incubation with human plasma.54 Finally, De Paoli and interaction of nucleic acids with the surface of NPs, no studies colleagues demonstrated that calf thymus histone H1 can bind have demonstrated the presence of cfDNA in the biomolecule onto carboxylated-multiwalled carbon nanotubes.55 corona formed when NPs come into contact with human Our data demonstrated that the corona-containing cfDNA plasma. The present study aimed to answer this fundamental levels were significantly higher in the biomolecule coronas question and establish whether cfDNA participates in the formed upon incubation with plasma samples obtained from formation of the biomolecule corona in a non-specific, untargeted ovarian cancer patients (both early- and late-stages) in compar- manner. In order to investigate this, we used a real-time qPCR- ison to healthy controls (Fig. 3). It has been widely reported that based approach to quantify cfDNA in the biomolecule corona of the total amount of cfDNA increases with disease progression lipid-based NPs upon incubation with human plasma. The NP in many different cancer types, such as colorectal, glio- corona samples underwent a two-step purification process prior to blastoma, colorectal and breast cancer.47,56–59 It is important cfDNA quantification (with and without an additional cfDNA to clarify that circulating DNA originating from the tumour purification procedure). Our data provide the first experimental (ctDNA) commonly accounts for only a small proportion of the evidence of the presence of cfDNA in the NP corona samples and total cfDNA, with the majority of DNA molecules released from show that the majority of cfDNA detected is associated with non-malignant cells.51,60 Moreover, cfDNA detected in indivi- the surface of liposomes and is not passively co-eluted during duals with cancer is commonly of heamatopoietic origin and purification (Fig. 2A–C). Direct quantification of cfDNA was can be attributed to increased white blood cell turnover and possible within complex lipid-based biomolecule corona samples chemotherapeutic- and/or radiation-induced cell death.51,56 without prior cfDNA extraction using the QIAamp circulating Overall our data here demonstrated that the total amount of nucleic acid extraction kit (QIAGEN). In addition, cfDNA was corona cfDNA increases in the presence of ovarian cancer, Fig. 4 Schematic representation of the formation of a biomolecule corona and the protein-mediated interaction of cell-free DNA (cfDNA) with the surface of PEGylated liposomes. Positively-charged histone protein complexed with DNA can bind directly to the negatively-charged liposomal surface. This journal is © The Royal Society of Chemistry 2020 Nanoscale Horiz., 2020, 5, 1476--1486 | 1481 View Article Online Nanoscale Horizons Communication which renders the need for further studies to estimate the affect their interaction with nucleic acids. Understanding the fraction of ctDNA by analysing tumour-specific mutations. This complexity of the biomolecular shell coating the surface NPs approach could offer significant advantages over current cfDNA is fundamental in developing new and effective biomedical purification methods, which lack the sensitivity required to applications. Only now, is the diagnostic and therapeutic This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. detect ctDNA in small volumes of human plasma in patients potential of the NP corona finally being realised, entering the with low tumour burden, especially pertinent to the challenge nanomedicine field into an exciting new era. of early cancer detection. Previous observations have shown that the composition of the biomolecule corona is directly affected by the presence Conclusion of a disease.9 Comprehensive comparison of ‘healthy’ and This study has shown that cell-free DNA is present in the Open Access Article. Published on 27 August 2020. Downloaded on 2/11/2021 12:49:42 PM. ‘diseased’ protein coronas has been proposed for the discovery biomolecule corona that forms around lipid-based NPs, upon of diagnostic biomarkers.9–12 For example, we have previously incubation with human plasma. The cfDNA content of the shown that protein corona quantitatively and qualitatively biomolecule corona could be directly quantified in the presence changed in the presence of tumorigenesis, with higher total other biomolecules (e.g. proteins) using conventional real-time amount of protein found to interact with intravenously qPCR assays. Furthermore, proteomic analysis of the biomole- injected liposomes recovered from melanoma and lung adeno- cule corona by LC-MS/MS revealed the presence of nucleosome carcinoma tumour-bearing mice in comparison to healthy complex proteins, suggesting an indirect protein-mediated controls.9 Further analysis revealed that histone H2A was interaction of cfDNA with NPs. Notably, the amount of cfDNA significantly upregulated in the in vivo lung adenocarcinoma was found to be significantly higher in the coronas formed in corona samples.9 Therefore, the increased amount of nucleosome- early- and late-stage cancer patient plasma samples compared related proteins identified in ovarian cancer patients in this to healthy controls, indicating a disease-specific biomolecule study is likely to extend to other cancer types and NP classes, corona formation. This work is thought to pave the way for as a general reflection of increased histone levels commonly future studies, to further understand the mechanism, type and seen in cancer.61–64 multi-molecular complex formation and adsorption of blood- In recent years, other cell-free nucleic acids, such as miRNAs, circulating nucleic acids onto the surface of NPs. Equally, our have received growing interest as disease biomarkers65 and study highlights the potential exploitation of the biomolecule although extensive characterisation of the NP corona total nucleic corona as a novel blood-analysis nanoscale tool. acid content was beyond the scope of this study, it remains an important avenue of future research. Blood-circulating RNA molecules are commonly complexed with proteins, including Experimental those forming the RNA-induced silencing complex (RISC).66 The Plasma samples potential protein-mediated interaction of RNA with plasma- Healthy human female pooled K2EDTA plasma samples were incubated NPs remains to be explored, but if confirmed would purchased from BioIVT (West Sussex, UK) (Lot #HMN2528). add another layer of complexity to the NP biomolecule corona. All ovarian cancer K2EDTA plasma samples were collected In addition, epigenetic analysis of ctDNA, such as differential by the MCRC Biobank (details provided in Table S1 and methylation profiles can also provide cancer-specific signatures.67 Fig. S2E, ESI†). Individual age- and sex-matched K2EDTA Intriguingly, methylated tumour DNA isolated from the plasma of plasma controls (female, 45–85 years old) were purchased from cancer patients has been shown to display a strong affinity to gold BioIVT (West Sussex, UK) (Table S1, ESI†). All plasma samples nanoparticles and forms the basis of a ctDNA detection tool.68,69 were stored at 80 1C. The molecular complexes of cell-free nucleic acids contained within the biomolecule corona need to be fully elucidated in Liposome preparation order to establish the scope for a sensitive blood-based biomarker HSPC : Chol : DSPE-PEG2000 (56.3 : 38.2 : 5.5) liposomes (Doxils enrichment tool. formulation) liposomes were prepared using the thin lipid film The molecular information contained within the NP corona method followed by extrusion as described previously.15 All is far richer than originally described and has been shown to liposome batches were diluted to 12.5 mM, with the same batch contain a diverse array of biomolecules including proteins, of liposomes used for group comparisons. The physiochemical lipids, metabolites and now cfDNA. This complex coating on characteristics of the liposome batches are shown in Fig. S1 the surface of NPs has the potential to be able to enhance nano- (ESI†). drug delivery and NP uptake, but perhaps most significantly, offers the potential to provide greater sensitivity for liquid biopsies. Further studies are required to determine whether Dynamic light scattering (DLS) for size and zeta-potential tumour-derived nucleic acids (ctDNA, miRNAs, RNAs) are measurements detectable in the biomolecule corona and whether NPs can Liposome size and surface charge were measured as described enrich the tumour-specific content obtained from human previously.15 Liposomes were diluted in distilled water and blood. Additional studies are also necessary to determine measured in size or capillary cuvettes using the Zetasizer Nano how the physiochemical properties of different nanomaterials ZS (Malvern, Instruments, UK). 1482 | Nanoscale Horiz., 2020, 5, 1476--1486 This journal is © The Royal Society of Chemistry 2020 View Article Online Communication Nanoscale Horizons Biomolecule corona formation (liposome plasma incubation combination with the robust Terra qPCR Direct SYBR Premix and purification) master mix. Liposome and plasma incubations and purifications were Mass spectrometry performed as described previously.15 In brief, 820 mL human This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. plasma and 180 mL PEGylated liposomes were incubated for In-gel digestion of corona proteins was performed prior to 10 min at 37 1C, shaking at 250 rpm. Unbound proteins and LC-MS/MS analysis, as described previously.15 Digested proteins other unknown biomolecules were removed by size exclusion were analysed by LC-MS/MS using an UltiMate 3000 Rapid chromatography (SEC) (Sepharose CL-4B columns (Sigma- Separation LC (RSLC, Dionex Corporation, Sunnyvale, CA) plus Q Aldrich)) followed by membrane ultrafiltration (Vivaspins Exactive Hybrid Quadrupole-Orbitrap (Thermo Fisher Scientific, columns (Sartorious, Fisher Scientific)). Samples were concen- Waltham, MA, USA) mass spectrometer system. Data were Open Access Article. Published on 27 August 2020. Downloaded on 2/11/2021 12:49:42 PM. trated to 100 mL for characterisation or downstream processing. analysed using Mascot (Matrix Science UK) in combination with For characterisation of individual chromatographic fractions, the SwissProt_2016_04 database (taxonomy human). Progenisis samples were concentrated to 100 mL using 1 000 000 molecular QI software (version 4.3.2, Proteome Software Inc.) was used for weight cut off (MWCO) Vivaspins membrane ultrafiltration relative protein quantification based on spectral counting and columns (Sartorious, Fisher Scientific). Plasma controls were statistical analyses (one-way analyses of variance (ANOVA)). subjected to the same purification process for comparison. Statistical analysis Circulating cell-free nucleic acid extraction Statistical comparisons of these data were performed using Cell-free nucleic acids were purified from ex vivo plasma GraphPad Prism v.8.2.0. For comparisons of three groups or samples, liposomal corona samples and plasma control samples more, one-way ANOVA tests were performed followed by the using a QIAamps Circulating Nucleic Acid Extraction kit and Tukey’s multiple comparison test (adjusted p values o 0.05 QIAvac 24 Plus vacuum manifold according to manufacturer’s were considered significant). For comparisons of two groups instructions (QIAGEN, Hilden, Germany). After an initial sample unpaired Student t-tests were performed (FDR-adjusted lysis step, cell-free nucleic acids were bound onto a silica-based p values o 0.05 were considered significant). All data averages purification column (QIAGEN mini column). Multiple washing were presented as mean  standard deviation (SD). steps were performed prior to elution of cell-free nucleic acids in buffer AVE (QIAGEN). All samples were eluted in a final volume Ethical approvals of 50 mL. This project has research ethics approval under the Manchester Cell-free DNA quantification Cancer Research Centre (MCRC) Biobank Research Tissue Bank Ethics (NHS NW Research Ethics Committee 18/NW/0092). Cell-free DNA was measured using two real-time quantitative All participants provided written informed consent to partici- PCR (qPCR) assays. The single-copy RNase P probe real-time pate in this study. assay was performed using TaqMans RNase P Detection Reagents kit (Life Technologies) and SensiFAST Probe Hi-ROX master mix (Bioline, Meridian Bioscience). All real-time qPCR Author contributions reactions included 7.5 mL of 2 SensiFAST probe mastermix, L. G., M. H., K. K., G. B. designed and performed all experi- 0.75 mL 20 RNase P primer/probe mix, 1.75 mL nuclease-free ments and took responsibility for planning and writing water (Ambion, Texas, USA) and 5 mL of sample. Cycling the manuscript. J. W. performed mass spectrometry sample conditions included (95 1C, 5 min)  1, (95 1C, 10 s; 60 1C, preparation and respective data analysis. J. R. provided access 50 s)  40 and were performed on a LightCyclers 96 (Roche, and information from the Christie NHS Hospital Biobank and Basel, Switzerland). supported the study. D. R. supervised and provided intellectual The multi-locus LINE-1 real-time qPCR assay was performed input to the genomics experimental work. G. B., C. D., K. K. and using primers described previously70 purchased from Inte- M. H. initiated, overall supervised, provided intellectual input grated DNA Technologies (desalted, 25 nmol scale) using a and contributed to the writing of the manuscript. All authors robust Terra qPCR Direct SYBR Premix master mix (Takara Bio, have given approval to the final version of the manuscript. USA). All real-time PCR reactions included 7.5 mL of 2 Terra qPCR Direct SYBR Premix master mix, 0.75 mL of each 10 mM (forward and reverse primers), 5.75 mL nuclease-free water Conflicts of interest (Ambion, Texas, USA) and 1 mL of sample. Cycling conditions included (98 1C, 2 min)  1, (98 1C, 10 s; 60 1C, 15 s; 68 1C, 30 s) There are no conflicts to declare.  35 and were performed on a LightCyclers 96 (Roche, Basel, Switzerland). Acknowledgements Sample input was either corona-coated liposomes, puri- fied cfDNA or plasma samples diluted 1 : 40. Plasma samples This research was funded by the NIHR Manchester Biomedical were only quantified using the LINE-1 real-time PCR assay in Research Centre (BRC). Research samples were obtained from This journal is © The Royal Society of Chemistry 2020 Nanoscale Horiz., 2020, 5, 1476--1486 | 1483 View Article Online Nanoscale Horizons Communication the Manchester Cancer Research Centre (MCRC) Biobank, UK. 19 S. Tenzer, D. Docter, S. Rosfa, A. Wlodarski, J. Kuharev, The role of the MCRC Biobank is to distribute samples and A. Rekik, S. K. Knauer, C. Bantz, T. Nawroth, C. Bier, therefore, cannot endorse studies performed or the interpreta- J. Sirirattanapan, W. Mann, L. Treuel, R. Zellner, tion of results. The authors also would like to thank the Faculty M. Maskos, H. Schild and R. H. Stauber, ACS Nano, 2011, This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. of Life Sciences Mass Spectrometry facility at the University of 5, 7155–7167. Manchester for their assistance. Finally, the authors would like 20 A. A. Kapralov, W. H. Feng, A. A. Amoscato, N. Yanamala, to thank the ovarian cancer patients and healthy volunteers for K. Balasubramanian, D. E. Winnica, E. R. Kisin, G. P. their blood donations, which supported this research. Kotchey, P. Gou, L. J. Sparvero, P. Ray, R. K. Mallampalli, J. Klein-Seetharaman, B. Fadeel, A. Star, A. A. Shvedova and References V. E. 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