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Programmable sequential mutagenesis by inducible Cpf1 crRNA array inversion

Mutations and genetic alterations are often sequentially acquired in various biological and pathological processes, such as development, evolution, and cancer. Certain phenotypes only manifest with precise temporal sequences of genetic events. While multiple approaches have been developed to model the effects of mutations in tumorigenesis, few recapitulate the stepwise nature of cancer evolution. Here we describe a flexible sequential mutagenesis system, Cpf1-Flip, with inducible inversion of a single crRNA array (FlipArray), and demonstrate its application in stepwise mutagenesis in murine and human cells. As a proof-of-concept, we further utilize Cpf1-Flip in a pooled-library approach to model the acquisition of diverse resistance mutations to cancer immunotherapy. Cpf1-Flip offers a simple, versatile, and controlled approach for precise mutagenesis of multiple loci in a sequential manner.

I n a large variety of biological and pathological processes, genetic mutations or alterations are often acquired in a sequential manner [1][2][3] . In evolution and speciation, the genomes of organisms acquire mutations constantly and are subjected to natural selection 4 . In genetically complex disorders such as cancer, multi-step mutagenesis is often a major obstacle for effective treatments. Cancers evolve through an ongoing process of mutation-selection balance, where initial mutations are selected for, or against, in vivo, followed by subsequent acquisition of additional mutations as the tumor grows 5 . Since the initial set of oncogenic "driver" mutations is generally what starts and sustains tumor growth, targeted molecular therapies are often chosen to specifically attack such oncogenic dependencies 5 . However, the selection pressures of treatment will favor secondary mutations that confer drug resistance, leading to relapse [6][7][8] . Thus, the process of cancer evolution by sequential mutagenesis stymies these therapies via continuous diversification and adaptation to the tumor microenvironment, eventually exhausting available treatment options 5 . Even with the advent of cancer immunotherapy, where checkpoint blockade is increasingly being utilized in the clinic, the acquisition of secondary mutations that abolish T cell receptor (TCR)-antigen-major histocompatibility complex recognition can still lead to immune escape and ultimately negate the effect of immunotherapy 9,10 . Thus, the ability to perform sequential and precise mutagenesis is critical for studying biological processes with multi-stage genetic events such as development and evolution, as well as the pathogenesis of complex diseases such as cancer.
From a genetic engineering perspective, stepwise mutagenesis or perturbation is a powerful technique for precise genetic manipulation of cells and live organisms. Multiple methods have been employed to achieve this end. In the pre-recombinant DNA era, stepwise perturbation was often done by multiple rounds of random mutagenesis using chemical or physical carcinogens followed by artificial selection. The subsequent discovery and application of recombinase systems such as Cre-loxP, Flp-FRT, and φC31-att enabled inducible genetic events 11 . In these systems, the DNA recombinase (i.e., Cre) specifically recognizes its target DNA sequence motif (i.e., loxP) and catalyzes recombination between two such target sites. Depending on the configuration of the target sites, targeted recombinases can be utilized for DNA excision, translocation, and/or inversion. However, the floxed genomic loci underlying Cre-based systems must be preengineered on a gene-by-gene basis. This process of generating new floxed alleles for each unique application is time and labor intensive, further limiting the feasibility of multiplexed Cre recombination.
More recently, precisely targeted and customizable mutagenesis was simplified by the discovery of RNA-guided endonucleases (RGNs) Cas9 12-14 and Cpf1 15,16 from clustered regularly interspaced short palindromic repeats (CRISPR) systems. RGNs can induce double-strand DNA breaks, subsequently generating insertions and deletions at the target site. This process is precisely targeted based on the sequences of CRISPR RNAs (crRNAs), which complex with RGNs to enable and guide their nuclease functions. Unlike with Cre recombination, CRISPR crRNAs can be easily transferred to target cells through transfection or viral vectors, thus obviating the need to pre-engineer the host genome for each target gene.
In contrast to Cas9, the most widely utilized RGN to date, Cpf1 is a single component RGN that does not depend on trans-activating RNA and can autonomously process crRNA arrays [15][16][17] . These features have made Cpf1 particularly attractive for multiplexed mutagenesis. In addition to several studies in mammalian systems, Cpf1-mediated mutagenesis and transcriptional repression have now been successfully applied in plants [18][19][20] . Furthermore, chemical modifications on Cpf1 mRNA and crRNAs have been identified that can improve cutting efficiency 21 . It was recently demonstrated that Cpf1 can also process crRNAs from mRNAs expressed by a Pol II promoter 22 , further enabling flexible transcriptional control.
Sequential mutagenesis using Cas9 was demonstrated in ex vivo organoid cultures 23 . However, this approach required sequentially introducing each sgRNA in culture, one at a time, limiting its broader applicability. In particular, the sequential introduction of different sgRNAs would be impractical for library-scale screening or any in vivo experimental designs. To our knowledge, conditional sequential mutagenesis using RGNs has not yet been demonstrated. Here we describe a flexible sequential mutagenesis system through inducible inversion of a single crRNA array (Cpf1-Flip) and demonstrate its simplicity in stepwise multiplexed gene editing in mammalian cells for modeling sequential genetic events, such as in cancer. We further apply Cpf1-Flip to model the acquisition of resistance mutations to immunotherapy in a pooled mutagenesis setting, demonstrating the feasibility of Cpf1-Flip for conducting sequential genetic studies. This system can be utilized for multi-step mutagenesis of any genes in the genome for interrogating complex genetic events with temporal control.

Results
Construction of a Cpf1 sequential mutagenesis system. When loxP sites are arranged such that they point towards each other, Cre recombination leads to inversion of the intervening sequence. However, this process leads to the complete regeneration of the loxP sites, thereby allowing Cre to continually catalyze DNA inversion. As continuous Cre-mediated inversion would be counterproductive in many applications, mutant loxP sites have been characterized that enable unidirectional Cre inversion 24,25 . When the mutant loxP sites lox66 and lox71 are recombined, they generate a wild-type loxP site and a double-mutant lox72. Cre has a substantially lower affinity for lox72, thus leading to mostly irreversible inversion of the floxed DNA segment.
We designed a U6 expression cassette containing two inverted BsmbI restriction sites, flanked by a lox66 sequence and an inverted lox71 sequence (Fig. 1a). In the same lentiviral vector, an EFS promoter drives the expression of Lachnospiraceae bacterium Cpf1 (LbCpf1, or Cpf1 for short) 15 and a puromycin resistance gene (EFS-Cpf1-Puro). After BsmbI restriction digest, the vector linearizes and allows for insertion of a crRNA array. To enable stepwise mutagenesis, we designed crRNA arrays in which the first crRNA is encoded on the sense strand, while the second crRNA is inverted. We hereafter refer to this construct as a crRNA FlipArray. Six consecutive thymidines (6xT) are present in cis at the 3′ end of each crRNA, terminating U6 transcription. Each crRNA is preceded by the LbCpf1 direct repeat (DR) sequence, which guides Cpf1 to process the crRNA array 16 .
Cre-mediated recombination of the lox66 and lox71 mutant loxP sites 24,25 leads to inversion of the FlipArray, generating a wild-type loxP and a double-mutant loxP, lox72. As the affinity of Cre recombinase for lox72 is substantially lower than for wildtype loxP, inversion of the FlipArray is mostly irreversible 24,25 . After inversion, the two crRNAs trade places and the second crRNA becomes expressed. Thus, in the absence of Cre, Cpf1 generates indels at the target site of the first crRNA; after Cre recombination, Cpf1 is directed to the target site of the second crRNA. We hereafter term this approach Cpf1-Flip. In short, the Cpf1-Flip system leverages CRISPR-Cpf1 mutagenesis and melds it with the inversion capabilities of Cre/lox66/lox71 to enable programmable two-step mutagenesis.
Sequential mutagenesis in murine and human cells. To demonstrate sequential editing of cancer genes, we first applied Cpf1-Flip to generate Nf1 and Pten mutations in a mammalian lung cancer cell line (KPD) 26 . We cloned in a FlipArray containing a spacer targeting Nf1 (crNf1) and an inverted spacer targeting Pten (crPten) (crNf1-crPten FlipArray, or NPF). We infected the cells with lentivirus containing EFS-Cpf1-Puro; U6-NPF (Fig. 1b). The pre-recombination construct was designed to only express crRNA targeting the first locus (Nf1) prior to the introduction of Cre. After 6 days of puromycin selection (1 week after the initial lentiviral transduction), we then infected the cells with lentivirus containing an EFS promoter driving the expression of Cre (EFS-Cre). Cre-expressing cells undergo inversion of the crRNA FlipArray, leading to sequential mutagenesis at the second locus (Pten) (Fig. 1c).
To detect Cre-mediated inversion of the FlipArray, we isolated genomic DNA from the NPF-expressing lung cancer cells before infection with EFS-Cre and 10 days after infection. We designed primers that would only generate a product if the FlipArray had successfully inverted (Fig. 2a). We also designed primers specific for the non-inverted FlipArray. These data demonstrated robust FlipArray inversion (Fig. 2b). Specifically, by D10 following EFS-Cre, the FlipArray inversion frequency was 79.07 ± 8.23% (mean ± s.e.m.) (Fig. 2c). In order to monitor the induction of functional FlipArray inversion at the transcript level, we also isolated total RNA from the double-infected KPD cells at various timepoints. After cDNA synthesis, we utilized inversion-specific primers to detect inverted crRNA FlipArray transcripts (Fig. 2d). The induction of inverted FlipArray transcripts steadily increased through the course of the experiment, illuminating the kinetics of Cre-mediated inversion of the FlipArray and its subsequent transcription. The low-levels of inverted FlipArray transcripts at baseline could be due to spontaneous inversion, or an artifact of the primer design.
We sequenced the target sites of crNf1 and crPten to determine whether the NPF construct had indeed created mutations in a controlled stepwise manner. Uninfected controls did not have any significant variants at crNf1 (Supplementary Data 1) or crPten target sites (Supplementary Data 2) (Fig. 2e, f, k). 7 days following the first lentiviral infection with EFS-Cpf1-Puro; U6-NPF, indels were found at the crNf1 target site, but not the crPten site ( Fig. 2g, h, k). Since the second crRNA is not transcribed prior to Cre recombination, this result affirms that inversion of NPF has not yet occurred at this time point. After another 10 days following infection with EFS-Cre lentivirus (17 days following the initial infection with EFS-Cpf1-Puro; U6-NPF), indels were found at both crNf1 and crPten target sites at high frequencies (Supplementary Data 3; Fig. 2i (Fig. 3a) (Fig. 3b). In this system, inversion efficiency was 85.42 ± 2.90% by 2 weeks following EFS-Cre (Fig. 3c). Next, to determine whether the Cpf1-Flip system had enabled sequential mutagenesis at the crDNMT1 and crVEGFA target sites, we performed deep sequencing. As anticipated, uninfected controls did not have significant mutations at either site (Supplementary Data 4, 5; Fig. 3d, e, j, k). Seven days after transduction with EFS-Cpf1; U6-DVF lentivirus, significant indels were found at the crDNMT1 target site but not at the crVEGFA target locus (Fig. 3f, g, j, k). The cells were then infected with EFS-Cre to cause FlipArray inversion, leading to expression of crVEGFA. 21 days after the initial transduction (14 days after EFS-Cre administration), significant indels were observed at both crDNMT1 and crVEGFA target sites (Supplementary Data 6; Fig. 3h-i). In these data, the DNMT1 cutting efficiency appeared to be consistently lower at D21 than at D7. This is likely a consequence of random sampling, as only a subset of the D7 cells were subsequently taken forward for Cre infection. In addition, it is possible that DNMT1 loss affects cell viability, given its crucial role in maintaining DNA methylation 27 . The cutting efficiency at crVEGFA was notably lower compared to crDNMT1. This contrast may be due to lower efficiency of the crRNA itself, as well as inefficiencies in FlipArray expression or subsequent crRNA array processing. Taken together, these results demonstrate that Cpf1-Flip is a flexible tool for sequential mutagenesis based on the Cpf1:crRNA complex, temporally controlled by Cre recombinase.
Modeling acquired resistance to immunotherapy with Cpf1-Flip. We next sought to apply Cpf1-Flip to model acquired resistance to immunotherapy in breast cancer cells (E0771 cell line). We designed a small pool of FlipArrays in which the first crRNA targets Nf1 while the inverted second crRNA targets a panel of immunomodulatory factors (Cd274, Ido1, B2m, Fasl, Jak2, and Lgals9; referred to as TSG-Immune FlipArray library).
These factors are thought to influence anti-tumor immunity and have been implicated in acquired resistance to checkpoint inhibitors. After pooled lentiviral transduction of E0771 cells with the TSG-Immune FlipArray library, we infected the cells with EFS-Cre lentivirus to induce FlipArray inversion (Fig. 4a). Upon Cremediated inversion, the second crRNA is expressed and triggers the knockout of various immunomodulatory factors, thus mimicking the sequential evolution of cancers in the face of immunotherapeutic pressures.
Targeted amplicon sequencing confirmed efficient mutagenesis of Nf1 (Fig. 4b), followed by mutagenesis of the immunomodulatory factors upon Cre-mediated FlipArray inversion (Fig. 4c) (Supplementary Data 7,8). Given the pooled nature of these experiments, lower population-level cutting efficiencies are anticipated at the second loci, as only a sixth of the total cell population, on average, is infected with a given FlipArray. The lack of consistent mutagenesis at the crB2m and crCd274 target sites may be intrinsic to the crRNA sequences themselves, a result of inefficient Cre infection/recombination and FlipArray processing, or simply a consequence of biased representation within the cell pool. Of note, we observed high cutting efficiencies at the Jak2 locus despite the pooled nature of the experiment. Since these cells were processed completely in parallel as a minipool, the observation that crJak2 and crLgals9 showed consistent mutagenesis points to intrinsic differences in crRNA targeting efficiencies as the key factor underlying the lack of consistent cutting by crB2m and crCd274. Collectively, these data demonstrate the potential of Cpf1-Flip to facilitate sequential genetic screens-for instance, to model the acquisition of resistance mutations to cancer immunotherapy.

Discussion
We introduced Cpf1-Flip, an inducible sequential mutagenesis system using invertible crRNA FlipArrays. As a proof-of-concept, we demonstrated sequential mutagenesis in both mouse and human cells, while additionally performing pooled sequential mutagenesis in a cancer cell line. These data revealed that the cutting efficiency of the second target loci can be low with certain crRNAs despite successful FlipArray inversion. The most likely explanation for the discordance between FlipArray inversion and subsequent mutagenesis of the second target locus is the differing efficiencies of the crRNAs themselves. This is corroborated by the variance observed across independent crRNAs in the pooled TSG-Immune library (Fig. 4), where consistent cutting efficiencies were observed at the Jak2 and Lgals9 target sites, but not at B2m or Cd274 sites. Moreover, cells with different crRNAs in a pool can undergo random drift or selection, further diverting their relative fractions and thereby indel frequencies. Nevertheless, the FlipArray library can be readout by barcoded PCR of the specific crRNA cassette followed by high-throughput sequencing. Thus, as with all CRISPR screens, pooled screen studies using Cpf1-Flip would require multiple independent Fli-pArrays targeting each gene/gene pair to ensure fair representation in the mutant pool. In the future, the use of optimized crRNA sequences 28 , improved FlipArray designs, and engineered Cpf1 enzymes will improve the consistency and efficiency of Cpf1-Flip. By altering the composition and length of the crRNA arrays within the FlipArray, one could readily engineer more complex CRISPR perturbation programs. Designs with two or more crRNAs within an invertible FlipArray at baseline would empower stepwise double knockouts (2 + 2, or quadruple knockouts as an end result) or higher dimensional sequential mutagenesis. Of note, the use of modified Cre systems such as CreER 29 , photoactivatable Cre 30 , and split-Cre 31 would provide even greater control of FlipArray inversion. Utilizing orthogonal recombinases and recognition sites in the crRNA array would allow for even more complex multi-step gene editing programs. Through the use of tethered Cpf1 variants, FlipArrays could also be potentially used for sequential and reversible gene activation [32][33][34] , repression [35][36][37] , or epigenetic modification [38][39][40] (Supplementary Fig. 1a). Given the scalability and flexibility of FlipArrays, conditional genetic studies for phenotypes that only emerge upon sequential genetic events can be performed using Cpf1-Flip either in culture or in vivo ( Supplementary Fig. 1b). Since new mutations are stochastically acquired by rare individual cells within tumors 41 , Cpf1-Flip could therefore be relevant for studying the dynamics of rare tumor subclones under varying selection pressures, such as immunotherapy.
All such applications of Cpf1-Flip and its derivatives could potentially be self-contained within a single viral vector, facilitating direct in vivo sequential genetic manipulations and functional studies.

Methods
FlipArray design and construction. The empty EFS-Cpf1-Puro; U6-FlipArray vector was constructed by modification of the pY109 lentiviral vector (Addgene plasmid #84740) 16 . After BsmbI digestion (FastDigest Esp3I, ThermoScientific) to linearize the U6 crRNA expression cassette, oligo cloning was performed to insert a lox66 sequence, a DR, two BsmbI sites, and an inverted lox71. The empty vector thus expresses LbCpf1 and puromycin resistance from an EFS promoter, while a U6 promoter drives expression of a lox66/lox71 flanked crRNA expression module containing two BsmbI sites. BsmbI digestion and oligo cloning was then used to insert FlipArrays into the empty vector. For a given pair of crRNAs, the following oligo overhangs were used for cloning: Following Qiagen PCR purification, 2 ng of the first PCR were used for the second inversion-specific or non-inverted-specific PCR. The following primers were used for detection of non-inverted or inverted FlipArrays: NPF_F: TCTTGTGGAAAGGACGAAACACCG NPF_R: TGCATACGCTATAGCTGCTTTTTTTTAAAAAATGGCA NPF_R_inv: TAAGCATAATGATGATGCCATTTTTTAAAAAAAAGCAG DVF_F: TCTTGTGGAAAGGACGAAACACCG DVF_R: GGGCTTTTTTAAAAAATAACAGACATGGACCATCAG DVF_R_inv: CTGATGGTCCATGTCTGTTATTTTTTAAAAAAGCCC PCR conditions: 98°C 2 min, 14 cycles of (98°C 1 s, 62°C 5 s, 72°C 2 s), 72°C 2 min, 4°C hold. PCR reactions specific to non-inverted and inverted FlipArrays were performed and analyzed simultaneously for each sample. Quantification was done on 2% E-gel using low-range quantitative ladder (ThermoFisher), and was normalized to the first PCR product. Full gel images are shown in Supplementary  Fig. 2a-c. Quantification of inverted FlipArray transcripts. KPD cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. For RNA extraction,~200,000 cells were isolated and spun down at 500 × g for 5 min. After a PBS wash, cells were resuspended in 450 µl TRIzol. 100 µl of chloroform was then added to each tube, followed by rigorous vortexing for 15 s and centrifuging at 12,000 × g for 10 min. The supernatant containing RNA was then purified using a Qiagen RNeasy Kit following the RNA cleanup protocol. cDNA was generated by reverse transcription with random hexamers. PCR detection of inverted crRNA FlipArray transcripts was done using the following primers: Inv_FlipArray_F: TGTAGATAGCGCTATAACTTCGTATAGC Inv_FlipArray_R: AAGCAGCTATAGCGTATGCAATC PCR conditions: 98°C 2 min, 34 cycles of (98°C 1 s, 56°C 5 s, 72°C 5 s), 72°C 2 min, 4°C hold.
Quantification of inverted FlipArray RNA abundance was done on 2% E-gel using low-range quantitative ladder (ThermoFisher), and was normalized to Cpf1 mRNA transcript abundance. The genomic DNA from~1000 cells was used for PCR with the NPF and DVF FlipArrays. For the TSG-Immune FlipArray library experiments, genomic DNA from~6000 cells were used to account for the pooled nature of the experiment. The resultant PCR products were used for Nextera library preparation following manufacturer protocols. Reads were mapped to the mm10 or hg38 genome using BWA-MEM 44 , with settings -t 8 -w 200. After identification of indel variants using the pileup2indel function in VarScan v2.3.9, a 1% variant frequency threshold was to identify high confidence variants for NPF and DVF experiments. A less stringent 0.2% variant frequency threshold was used for the TSG-Immune experiments due to their pooled nature. All variant calls are detailed in Supplementary Data. Sample size determination. No specific methods were used to predetermine sample size.
Blinding statement. Investigators were blinded for sequencing data analysis with generic sample IDs, but not blinded for PCR or RT-PCR.
Code availability. Custom scripts will be available to the academic community upon reasonable request Data availability. The FlipArray base vector has been submitted to Addgene (ID: 109349) and is available to the academic community. Cell lines, and additional data will be available to the academic community upon reasonable request. The genomic sequencing data sets generated during the current study are available in NCBI SRA under accession SRP136201.


extfieldsofstudy: Chemistry Medicine

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LC–ESI–IT-MS/MS and MALDI-TOF Approach: Identification of Natural Polymers from Rhizophora mangle Barks and Determination of Their Analgesic and Anti-inflammatory Properties

Abstract We recognize the chemical composition of the acetonic extract of Rhizophora mangle barks (AERM) using mass spectrometry analysis [liquid chromatography (LC)–ESI–IT-MS/MS and matrix-assisted laser desorption/ionization-time of flight-MS (MALDI-TOF)]. Analgesic activity was evaluated by formalin and tail-flick experimental assays. Anti-inflammatory activity was performed by paw edema test induced by carrageenan and 48/80 compounds. The first series of experiments involved [LC]–FIA–IT-MS/MS with 11 separated catechins derivatives until degree of polymerization 3 (DP3). The spectra obtained by MALDI-TOF analysis of the AERM presented two homologous series: one based on polymers of m/z 288 Da increments (up to DP12) and another series based on polymers of m/z [288 + 162] Da increments (up to DP11). In addition to these series of flavan-3-ol, each DP had a subset of masses with a variation of − 16 Da (homologous series of afzelechins—m/z 873–3465 Da) and + 16 Da (homologous series of gallocatechins—m/z 905–3497 Da). A similar pattern with homologous series of gallocatechins and afzelechins could also be observed for a fifth and a sixth monohexoside series: glucogallocatechins (m/z 779–3371) and glucoafzelechins (m/z 747–3339). The intraperitoneal administration of different doses of AERM (50, 150 and 300 µg mL−1) have a morphine-like effect and intense anti-inflammatory activity. Graphical Abstract

Introduction
Rhizophora mangle L. (Rhizophoraceae) is a pioneer plant that dwelleth the estuarine ecosystem. Mangrove plants are potential sources of biologically active compounds with wide application in ethnopharmacology practices. These species dwell in stressful environmental conditions (salinity, tidal fluctuations, and anoxic soil), and are adapted to this harsh ecosystem having many substances that protect them from these conditions [1].
In traditional medicine, R. mangle is commonly used to treat diarrhea, ulcers, inflammation, tumors, diabetes, and pain [1,2]. Pain is the major reason for seeking medical consultations and represents high medical and economic costs to the community [3]. Opioids are the most used resource to treat this condition, but the continuous use of this compound can generate dependence and side effects, such as gastrointestinal problems and respiratory depression. Therefore, the search for new analgesics, especially those of natural origin, is a need and a challenge for medicinal chemistry [4].
Inflammatory diseases, despite being described many years ago, have no fully effective treatment, there are many side reactions caused by anti-inflammatory drugs. Therefore, a significant commercial interest to discover new anti-inflammatory drugs with fewer side effects has been observed nowadays [5]. Corticosteroids and nonsteroidal anti-inflammatories remain the major therapeutic resources for inflammation control [6], but these drugs induce significant side effects, especially when used for a prolonged period, as overload kidneys and liver, osteoporosis and anaphylactic shock [7].
The polyphenolic compounds such as proanthocyanidins (PAs-tannins) consisting of oligomers and polymers by flavan-3-ol units the are most abundant in Rhizophora [8]. Due to its variable degree of polymerization (DP), the isolation and identification of the PAs require the use of complex steps of separation and purification. Additionally, PAs are highly unstable compounds with complex molecular structures, thermolabile and photosensitive. Therefore, mass spectrometry (MS) techniques based on matrix-assisted laser desorption/ionization-time of flight-MS (MALDI-TOF-MS) and liquid chromatography coupled to the tandem MS (LC-MS) has been attracting attention because of their ability to reliable analysis. In this context, the purpose of the present study was to characterize the chemical composition of the acetone extract of R. mangle bark's (AERM) and evaluate the analgesic and anti-inflammatory activity, which can be an excellent alternative for the development of new drugs.

Results and Discussion
Previous studies carried out with species of the genus Rhizophora and Rhizophoraceae family have demonstrated that these specimens are important sources of polyphenolic compounds (tannins) [2,8]. MS experiments were performed using LC-MS system equipped with an ESI source and an Ion Trap analyser was made to investigate the presence of different compounds with the same molecular weight and then to perform a qualitative analysis on the PAs constituents occurring in the AERM.
In order to recognize the main class of compounds, we performed LC-ESI-MS/MS analysis. The chromatographic separation was optimized to obtain an excellent separation. The LC-ESI-MS/MS obtained has satisfactory chromatographic resolution (Fig. 1), with 11 separate compounds and their structure proposed in Table 1. Compounds 1, 3, 7 and 8 were identified as catechin (CA) dimers based on their [M-H]precursor ion at m/ z 577, as well as after MS 2 fragmentation, which resulted in the products ions at m/z 425, m/z 407, m/z 272, m/z 298 and m/z 228, consistent with the data reported in the literature for CA dimmers [9]. Compound 2 (m/z 723, [M-H] -) was assigned as a CA dimer with one deoxyhexose moiety; MS 2 fragmentation of this precursor ion led to the products ions at m/z 577, m/z 571 and m/z 269 [10]. There are two isomers of CAs: (?)-CA, and (-)-epicatechin (EC), they are considered geometric isomers, where CA in a trans form and EC in a cis form [11]. Their structure consists of two benzene rings (A and B rings) connected through a pyran ring C. The subtle difference in molecular structure may result in drastic differences in pharmacological processes and therapeutic efficacy [12]. Besides we cannot confirm the presence of these isomers by LC-MS/MS analysis, we suggest, that AERM possess these compounds. We detected for the compounds 1, 3, 7, 8 and compounds 6, 9, 10 and 11 different retention times and same m/z, thus strongly suggesting the presence of isomers.
CAs are classified as flavan-3-ol monomers and have the capacity to polymerize, forming oligomeric compounds with high molecular weight [13]. The polymeric character is evidenced by the chromatogram; however, the characterization of condensed tannins was limited with ESI-IT-       MS/MS experiments. To solve this bottleneck, analysis using MALDI-TOF MS was performed to establish the DP to the PAs and its glucosylated forms. The spectra obtained by MALDI-TOF analyzes of the AERM presented two main homologous series: one with 288 Da increments and another one with 288 ? 162 Da increments, corroborating with those found using LC-ESI-IT-MS/MS technique. The series containing 288 Da increments were attributed to the polymeric CAs, according to formula 290 ? 288 (n -1) ? 23 (n = DP; ? 23 Da was attributed due to sodium adducts). Sodium adducts with up to DP12 (3481.673 Da) were found, even with the decrease in the intensity of the intercepted ions (Fig. 2a). In addition to this first series of flavan-3-ol, each DP had a subset of masses with a variation of -16 and ? 16 Da (Fig. 2a). These subsets can be explained by heteropolymers of repeating flavan-3-ol units containing an additional hydroxyl group (D ? 16 Da), characterizing a homologous series of gallocatechins (m/ z 905.145-3497 Da) (Fig. 2a). The other series consists in heteropolymers of repeating flavan-3-ol units without a hydroxyl group (D -16 Da), that is, a homologous series of afzelechins (m/z 873-3465 Da) (Fig. 2a).
MALDI-TOF MS experiments indicated the occurrence of a mixture of PAs and its monohexoside derivatives, which presented a variable DP (from 2 up to 12). Each precursor ion of the condensed tannins from AERM was always followed by mass signals at 162 Da, suggesting the presence of a series of monohexoside anthocyanidins, that can be explained by the formula 290 ? 288 (n -1) ? 162 ? 23 (Fig. 2b). Condensed tannins with a hexose group with DP2-11 (m/z 763.110-3355.615) were detected in AERM (Fig. 2b). Species with up to DP11 CatHex (m/z 763.110-3355.615 Da) could be observed (Fig. 2b).
The same pattern with homologous series of gallocatechins and afzelechins could also be observed for monohexoside series, thus evidencing that the extract also possesses a series of monohexoside gallocatechins (m/ z 779-3371) as well as a series of monohexoside afzelechins (m/z 747-3339). The peaks with the highest intensities in MALDI-TOF MS of the AERM were summarized in Table 2.
Rhizophora species are traditionally used for the treatment of pain [14], but no scientific support corroborates this action, especially for R. mangle. There are already works evidencing the positive therapeutic effect of R. mangle in the treatment of gastro intestinal diseases [15], which cause a high sensation of pain [16]. Banerjee et al. reported a significant reduction in pain severity and frequency of painful episodes, in patients who underwent alternative treatment with extracts rich in grape PAs, reported a significant reduction in the use of narcotic analgesics. The mechanism of action by which tannins act in the pain treatment is still poorly explained, but it is a reality [17]. In order to evaluate the effect of AERM in the treatment of pain, we performed experimental tests with formalin and tail-flick assays.
Treatment of animals with AERM at doses of 50, 150 and 300 lg kg -1 did not induce any sign of intercurrence in the animals. This test showed that the doses do not interfere in an animal's behavior. The formalin test was performed to contribute to the tail-flick, whilst the former is better known to evaluate pain threshold or reflex through acute cutaneous stimulation, formalin injection induces a state that better approximates to clinical conditions, therefore evaluating tonic pain. Thus, in the first phase of the test, formalin predominantly evokes activity in C fibers, substance P and bradykinin, whilst histamine, serotonin, prostaglandins (PGs), and bradykinin are involved in the second phase [18]. Therefore, the formalin test allows the evaluation of analgesic effect in peripheral inflammatory processes, that is evoked especially in the second phase of the test. The stimulus provided by subcutaneous injection of formalin-induced a behavioral response in animals treated with formic acid, in contrast to animals treated with morphine and AERM compounds at different concentrations, which had almost no behavioral response to formalin injection in both phases of the formalin test. Therefore, the formalin test showed that there is no difference between the  analgesic effect of morphine and bark compounds in the lowest concentration tested, even in a test that provides a long-lasting pain stimulus such as the formalin test. Despite the dose-response tendency presented, there were no differences between the extract's concentrations tested in relation to the biological activity ( Fig. 3; Table 3).
Tests with more phasic stimuli, such as the tail-flick tests, provide an evaluation of antinociceptive effect in acute pain state. In this scenario, the response to thermal stimuli was evaluated using the tail-flick test. As it was shown in Fig. 4 and Table 4, both the effect of morphine as well as the bark compounds in the lowest concentration, were substantially greater than in control (saline-treated animals). This result indicates that the AERM compounds had a spinal effect similar to morphine, though the exact mechanism is still unknown. The reason behind this morphine-like effect presented by the AERM may be due to the action of CAs, as it was shown previously that these substances act like blockers for sodium channels in neuronal cells [19]. Hence, this morphine-like effect observed for AERM has a significant clinical importance, as it could be used as a potential substitute for the morphine. Considering all the morphine's adverse effects (sedation, nausea, and pruritus) [15], the use of the AERM could improve the treatment of pain, especially chronically.
Pain may originate through an inflammatory process, several mediators such as cytokines, bradykinins and PGs are responsible for pain [20]. Our research group evidenced the anti-inflammatory activity of the ethyl acetate and butanol fractions of the AERM [21]. A scientific basis underpinned by the compounds of R. mangle may be helpful to explain its antinociceptive and anti-inflammatory effects. Pharmacological test of paw edema induced by carrageenan showed an anti-inflammatory potential, since there was the statistical difference (two-way analysis of variance, ANOVA with Bonferroni post test, F = 61.46, p \ 0.01, Df = 5) (Fig. 5). Also, paw edema induced by compound 48/80 also demonstrate a large inhibition when AERM was injected (two-way ANOVA with Bonferroni post test, F = 43.00, p \ 0.01, Df = 5) (Fig. 6).
The acute inflammatory response is characterized by an increase in vascular permeability and cellular infiltration leading to edema formation, because of extravasation fluid and proteins and accumulation of leukocytes at the inflammatory site [22]. Carrageenan-induced paw edema is a widely-used test to determine the anti-inflammatory activity and induced cyclo-oxygenase-2 (COX-2) maximal expression 1 h from carrageenan local injection. However, compound 48/80, like other basic secretagogues, causes mast cell degranulation via direct activation of Gi proteins (sensitive to Pertussis toxin), releasing oxygen reactive species (ROS). Degranulation of mast cells results in the release of stored autacoids, such as histamine, as well as the de novo synthesis of arachidonic acid (AA), cytokines and chemokines. Upon mast cell degranulation, AA is released and metabolized through COX or 5-lipoxygenase (5-LO) pathways to yield, respectively, PGs and thromboxane, or leukotrienes [23,24]. So, phenolic compounds as found in R. mangle could be actin by controlling the mast degranulation as ROS scavenger and might inhibit specifically enzymes from the inflammatory process as COX-2 [25].   Furthermore, Pinus pinaster is also rich in polyphenolic compounds, such as afzelechins and galocatechins, same as found in R. mangle, and has been shown to have similar analgesic and anti-inflammatory effects as R. mangle [26]. Pinus pinaster cause inhibition of cyclo-oxygenase (COX-1 and COX-2) activity in serum samples of human volunteers. COXs enzymes are the major targets for the treatment of inflammation, fever and pain, due to its function, that is synthesis of mediators, such as PGs, prostacyclin, and thromboxane. Which suggests, that the COX blockage may also play a role in the analgesic and anti-inflammatory effect of R. mangle due to polyphenolic compounds activity.
Several anti-inflammatory drugs on market are known, however, they reveal side effects such as liver and kidney commitment [27]. Other species by genus Rhizophora have anti-inflammatory activity [28]. In R. mangle, inhibition of PLA2 and COX-2, enzymes related to inflammation process, demonstrated by in vitro tests [14], which reiterates the results revealed in this study. Kanagaratnam et al. [29] reported that CAs may act as an anti-inflammatory because they reveal antibacterial activity; which probably supports the results found in this work. Another mechanism capable of generating pain can originate in response to tissue damage or physiological stress, including oxidative stress [30]. The condensed tannins present in R. mangle have excellent free radical sequestration [21], thus, the antioxidant capacity of this extract can aid in the maintenance of pain and inflammation.

Conclusions
This research showed the potential of UPLC coupled to ion trap tandem MS and MALDI-TOF analysis for the rapid and sensitive detection of natural phenolic polymers in the acetone extract from the barks of R. mangle. This technique allowed detecting the presence of a series of anthocyanidins in less than 5 min of analysis. MALDI-TOF MS allowed characterizing all the polymeric substances present in the extract, revealing the presence of a series of condensed tannins based on CAs, gallocatechins and afzelechins. Besides, these series were accompanied by their respective glucosilated derivatives. The identification of these compounds has provided relevant data for fully characterize the secondary metabolites present in these complex vegetal extracts. The chemical characterization of the extract also helped to understand the results observed in the formalin, tail-flick and paw edema assays using experimental models, which demonstrated the antinociceptive and anti-inflammatory effects of the AERM.

Preparation of Plant Extract (AERM)
Fresh barks of R. mangle were washed, shade dried, powdered in a knife mill and sieved through a #60 mesh sieve. The powder (50 g) was extracted with 0.5 L acetone:water (70% v/v) and macerated for 7 days at room temperature (24°C), protected from light. The macerate was filtered through Whatman No. 1 filter paper and concentrated in a rotating flash evaporator at a temperature not exceeding 35°C. The extract was lyophilized and stored in amber glass flasks, stored in a freezer (-40°C) and resuspended with distilled water for use in pharmacological experiments. The percent yield of the lyophilized powder was 7% of the dry plant material.

LC-ESI-MS/MS Analysis
MS analyses were carried out in a Thermo Finnigan (San Jose, CA, USA) LCQ mass spectrometer equipped with an electro-spray ionization source, ion-trap analyzer and Xcalibur software for data processing. For LC-MS analysis: column Phenomenex, 300 lL min -1 , followed by chromatographic using MeOH and 0.002% formic acid in water was added using a linear gradient from 0 to 100% MeOH over 5 min. The mass spectrometer was operated in the negative ion mode under the following conditions: flow 5 lL min -1 ; the capillary temperature 270°C; 80 arbitrary units of nitrogen; gas assist with 5 arbitrary units. All solvents used in these analyzes have a mass grade, purchased from Sigma.

MALDI-TOF
For the analysis using MALDI-TOF, 1.0 mg of the powdered vegetable material was diluted in 500 lL of MeOH containing 2,5-dihydroxybenzoic acid (DHB -125 nm) and sodium iodide (1 nM) with the addition of 1% trifluoroacetic acid (v/v), purchased from Sigma. This solution was placed for 1 min by vortexing. After this, 1 lL of the solution suspension has been deposited on MALDI plates, made of stainless steel and allocated to air dry prior to analysis. The analysis of MALDI-MS was carried out a Voyager DE-RP coupled to the spectrometer flight time mass (TOF), equipped with a nitrogen laser (k = 337 nm, pulse width 3rd). The intensity of the laser beam was experimentally attenuated slightly above the threshold level for forming ions from the tannin components. The ions generated by laser desorption were introduced in the flight tube with a 20 kV accelerating voltage in positive linear mode. The delay time is 100 ns. All mass spectra were acquired by averaging 100 individual laser shots [31]. The animals were kept in standard environmental conditions: temperature (23 ± 2°C), light control (12 h light cycle) and provide water and food ad libitum (standard rodent diet), except during the experimental period. All animals were acclimated for a period of 5 days before the start of the experimental assays.

Tail-Flick Assay
Tail-flick test was evaluated of central analgesic activity [35]. The latency period is used as a determination index of nociception. Data collected from mice with latency lower than 2 s were not used, and 6 s was the maximum latency tolerance. Animals were divided into five groups (n = 6), Control (saline solution), Morphine (50 lg kg -1 , i.p.) and AERM (50, 150 and 300 lg kg -1 , i.p.), an analgesymeter (Insight EFF 300L), with the tail docked in apparatus with the light thermal source was used. The latency of tail withdrawal response was determined 0, 30, 60, 120 e 180 min after the AERM administration, inject peritoneally.

Statistical Analysis
All data were expressed as a mean ± standard error of the mean. Statistical analyses were performed by using the Statistical Package for Social Science version 15.0 (SPSS) and GraphPad Prism 5.0. The statistical significance was determined using a two-way ANOVA, followed by Bonferroni post hoc test, a level of 5% (p \ 0.05) was considered to be statistically significant.


extfieldsofstudy: Biology Medicine

text:
16S rRNA Gene Sequencing for Deciphering the Colorectal Cancer Gut Microbiome: Current Protocols and Workflows

The human gut holds the densest microbiome ecosystem essential in maintaining a healthy host physiology, whereby disruption of this ecosystem has been linked to the development of colorectal cancer (CRC). The advent of next-generation sequencing technologies such as the 16S rRNA gene sequencing has enabled characterization of the CRC gut microbiome architecture in an affordable and culture-free approach. Nevertheless, the lack of standardization in handling and storage of biospecimens, nucleic acid extraction, 16S rRNA gene primer selection, length, and depth of sequencing and bioinformatics analyses have contributed to discrepancies found in various published studies of this field. Accurate characterization of the CRC microbiome found in different stages of CRC has the potential to be developed into a screening tool in the clinical setting. This mini review aims to concisely compile all available CRC microbiome studies performed till end of 2016 and to suggest standardized protocols that are crucial in developing a gut microbiome screening panel for CRC.

INTRODUCTION
The human gut harbors an enormous, diverse, and dynamic microbiome, consisting primarily of bacteria and archaea, as well as fungi, protozoa and viruses. There are at least 100 trillion (10 14 ) microbial cells in the human gut, almost outnumbering the eukaryotic cells that reside together (Whitman et al., 1998;Costello et al., 2012;Sender et al., 2016). The gut microbiome is known to play a vital role in health, contributing toward the host's energy harvest and storage via various metabolic functions (Gill et al., 2006). In good health, our gut microbiota is mainly subdivided into two categories; commensal symbionts and commensal pathobionts. Commensal bacteria has been acknowledged to be important for host physiology through provision of essential nutrients and providing protection against colonization by opportunistic pathogens (Hooper and Gordon, 2001).
The gut microbiota has been typically controlled by environmental factors such as adoption of westernized diet and lifestyle (David et al., 2014). Incidentally, there is growing evidence to suggest that environmental factors such as obesity and diet are associated with the pathogenesis of colorectal cancer (CRC). As the mechanism of sporadic CRC is still poorly understood, an individual's gut microbiome landscape may reflect his or her dietary patterns which can either promote or protect against CRC (Bultman, 2017).
Bacterial composition in the gut helps to maintain its host's mucosal and systemic immunity homeostasis, avoiding any immunity trigger that might lead to physiological impairment. A shift of gut commensal microbiota toward opportunistic pathogens is a condition designated as dysbiosis (Barman et al., 2008). A few studies suggested dysbiosis as the scenario that will impact numerous physiological functions and that this will serve as a primary driver for inflammation in the colon leading to increased risk for CRC (Nistal et al., 2015). Many recent studies have started to disclose that the gut microbiome plays a role in oncogenesis, where their interaction with the immune system might either maintain a healthy host or drive tumor progression (Gagliani et al., 2014).
Unlike gastric cancer which is solely associated with Helicobacter pylori infection, metagenomics studies showed that fecal and mucosal samples of CRC patients and non-CRC individuals are enriched in different microbiome composition (Uemura et al., 2001;Thomas et al., 2016;Flemer et al., 2017). Most studies reported Bacteroides, Fusobacterium, and Peptostreptococcus as the more prominent genera in CRC samples compared to controls (Table 1). CRC patients also showed an increase in the abundance of Gemella, Parvimonas, and Porphorymonas (Chen et al., 2012;Allali et al., 2015;Sinha et al., 2016).
In conjunction with the acknowledgement of gut microbiome contribution toward health and disease, developments in next-generation sequencing provided many breakthroughs for taxonomic, phylogenetic or functional profiling of the gut microbiome. A metagenomics approach toward gut microbiome profiling will confer the added advantage of not only metagenome community characterization, but also provides answers on its physiological impact to the human host. Furthermore, via metagenomics sequencing, measurements of bacterial taxa abundance within a sample as well as identification of dysbiosis events within its tumor microenvironment could be carried out. Indeed, the Metagenomics of Human Intestinal Tract (MetaHIT) project was initiated to study associations between genes of the gut microbiome with health and disease (Qin et al., 2010).
Nevertheless, despite similar approaches being used in several gut microbiome studies on CRC patients, dissimilarities in results were still apparent ( Table 1). These dissimilarities may either be caused by differences in the patients' dietary customs specific to a certain geographical location (Figure 1) or caused by the technical aspects of next-generation sequencing experiments due to variations in sample handling and processing, and bioinformatics analysis pipelines. Lack of standardization in human microbiome studies may cause repetitive discrepancies if no baseline protocol is available. To this end, recently, The Microbiome Quality Control (MBQC) project (http://www. mbqc.org/)has been initiated to expand and encourage open sharing of standard operating procedures and best practices in the metagenomics field (Sinha et al., 2015).
To elucidate the gut microbiome landscape in tumor microenvironments, specific study design and experiment protocols are required for metagenomics sequencing and analyses. In this review, we discuss the culture-independent application of 16S rRNA gene sequencing in cataloguing the CRC gut microbiome. This review will also cover the assessment of 16S rRNA gene sequencing of the CRC gut microbiome, covering steps from nucleic acid extraction, sample preparation, selection of hypervariable regions, sequencing platforms, and the determination of algorithms for bioinformatics analyses, which we believe will provide an insight into CRC gut microbiome characterization via the next-generation sequencing approach.

HIGH-THROUGHPUT MICROBIOME SEQUENCING
Advances in high-throughput sequencing technologies have enabled researchers to explore microbiome complexity associated with the human body and diseases. In the past decade, application of high-throughput DNA sequencing to profile the genomic composition of a microbial community in a culture-independent manner has expanded immensely. This high resolution molecular sequencing technique, designated as metagenomics, can be further sub-divided into two different approaches, namely 16S rRNA gene and shotgun metagenomics sequencing. The 16S rRNA gene sequencing approach relies on sequencing of the 16S ribosomal RNA (rRNA) gene as the genetic marker to study bacterial phylogeny and taxonomy. This genetic marker contains conserved hypervariable regions which can be used for bacteria identification. Selection of hypervariable regions for sequencing as well as amplicon primer design are important for 16S rRNA gene sequencing as these factors might contribute to differences in the results.
On the other hand, whole genome or shotgun metagenomics sequencing, an alternative to 16S rRNA gene sequencing, refers to massive parallel sequencing of DNA samples. This technique also identifies gene functions of the sequenced microbiome. Shotgun metagenomics sequencing involves random fragmentation of DNA, sequencing of these fragments, followed by reconstruction and assembly of overlapping sequences into a continuous sequence (Fraher et al., 2012). Researchers have used shotgun metagenomics sequencing to discover interactions between microbiota and its host. The disadvantages of this technique are that it is costly and it produces a huge amount of data that requires advanced bioinformatics analyses.

16S rRNA GENE SEQUENCING AND ITS IMPACT ON HUMAN GUT MICROBIOME
Many studies have employed 16S rRNA gene sequencing to profile the gut microbiota composition (Gill et al., 2006;Huse et al., 2012;Yatsunenko et al., 2012). The 16S small subunit ribosomal gene is an exclusive housekeeping gene in prokaryotes which can be used to determine microbial communities within samples; it is highly-conserved and contains hypervariable regions ranging from region V1 to V9. Sequencing of the 16S rRNA gene requires amplification of a selected variable region via PCR using a variety of "universal" primers followed by sequencing. The V4 region of 16S rRNA gene has been highly recommended as the gold standard for profiling of  (Qin et al., 2010;Lozupone et al., 2013). Besides selection of a suitable hypervariable region, compatibility of amplification fragment lengths with the read length capacity of the intended sequencing platform has to be confirmed. The number of microbiome studies has increased enormously in concert with technological developments of DNA sequencing that facilitate culture-and cloning-free analyses. When it was first released in 2005, the first next-generation sequencer (Roche 454 pyrosequencer), can only sequence ∼120 bases of bacterial genome in a single run (Margulies et al., 2005). In recent years, this technology has enabled the coverage of up to 1000 bp and can span multiple hypervariable regions of the 16S rRNA gene. Due to its high resolution and cost-effective approach, 16S rRNA gene sequencing has become the commonest approach for microbial community profiling of the human gut.

16S rRNA GENE SEQUENCING AS A MOLECULAR SCREENING TOOL FOR CRC
Guaiac fecal occult blood test (gFOBT) and the immunochemical-based fecal occult blood test (iFOBT/FIT) are current screening tools for CRC via detection of gastrointestinal bleeding. Nevertheless, these tests are not specific for CRC as pathologies such as ulcerative colitis and polyps could also cause bleeding in the gut. Gut microbiome profiling studies based on 16S rRNA gene sequencing has identified bacterial genus frequently associated with CRC, including Fusobacterium, Bacteroides, and Peptostreptococcus (Nakatsu et al., 2015;Baxter et al., 2016;Flemer et al., 2017; Table 1). In particular, the role of F. nucleatum in CRC tumorigenesis and metastasis has been consistently reported in animal models and cell culture experiments (Kostic et al., 2013;Rubinstein et al., 2013). Recent, additional studies confirmed the importance of F. nucleatum in CRC, where the bacteria was shown to modulate tumorigenesis in the colon via miRNA-21 expression, which subsequently suppresses the immune response and activate oncogenic pathways (Nosho et al., 2016;Yang et al., 2017). Zackular et al's study demonstrated the feasibility of using microbial biomarkers such as F. nucleatum for CRC screening (Zackular et al., 2014). In addition, Liang et al established a species-level microbiome panel that could distinguish between CRC patients and healthy individuals of the Hong Kong population with greater accuracy and sensitivity than the current screening kit (Liang et al., 2017). Recently, it has been suggested that FOBT coupled with 16S rRNA gene sequencing will serve as a better screening approach for CRC, where stool-containing buffer samples from the FOBT kits could be used for sequencing (Liang et al., 2017;Taylor et al., 2017). To this end, standardized workflows for 16S rRNA gene sequencing will be crucial to produce results which are accurate and reproducible.  (Kostic et al., 2012) 12 (Sobhani et al., 2011) 13 (Gao et al., 2015) 14 (Geng et al., 2013) 15 16 (Wang et al., 2012) 17 (Nakatsu et al., 2015) 18 (Zeller et al., 2014).

SAMPLE COLLECTION AND STORAGE
An important but seldom emphasized aspect of 16S rRNA gene sequencing studies is sample integrity. For gut microbiome profiling studies, biopsy, surgical tissues and stool samples are the common biospecimens collected for characterization. Among these, stool samples were the first to be used for 16S rRNA gene sequencing to study the CRC microbiome. Subsequent studies used swabs, surgical or biopsy tissues as the starting material with different sample handling procedures. Minimization of sample contamination ensures that each biospecimen retains as much as possible of its original microbiome and contributes toward accurate results in gut microbiome profiling. Nevertheless, studies comparing methods of biospecimen handling remain few. Many published studies did not comprehensively disclose methods used for sample handling. Sobhani et al published the first 16S rRNA gene sequencing study on CRC microbiome in 2011. Their protocol used stool samples; samples were placed in a sterile carrier box, transported to the laboratory within 4 h and later stored at −20 • C prior to DNA extraction. Subsequent studies published improvements in handling the biosamples and transportation protocols, including steps such as transportation of samples on ice (Wu et al., 2010) and storage at −80 • C to preserve sample integrity (Consortium, 2012).
Stool samples require non-invasive techniques for collection and they are sufficient for researchers to obtain an overview of the gut microbiome spectrum in our colon. These samples could be studied using leftover stool-containing buffer from used FOBT and FIT cartridges (Baxter et al., 2016;Taylor et al., 2017). However, stool samples might not be able to provide a clear picture for studies exploring site-specific microbiome which would require a tissue biopsy. On the other hand, despite the invasiveness of tissue biopsy, these samples would enable indepth studies into tumor-specific microbiota in comparison to stool sampling.
Short and long-term storage conditions post-transportation of samples is important to minimize differences in the microbiome spectrum caused by storage conditions. Sample transportation and storage conditions for fecal samples used in CRC gut microbiome characterization have been studied. Low temperature transportation, such as on ice or dry ice, ethanol-stored and long-term storage in −80 • C are strongly recommended to maximize microbiome recovery within the sample prior nucleic acid extraction (Choo et al., 2015;Fouhy et al., 2015;Gorzelak et al., 2015;Blekhman et al., 2016). This also applies to biopsy and surgical tissue samples. Tissue handling protocols such snap-freezing cryovials in liquid nitrogen or usage of RNAlater medium will ensure optimum conditions for sample storage prior to nucleic acid extraction, leading to better microbiome recovery.
From the available literature, seven studies on CRC profiled the fecal microbiome, while others used colon tissue samples for their analyses (Table 1). Some studies investigated both fecal and tissue samples from the same individual to differentiate the microbiome compositions ( Table 1). The authors from these studies applied various approaches for sample handling and storage of the biospecimens. For studies using fecal samples, samples were all transported fresh and on ice to the research laboratory in <24 h; all samples except the ones used in a particular study were stored in −80 • C. For the exception, DNA was directly extracted from samples and stored at −20 • C until library preparation (Weir et al., 2013). On the other hand, for studies using tissue samples as the starting material, samples were usually snap-frozen in liquid nitrogen. Flemer et al. and Dejea et al. chose to use RNAlater as a preservative medium to ensure maximum recovery of nucleic acids; while the studies by Burns et al. and Gao et al. performed nucleic acid extraction right after sample collection (Table 2). Overall, perhaps due to the fact that 16S rRNA gene sequencing studies are still new, long-term effects of sampling and storage conditions for gut microbiome specimens have not been described.

METHODS IN NUCLEIC ACID EXTRACTION FOR PERCEIVING THE GUT MICROBIOME COMPOSITION
Nucleic acid extraction of samples is a simple but critical step in microbiome studies. In recent years, a few debates have risen about the best isolation protocol which will give the most accurate representation of the microbial spectrum. Most isolation protocols comprise of three basic steps that include cellular lysis, non-DNA macromolecule elimination together with DNA detachment and collection. In general, cell lysis protocols have received the most scrutiny, as complete cell disruption achieved from either enzymatic and/or mechanical processes will enable subsequent comprehensive DNA isolation, and vice versa. Gram positive organisms require stronger lysis conditions due to their thicker cell walls, unlike gram negative organisms which require only gentle lysis (Brown et al., 2015). Several studies have been carried out with modifications of the nucleic acid extraction protocols compared with earlier published studies. These modifications include incorporation of additional procedures such as mechanical homogenization of cells with glass or silica beads, enzymatic lysis reaction with lysozyme, or a combination of both mechanical and enzymatic reactions ( Table 2). Once cellular lysis has been accomplished, DNA clean-up, concentration, and elution were routinely carried out.
On the other hand, there were studies that achieved success in microbiome profiling without modifications toward standard lysis protocols (Table 2). Nevertheless, it should be noted that no isolation protocol works equally well on different sample types or produces completely unbiased results. The prevention of sample contamination during nucleic acid extraction is also vital to eliminate DNA from non-indigenous microbes. Proper sample handling such as working in clean laboratory environments and using commercially available DNA/RNA-free nucleic acid extraction reagents will decrease the risk of contamination. The operator should also don proper attire, gloves and face mask to protect samples from contamination with their own microbiota.

SELECTION OF UNIVERSAL 16S rRNA GENE PRIMERS, SEQUENCING TECHNOLOGIES AND DATABASES
As described in an earlier section of this manuscript, the 16S rRNA gene consists of nine hypervariable conserved regions (V1 to V9) separated by ten highly conserved regions (Cox et al., 2013). The first 16S rRNA gene sequencing of CRC gut microbiome was completed 6 years ago using stool samples from 6 CRC patients (Sobhani et al., 2011). The study targeted the 16S rRNA V3/V4 regions, while its sequencing analyses relied on the database from the Ribosomal Database Project (RDP) using the RDP classifier (Cole et al., 2005). Similar studies targeting the same hypervariable regions were carried out a few years later using larger sample sizes and different biospecimens such as endoscopic biopsies and surgical tissues (Ahn et al., 2013;Flemer et al., 2017). Only very few studies were carried out to compare sequencing results obtained using primers targeting different hypervariable regions; most studies were done via sequencing of either one of the V3 or V4 regions, otherwise, a combination of two or more 16S rRNA gene hypervariable regions, whereby the most commonly used were the V3/V4 regions. Nevertheless, one study showed that while choice of primers had considerable effect, usage of matched primers on different sequencing platforms yielded little difference in results. (Tremblay et al., 2015). Differences obtained from the results of the various studies are conceivably due to factors such as selection of 16S rRNA gene primers and number of sequencing reads produced, as well as differences in classification techniques and bioinformatics analysis parameters. There is currently still no consensus on the best approach (Caporaso et al., 2011;Mizrahi-Man et al., 2013;Zheng et al., 2015).
Earlier gut microbiome studies in CRC were performed using the Roche 454 pyrosequencing technology (Table 2). Recently, the use of the Illumina MiSeq sequencer with paired-end reads and enhanced sequencing chemistry for microbiome studies has also increased (Caporaso et al., 2012). A study has been carried out to compare sequencing results produced between benchtop sequencers commonly used for 16S rRNA gene sequencing studies, including the Illumina MiSeq, Ion torrent Personal Genome Machine (PGM) and 454 GS Junior. Illumina MiSeq was found to generate data of the highest quality with almost no indel error compared to the other platforms (Loman et al., 2012).
Another important parameter in microbiome research is bioinformatics analysis. Most CRC microbiome studies rely on either the Greengenes or RDP database ( Table 1; DeSantis et al., 2006;Cole et al., 2009). To the best of our knowledge, only RDP and SILVA are frequently updated; the Greengenes database has not been updated since 2013. On the other hand, the RefSeq Targeted Loci has been proposed to be the "gold standard" by the National Centre for Biotechnology Information (NCBI) for 16S rRNA gene sequencing data analysis (Tatusova et al., 2015). The NCBI database is also recommended for both 16S rRNA gene and shotgun metagenomics sequencing studies (Balvočiute and Huson, 2017). In addition, the SILVA database which covers phylogenies for small subunit rRNAs (16S for prokaryotes and 18S for eukaryotes) is also a resource for-aligning and/or quality-checking of rRNA sequence data (Pruesse et al., 2007;Quast et al., 2013). Recently, the EzBioCloud Genome database (previously known as EzTaxon) has been officially released. This database is well-curated; however, as it was recently launched, the database has yet been adapted in any 16S rRNA gene sequencing studies (Yoon et al., 2017). Despite the availability of numerous databases for 16S rRNA gene sequencing data analysis, a single, standardized database for this purpose is still unavailable. The selection of appropriate computational tools for 16S rRNA gene sequencing dataset analysis is also crucial. Three commonly used bioinformatics pipelines were evaluated to determine the most precise tool available for unraveling the microbiome landscape via 16S rRNA gene sequencing (Plummer et al., 2015). Nevertheless, the comparison of findings from currently available studies showed that different results were obtained when analyses were run using different software or different databases. For all CRC gut microbiome studies carried out so far, most datasets were analyzed using Quantitative Insight into Microbial Ecology (QIIME) and MOTHUR (Schloss et al., 2009;Caporaso et al., 2010). Both tools were found to be precise for 16S rRNA gene sequencing dataset analysis.

CONCLUDING REMARKS AND FUTURE PERSPECTIVES
Initiatives on method standardization to study the human microbiome has been proposed by many research organizations such as the International Human Microbiome Standard (IHMS), MBQC and the well-known HMP project which acts as a baseline reference. In this mini review, we compiled and concisely compared all CRC 16S rRNA gene sequencing studies that have been carried out until 2016. As far as we know, no study is completely similar to another in terms of sample type and laboratory transfer, sequencing platform and primers, and bioinformatics database and analysis; therefore, the reproducibility of results obtained from a specific workflow could not be determined. Nevertheless, from our compilation, we found that most studies used the following workflow for CRC 16S rRNA gene sequencing: DNA extraction with mechanical homogenization, sequencing of the 16S rRNA V3/V4 regions, OTU picking using either the QIIME or MOTHUR software, and microbial classification against the Greengenes or RDP database. This commonly-used workflow was found to provide good quality sequencing reads and a comprehensive profile of CRCassociated gut microbiome. The reproducibility of 16S rRNA gene sequencing results from a specific workflow could only be tested via replicate experiments using identical workflow on the same DNA sample; this research approach could be further explored in future studies to build a sensitive, specific and noninvasive CRC molecular screening tool based on 16S rRNA gene sequencing.

AUTHOR CONTRIBUTIONS
M-AO and HN drafted and wrote this manuscript. HN, N-SA, S-FC, and RJ were responsible for idea conception, critical evaluation, and manuscript review.


extfieldsofstudy: Medicine Chemistry

s2fieldsofstudy: Biology Chemistry

text:
Colloidal hydroxyethyl starch for tumor-targeted platinum delivery

Cis-platinum has been widely used as a first-line chemotherapy agent in clinics for more than 40 years. Although considerable efforts have been expended for developing platinum-based nano drug delivery systems (NDDS) to resolve the problems of low water solubility, short half-life, and severe side effects of cis-platinum, it remains challenging to apply these nanoplatforms to cancer treatments in clinics on account of the issues related to safety, complex fabrication procedures, and limited cellular uptake. Herein, we constructed a novel cis-platinum delivery system with hydroxyethyl starch (HES), which is a semisynthetic polysaccharide that has been used worldwide as colloidal plasma volume expanders (PVE) in clinics for several decades. By combining TEM, AFM, and DLS, we have found that HES particles are colloidal nanoparticles in solution, with diameters ranging from 15 to 40 nm as a function of molecular weight. We further revealed that HES adopted a hyperbranched colloidal structure with rather compact conformation. These results demonstrate that HES is a promising nanocarrier to deliver drug molecules. Taking advantage of the poly-hydroxyl sites of HES, we constructed a novel HES-based cis-platinum delivery nanoplatform. HES was directly conjugated with cis-platinum prodrug via an ester bond and decorated with an active targeting molecule, lactobionic acid (LA), contributing toward higher in vitro antitumor activity against hepatoma carcinoma cells as compared to cis-platinum. These results have significant implications for the clinically used plasma volume expander-HES and shed light on the clinical translation of HES-based nano drug delivery systems.

Introduction
Cis-platinum is a famous anticancer chemotherapeutic agent found by Barnett Rosenberg and his colleagues in 1969. 1 Since it was rst approved by the FDA in 1978, cis-platinum has been widely used in clinics to treat many kinds of cancers (e.g., breast, liver, ovarian, head and neck cancers). 2,3 Aer its cellular uptake either by passive diffusion or by copper transporters, cisplatinum attacks DNA, blocks replication, and induces cell apoptosis. 4 Owing to this unique antitumor mechanism, cisplatinum-one of the most common chemotherapeutic agents-has excellent anticancer efficacy and non-cross drug resistance. However, the water solubility of cis-platinum is not appreciable, which hinders its cellular uptake and antitumor activity. It has also been reported that almost 65-98% of cisplatinum is preferentially combined with proteins in the plasm aer intravenous administration. 5 This protein combination process can further hinder cis-platinum from entering into cancer cells and reduce antitumor efficiency. This nonspecic combination can also affect the function of proteins and induce some serious systemic toxicity, such as nephrotoxicity, myelosuppression, and neurotoxicity. 6 Therefore, developing platinum-based drugs with improved cellular uptake, higher antitumor efficacy, and reduced side effects is still a pressing unmet need. [7][8][9][10][11] With the development of nanotechnology, a series of nano drug delivery systems (NDDS) have been developed for cancer chemotherapy to simultaneously prolong drug circulation, enhance tumor accumulation, increase specic cancer cell uptake, improve antitumor efficiency, and reduce side effects. 12 Nanoparticles with good biocompatibility can stealthily circulate in the blood and selectively accumulate at the tumor site by the enhanced permeation and retention (EPR) effect or active tumor targeting with specic ligand modifying the surface of the nanoparticles. 13 A specic ligand can improve the receptor-mediated endocytosis by targeting receptors overexpressed by cancer cells. 14 The loaded drugs are encapsulated or conjugated within the nanoparticles to achieve superior water solubility and sustained or stimulated drug release. To take advantage of these properties of nanoparticles, several NDDS have been designed to deliver cis-platinum, such as micelles, [15][16][17] dendrimers, 18,19 nanoscale coordination polymers, 20,21 nanogels, 22 and inorganic nanoparticles. 23,24 However, it remains challenging to apply these nanoplatforms to clinical cancer treatment on account of the following reasons. First, there are certain safety concerns about most nanoplatforms that are undegradable. The accumulation of these materials in the human body may induce toxicity in normal tissues. 25,26 Second, the fabrication process requires complex procedures, tedious chemical purications, and/or harsh conditions that can result in reduced efficacy or increased costs. Third, these stealth nanoparticles capable of long circulation are still hindered by compromised cellular uptake, resulting in limited antitumor efficacy. Therefore, constructing a nanoplatform with biodegradable materials and tumor-specic ligands for cis-platinum delivery by simple and robust procedures can facilitate the clinical translation of cis-platinum-loaded NDDS.
Hydroxyethyl starch (HES) is a semisynthetic polysaccharide and used as one of the rst-line colloidal plasma volume expanders (PVE) in clinics for decades, showing commendable physiochemical properties, biocompatibility, and biodegradability. As an FDA-approved biocompatible polymer, HES has also been used widely in drug delivery systems to improve drug solubility and stability, extend half-life time, and achieve passive cancer targeting. Taking advantage of the abundant hydroxyl groups on the polysaccharide, HES can conjugate with drug molecules, specically targeting ligands or hydrophobic chains. To improve the water solubility and antitumor efficacy, HES has been successfully conjugated with drug molecules, such as doxorubicin, 27,28 5-Fu, 29 10-CPT, 30,31 paclitaxel, 32 docetaxel, 33 and methotrexate 34 for cancer chemotherapy. Hence, employing HES to conjugate with cis-platinum could be a promising way to potentiate the anticancer efficacy of cisplatinum. Most importantly, developing a cis-platinum delivery platform based on HES has promising clinical application prospects because the utilization of HES has been approved by numerous clinical practices.
Herein, we constructed a novel cis-platinum delivery system with clinically used HES. First, to conjugate with HES, cisplatinum was oxidized into Pt(IV) and functionalized with a carboxyl group, as previously reported. 35 Then, by two simple esterication reactions, the cis-platinum prodrug, Pt-COOH(IV), and the active targeting molecule, lactobionic acid (LA), were linked with HES via ester bonds. Finally, aer this simple fabrication process, LA-HES-Pt was obtained. The water solubility of cis-platinum can be signicantly improved aer conjugation with HES. According to our previous reports, HES is a nanoparticle that can passively target a tumor site by the EPR effect. Moreover, LA can actively target hepatoma cells with a high-level expression of ASGPR, promoting cellular endocytosis. 14 Aer entering into cancer cells, conjugated Pt(IV) is reduced to Pt(II) because of GSH, ascorbic acid, thioalcohol, and low pH. Hence, cis-platinum can be released, leading to DNAinduced apoptosis. 15 This simple fabrication process and FDAapproved raw materials (cis-platinum, HES, and LA) endow LA-HES-Pt with high druggability and signicant clinical translation potential. To the best of our knowledge, this is the rst study in which HES has been used to deliver cis-platinum in cancer chemotherapy.

TEM characterization
HES samples (200 mg mL À1 ) were dripped onto the copper mesh, dried in air, and stained with 0.2% phosphotungstic acid for 30 s and characterized with TEM at 80 KV acceleration voltage. HES-Pt and LA-HES-Pt samples were dripped on the copper mesh, dried in air, and directly observed with TEM.

AFM characterization
HES samples were dissolved in deionized water and diluted to 20 mg mL À1 . The diluted solutions were dripped with 10 mL on fresh mica, dried in air, and characterized by AFM in the Sca-nAsyst mode.

GPC characterization
Different HES samples dissolved in an acetate buffer with a concentration of 10 mg mL À1 were characterized by GPC. The mobile phase was acetic acid/sodium acetate buffer and the ow rate was 0.5 mL min À1 . The column temperature was controlled at 35 C. The wavelength of the laser detector was set at 658.0 nm.

Synthesis of c,t,c-[PtCl 2 (OH) 2 (NH 3 ) 2 ]
Briey, cis-platinum (1.00 g, 3.33 mmol) was suspended in deionized water (25.0 mL). To the solution, H 2 O 2 (35.0 mL, 30.0 mmol) was added and the resulting mixture was stirred at 50 C for 1 h. Then, the precipitate was isolated by ltration and sequentially washed with water, ethanol, and ether. The obtained precipitate was dried under vacuum to give a yellow powder (816 mg; yield: 73.3%).

Synthesis of c,t,c-[PtCl
Succinic anhydride (150.1 mg, 1.5 mmol) was added to a solution of c,t,c-[PtCl 2 (OH) 2 (NH 3 ) 2 ] (500 mg, 1.5 mmol) in DMSO (40 mL) and the resulting mixture was stirred at room temperature for 12 h. Then, the reaction mixture was precipitated with ether. The precipitate was isolated by centrifugation, washed with acetone and ether thrice, and dried under vacuum to give a yellow powder (590 mg; yield: 90.6%).

Synthesis of LA-HES-Pt
LA (100 mg, 0.28 mmol) was dissolved in DMF (20 mL). To the solution, HES-Pt (100 mg; glucose unit: 0.52 mmol), EDCI (268.4 mg, 1.4 mmol), and DMAP (85.5 mg, 0.7 mmol) were added and the resulting mixture was stirred at 45 C for 48 h. Then, the reaction mixture was dialyzed against deionized water for 3 days and lyophilized (109 mg; yield: 54.5%). Based on the 1 H NMR spectrum, we calculated that there are around 104 LA molecules on each HES nanoparticle.
The platinum loading content (DLC) of HES-Pt and LA-HES-Pt were determined by an atomic absorption spectrometer, which was calculated as where Wt (loaded Pt) is the weight of the loaded platinum and Wt (Pt-loaded conjugates) is the weight of Pt-loaded conjugates. From the drug loading content, we calculated that there are around 23 cisplatin molecules in LA-HES-Pt.

Cellular uptake
To investigate the cellular uptake mechanism of LA-modied HES (LA-HES), ASGPR-overexpressed HepG-2 cells were selected, whereas 4T1 cells without ASGPR expression were selected as the negative control. HES and LA-HES were labeled by uorochrome Cy5 for confocal imaging and ow cytometry analysis. Briey, HepG-2 and 4T1 cells were seeded into glass bottom cell culture dishes at 5 Â 10 4 cells per well. Aer incubating with HES-Cy5 or LA-HES-Cy5 (200 mg mL À1 ) for 12 h, the medium was removed and the cells were washed with PBS buffer (pH 7.4, 6.7 mmol L À1 ) thrice. Then, the cells were xed with 4% paraformaldehyde and stained with DAPI for confocal imaging. To investigate the competitive inhibition effect of LA on the cellular uptake of LA-HES-Cy5, HepG-2 cells were preincubated with LA (500 mg mL À1 ) for 4 h followed by incubation with HES-Cy5 or LA-HES-Cy5 (200 mg mL À1 ) for 12 h. Then, the cells were xed with 4% paraformaldehyde and stained with DAPI for confocal imaging. The cellular uptakes of HES-Cy5 and LA-HES-Cy5 were also quantied by ow cytometry. The cellular uptakes of HES-Pt and LA-HES-Pt were quanti-ed by ICP-OES. Briey, HepG-2 cells were seeded into 12-well plates at a cell density of 2 Â 10 5 cells per well. Aer incubating the cells with cis-platinum, Pt-COOH, HES-Pt, and LA-HES-Pt for 6 h, the medium was removed and the cells were washed with PBS thrice. Then, the cells were harvested and counted. The cell suspensions were digested with a mixture of nitric acid (65-68%, 2.0 mL) and perchloric acid (70-72%, 0.15 mL) at 320 C for 30 min. Aer cooling to room temperature, the above solutions were diluted to 10 mL with deionized water and measured by ICP-OES at 214.4 nm.

Biocompatibility assessment
The murine broblast cell line, 3T3, was used to evaluate the biocompatibility of HES. Briey, 3T3 was seeded into a 96-well plate with a cell density of 5 Â 10 3 per well. Aer culturing overnight, different HES samples were incubated with 3T3 cells at concentrations ranging from 0.1 mg mL À1 to 1000 mg mL À1 for 24 h. Then, MTT assays were used to determine the cell viability, as previously reported. Cells incubated only with the medium were used as the reference for 100% viability.
In vitro antitumor activity MTT assays were used to evaluate the in vitro cytotoxicity of cisplatinum, Pt-COOH, HES-Pt, and LA-HES-Pt against human hepatoma cells, HepG-2. Briey, HepG-2 cells were seeded into 96-well plates with a cell density of 5 Â 10 3 per well. Aer culturing overnight, 4-well cells were incubated with different materials at various concentrations. Aer 24 h incubation, MTT assays were used to determine the cell viability as previously reported. Cells incubated only with the medium were used as the reference for 100% viability.

Statistical analysis
All the data were presented as the mean value AE standard deviation (SD). Statistical analysis was performed by the Statistical Product and Service Solutions (SPSS) program with independent samples T-test. The statistical signicance was established at p < 0.05. *p < 0.05, **p < 0.01, ***p < 0.001.

Results and discussion
Derived from waxy maize that contains more than 95% amylopectin, HES is a semisynthetic polysaccharide and has been used as the rst-line colloidal PVE for several decades in clinics. 36 HES can be categorized into various classes based on its molecular weight, molar substitution of hydroxyethyl, and hydroxyethylation substitution pattern (C2/C6 ratio). 37 These parameters affect the endogenous a-amylase, which selectively cleaves a-(1,4)-glycosidic bonds but not the branching a-(1,6)glycosidic bonds and mediates the degradation of HES in blood, thereby determining the in vivo pharmacokinetics of HES and facilitating the tailoring of the in vivo fates of HES by simply modulating these parameters. Although safety concerns have been raised for acute kidney injury and increased risk of mortality when HES is administered to seriously ill patients requiring acute volume resuscitation (particularly to patients with severe sepsis and those in intensive care), 38-41 HES has extremely low immunogenicity, presumably owing to the common structural features between HES and glycogen, both of which are branched polysaccharides. 42 A good manufacturing practice, high water solubility, tailorability, biocompatibility, biodegradability, and well-dened and proven in vivo safety features not only facilitate HES to be widely applied in clinics as PVE, 36,43-45 cryoprotectant, 46-49 organ preservation solutions, 50 granulocytes separation solutions, 51-53 and cell culture medium, 54 but also to be a promising drug carrier with promising clinical translation potential. [55][56][57] To this end, various drug delivery systems have been recently developed based on HES, including HES and small drug conjugates; 27-30,32,34,58 HES and protein conjugates; 56,59 HES-derived nanocolloidosomes; 60 and HES-based nanoparticles, 61-67 capsules, 68-71 and hydrogels. [72][73][74][75] Although HES has been applied in clinics for over 50 years, its fundamental conformation, structure, and morphology are largely unknown. To retain the branched structure of amylopectin, hydrodynamic diameters around dozens of nanometers have been frequently measured in HES solutions with DLS. 28,34,58,59,76 TEM and AFM imaging has revealed that HES exhibits a colloidal structure in self-assembled nanoparticles 67,77 and conjugates. 60 Nonetheless, the conformation and structure of a single HES particle remains an enigma. Without such information, correlating the structure of HES with clinical uses and safety concerns is impossible, and the rational clinical applications of HES and clinical translations of HESbased drug delivery systems will be limited. Initially, the conformation and structure of single HES, with different molecular weights, will be systematically scrutinized by a combination of DLS, TEM, AFM, GPC, and NMR.

HES conformation
To comprehensively understand the conformation of HES, HES with different molecular weights, namely, 480 kDa, 200 kDa, 130 kDa, 70 kDa, and 25 kDa, have been studied by a combination of GPC and DLS. As shown in Table 1 The conformational coefficient a is a good indicator of the conformation of macromolecules and can be obtained with the following equation: taking the logarithm of both the sides of eqn (1): where R g is the radius of gyration, k is the proportionality constant, and M is the molar mass of the individual polymer molecule. Therefore, a can be obtained by measuring R g and molar mass. Fig. S2  The a values of our HES samples are slightly lower than that of random coils, indicating that HES has a relatively compact conformation rather than a linear one. Our results are in good agreement with those reported by others. 79 Further, rratio is another indicator for the conformational quantication of macromolecules. The r-ratio is known as the ratio of the gyration radius to the hydrodynamic radius (R g /R h ); these radii can be measured by GPC and DLS, respectively. As shown in Table 2, the r-ratio values of our HES samples are between 1.04 and 1.13. It was reported that the r-ratio is equal to 0.778 for hard spheres, 0.977 for dendrimers, 1.225 for a hyper-branched polymer, and 1.73 for a random coil of linear macromolecules in a q-solvent. 82 The r-ratio values of our HES samples are between dendrimers and hyper-branched polymers, indicating that HES has a relatively compact conformation. 42 Meanwhile, the 1 H NMR spectra of HES were investigated to conrm that HES is a hyperbranched polymer, and there are many hydroxyl sites that can be modied ( Fig. S3 and S4 †). Collectively, the 1 H NMR spectra, a, and r-ratio corroborate that HES can adopt a compact conformation.

HES morphology
The size distributions of HES in an aqueous solution have been reported in previous studies. 34,76,77 However, the morphology of HES is largely unknown, particularly that of a single HES particle. TEM and AFM have been widely employed to directly characterize the morphology of macromolecules. 77,83 Here, TEM and AFM were utilized to characterize the morphology of HES 480/0.5, HES 200/0.5, and HES 130/0.4. HES was negatively stained by phosphotungstic acid and characterized by TEM at a low acceleration voltage (80 kV). Fig. 1A shows that HES particles are nearly spherical (white) for each single HES. Moreover, all the HES samples have a spherical shape. The AFM images consistently show that HES are nearly spherical particles (Fig. 1C). These results are consistent with the relatively compact conformation of HES and indicate that the HES nanoparticles are intrinsic. The diameters of HES determined by TEM and AFM are in accordance with those determined by DLS (Fig. 1B and D (Fig. 1B). The AFM results are consistent with the TEM results (Fig. 1D) (Table 2). Further, we found that the hydrodynamic diameter of HES decreased with increasing concentration, suggesting the hydrodynamic diameter of HES is dependent on the osmotic pressure and implying that HES particles are so nanoparticles that can be squeezed by osmotic stress (Fig. S5 †). The DLS, TEM, and AFM images corroborate the fact that the size of HES increases with its molecular weight, and the diameters of HES 480/0.5, HES 200/ 0.5, and HES 130/0.4 are in the range of 10-40 nm, which is benecial for prolonged in vivo circulation and passive tumor targeting via the EPR effect. 28,76 The diameters of HES with different molecular weights and degrees of substitution were also measured by DLS, AFM, and TEM in previous studies. 28,34,60,66,76 However, integrating GPC, DLS, and TEM with AFM for uncovering the structure and morphologies of HES has been seldom reported. The combination of various advanced characterization techniques facilitates the complete appreciation of HES. The revealed colloidal structure of HES might correlate with acute kidney injury when administered as PVE in clinics, as branched HES might get stuck in the glomeruli of kidneys. Nonetheless, more systematic studies are required, particularly for undertaking clinical trials. Taken together, the GPC, DLS, TEM, and AFM results corroborate the colloidal structure of HES.

Pt delivery system based on HES nanoparticles
Based on the understanding that HES particles are intrinsic nanoparticles with a suitable size for in vivo circulation and passive tumor targeting, we constructed a novel prodrug of cisplatinum further modied with LA, which can specically bind to the ASGPR overexpressed on the surfaces of hepatoma cells, HepG-2. 60 There are many advantages of this drug delivery system (LA-HES-Pt). First, HES has excellent solubility and good biocompatibility and biodegradability, which can improve cisplatinum solubility and stability. 31,33,63,84 Second, LA-HES-Pt can attain passive cancer targeting due to the EPR effect of HES, and it can also be specically taken up by hepatoma cells via ASGPR-LA-mediated endocytosis pathways. Third, HES, cisplatinum, and LA have been approved for clinical practice, which endows LA-HES-Pt with high druggability. 64 Last, but not the least, platinum is a high-atomic-number element, which  facilitates the direct detection of HES under TEM observations without the need for staining. As shown in Fig. 2A, LA-HES-Pt was synthesized through three simple steps. Pt-COOH, a rami-cation of cis-platinum, was rstly prepared according to a previously method reported. 35 Then, HES-Pt was synthesized via an esterication reaction between the carboxyl group of Pt-COOH and hydroxyl group of HES. Finally, HES-Pt was modied with galactose moieties via an esterication reaction between the carboxyl group of LA and hydroxyl group of HES. Several studies have shown that HES 200/0.5 exhibits prolonged in vivo circulation and excellent tumor accumulation. 28,30 Here, HES 200/0.5 was selected as the starting material to synthesize LA-HES-Pt. The successful synthesis of HES-Pt and LA-HES-Pt was conrmed by 1 H NMR, FT-IR, and UV (Fig. 2B, C and S6A-C †).
The 1 H NMR spectra of Pt-COOH show the characteristic signals of succinyl at 2.50-2.62 ppm (Fig. S6A †) and the FT-IR spectra of Pt-COOH are in accordance with those reported in the literature 15,85 (Fig. S6B †), suggesting the successful synthesis of Pt-COOH. The 1 H NMR spectra of HES-Pt also show the characteristic signals of succinyl (Fig. 2B) and the FT-IR spectra of HES-Pt show the characteristic band of an ester bond at 1718 cm À1 (Fig. 2D), indicating that Pt-COOH is successfully conjugated onto HES through the ester bond. When compared with HES-Pt, the characteristic signal of LA at 4.50 ppm appears in the 1 H NMR spectra of LA-HES-Pt (Fig. 2C), suggesting the successful synthesis of LA-HES-Pt. Collectively, 1 H NMR and FT-IR effectively demonstrate the successful synthesis of HES-Pt and LA-HES-Pt. The platinum loading content of HES-Pt and LA-HES-Pt are 6.4% and 2.6%, respectively, as determined by the atomic absorption spectrometer. The size distribution of HES, HES-Pt, and LA-HES-Pt are measured by DLS (Fig. 2E); their hydrodynamic diameters are 18.09 AE 3.89, 19.25 AE 3.94, and 20.00 AE 2.94 nm, respectively (Table 3), indicating the insignicant size changes aer Pt-COOH and LA conjugation. Due to the high atomic number of platinum, platinum can effectively absorb the incident electrons in TEM imaging, resulting in high contrast with HES.
Aer coupling with platinum, the resultant conjugate can be easily characterized by TEM without staining. HES-Pt based on HES 130/0.4, HES 200/0.5, and HES 480/0.5 were characterized by TEM (Fig. S7 †). HES-Pt exhibits nearly spherical morphology and the size increases with the molecular weight of HES, which is consistent with the results of HES characterized by TEM and AFM (Fig. 1). Further, the TEM image of LA-HES-Pt shows that LA-HES-Pt exhibits nearly spherical nanoparticles (Fig. 2F). Moreover, the XRD spectra of HES, HES-Pt, and LA-HES-Pt show an amorphous characteristic, indicating that the morphology of those samples characterized by TEM and AFM exhibit single molecular, and not crystalline, morphology (Fig. S6D †). The average diameter of LA-HES-Pt determined from the TEM image is 19.35 AE 2.50 nm (Fig. 2G), which is consistent with the hydrodynamic diameter determined by DLS. The zeta potentials of HES, HES-Pt, and LA-HES-Pt in PBS buffer are 1.35 AE 2.03, À1.31 AE 0.04, and À2.06 AE 0.57 mV, respectively, indicating that  they are neutral. Collectively, these results conrm that HES-Pt and LA-HES-Pt are intrinsic nanoparticles, like HES. To investigate the cellular uptake of HES and LA-modied HES (LA-HES), ASGPR-overexpressed HepG-2 cells were chosen, and 4T1 cells without ASGPR expression were chosen as the negative control. Fluorescent dye Cy5 was conjugated onto HES and LA-HES for confocal imaging and ow cytometry analysis. Human liver cancer HepG-2 cells with ASGPR overexpression were incubated with Cy5-labeled HES and LA-HES for 12 h. As expected, an increased Cy5 signal was observed in the LA-HES-Cy5 group when compared with that in HES-Cy5 by confocal laser scanning microscopy (CLSM) (Fig. 3A). This indicated that increased HES was taken into HepG-2 cells aer modication with LA. Meanwhile, this uptake could be inhibited by the preincubation with additional free LA by competition uptake (Fig. 3A). Quantitative ow cytometry measurement further revealed that the cellular uptake of HES was enhanced by 4.1 times by the modication of LA molecules ( Fig. 3B and C), while the endocytosis of HES-Cy5 was not affected by the preincubation of free LA with HepG-2 cells. These results suggest that the uptake of LA-HES-Cy5 by HepG-2 cells is via the ASGPR-LA-mediated endocytosis pathway. The cellular uptake of LA-HES-Cy5 is similar to that of HES-Cy5 on ASGPR-negative 4T1 cells (Fig. S8A-C †), further conrming the ASGPR-LA-mediated endocytosis of LA-HES-Cy5 on HepG-2 cells.
Since it is reported that the modication of hydrophilic HES inhibits the cellular uptake of drug conjugates, 28,34 several targeting molecules have been used to improve the cellular uptake of HES-based drug delivery systems, such as iRGD-HES, 67 FA-HES (folic acid), 86 and Gal-HES (galactosamine). 60 Further, it has been reported that the cellular uptake of nanoparticles modied by LA (galactose moiety) is high on ASGPR-positive cancer cells via ligand-receptor-mediated endocytosis. 14 Herein, we successfully conjugated LA with HES and investigated its cellular uptake properties. Taken together, these results show that LA modication can promote the cellular uptake of HES, particularly on ASGPR-overexpressed cells due to ASGPR-LA-mediated endocytosis.
To study the cellular uptake of HES-Pt and LA-HES-Pt, the intracellular platinum content was measured by ICP-OES (Fig. 4A). HepG-2 cells were incubated with cis-platinum, Pt-COOH, HES-Pt, and LA-HES-Pt (10 mg mL À1 of platinum) for 6 h, and the intracellular platinum content was determined there-aer. To be specic, the intracellular platinum content of cisplatinum, Pt-COOH, HES-Pt, and LA-HES-Pt determined by ICP-OES are 0.02 AE 0.01 mg, 0.01 AE 0.01 mg, 0.06 AE 0.01 mg, and 0.26 AE 0.01 mg for every 10 5 cells, respectively (Fig. 4A). Pt-COOH exhibits a reduced cellular uptake as compared to cisplatinum, which can ascribe to the enhanced hydrophilicity of Pt-COOH, preventing the interaction between Pt-COOH and cell membranes. 16 HES-Pt exhibits enhanced cellular uptake as compared to both Pt-COOH and cis-platinum, implying the advantage of conjugation with HES. LA-HES-Pt exhibits the highest cellular uptake, with intracellular platinum content 15.6 times higher than that of cis-platinum and 4.3 times higher than that of HES-Pt. These results surprised us with a signicant improvement in the platinum uptake for 15.6 times when compared with that of cis-platinum. When compared with the previously reported platinum drug delivery systems in which platinum uptake was improved by 9.8 times by micelles 15 and  The in vitro cytotoxicity values of HES-Pt and LA-HES-Pt against HepG-2 cells were nally evaluated by MTT assays (Fig. 4B). Cis-platinum, HES-Pt, and LA-HES-Pt exhibit cytotoxicity in a dose-dependent manner. LA-HES-Pt exhibits the highest cytotoxicity against HepG-2 cells, with IC 50 value that is 5.3-fold lower than that of cis-platinum and 4.4-fold lower than that of HES-Pt ( Fig. S10 and Table S2 †). To be specic, the IC 50 values of cis-platinum, HES-Pt, and LA-HES-Pt were 11.34 mg mL À1 , 9.40 mg mL À1 , and 2.14 mg mL À1 , respectively. Pt-COOH shows no cytotoxicity by MTT assay. Meanwhile, the biocompatibility of HES is also conrmed with MTT assay against murine broblast 3T3 cells (Fig. S9 †).
Although several platinum drug delivery systems have been developed, only a few of these reported systems are more efficient than the cis-platinum system. 15 It is mainly on the account of limited cellular uptake. Herein, our results have indicated that LA-HES-Pt have the highest cytotoxicity to HepG-2 cells. This can be ascribed to the efficient cellular uptake by ASGPR-LA-mediated endocytosis (Fig. 4A). The platinum conjugated on LA-HES-Pt was reduced to cis-platinum by GSH, ascorbic acid, thioalcohol, and low pH aer entering into the cell, leading to DNA-induced apoptosis. 15 HES-Pt also exhibits enhanced cellular uptake as compared to cis-platinum, but the cytotoxicity of HES-Pt is similar to cis-platinum, which may be ascribed to the insufficient intracellular Pt release of HES-Pt. 15,16 Due to this limited cellular uptake, Pt-COOH exhibits minimal cytotoxicity, which is also consistent with previous reports. 15 When compared with previous reports, our results also show the best in vitro anti-hepatoma efficiency for platinum drug delivery systems. Taken together, based on our rational design, the in vitro antitumor activity of LA-HES-Pt against HepG-2 cells is signicantly enhanced when compared with cis-platinum.

Outlook and concluding remarks
In summary, a novel cis-platinum delivery nanoplatform, LA-HES-Pt, is constructed based on clinically used colloidal PVEs, namely, HES, to enhance the cellular uptake and cytotoxicity of cis-platinum. GPC, DLS, TEM, and AFM corroborate that HES particles are spherical. In vitro experiments veried that LA-HES-Pt can actively target HepG-2 cancer cells with a highlevel expression of ASGPR and promote cellular endocytosis to exert better antitumor effects than cis-platinum. Remarkably, the intracellular platinum content increases 15.6 times and IC 50 decreases 5.3-folds for LA-HES-Pt when compared with cisplatinum. By revealing the structure of clinically used PVE, namely, HES, this study has signicant clinical implications, paving the road for clinical translations of HES-based NDDS. Since HES, cis-platinum, and LA have been approved for clinical practice, the reported LA-HES-Pt has signicant clinical translation potential and warrants further in vivo investigations.

Conflicts of interest
There are no conicts to declare.