AGAMOUS-like genes. We performed functional analysis of AGL19 and AGL24 genes related to bolting and flowering using CRISPR/Cas9-mediated Chinese cabbage transformation. Single-guide RNA (sgRNA) sequences were created with a low off-targeting probability to construct gene-editing vectors. Agrobacterium-mediated transformation was conducted, and tentative E0 AGL-edited lines were analyzed using molecular biotechnological methods. Two AGL19-edited lines with nucleotide sequence mutations in the target sequence of the AGL19 genes and four AGL24-edited lines with nucleotide sequence mutations in the target sequence of the AGL24 genes showed particularly late bolting compared to the inbred line ‘CT001.’ Generational progression using bud pollination obtained T-DNA-free E1 AGL-edited lines, which also showed late bolting. The loss of function of the AGL protein was caused by the occurrence of an indel mutation in the AGL19 and AGL24 genes, which results in an early stop codon. Furthermore, frameshift mutations led to structural changes and the introduction of an early stop codon in the AGL19 and AGL24 proteins. Our results indicate that CRISPR/Cas9-mediated editing of AGAMOUS-like genes results in a late-bolting phenotype and that CRISPR/Cas9 is a useful technology for analyzing gene function in Chinese cabbage (Brassica rapa ssp. pekinensis).Due to the sudden change in temperature in spring, Chinese cabbage, a leafy vegetable cultivated for consumption, loses its commercial value due to the onset of bolting—the phenomenon of switching from vegetative to reproductive growth. In this study, we applied clustered regularly interspaced short palindromic repeats/(CRISPR)-associated system 9 (CRISPR/Cas9) technology to analyze  Brassica, including rapeseed, cabbage, and Chinese cabbage, are cultivated on a large scale worldwide. Chinese cabbage is one of the three major vegetable crops cultivated in Korea. Chinese cabbage is an important crop that supplies vitamins, calcium, and minerals, and new varieties are currently being developed [Crops belonging to the genus eveloped . Chineseeveloped . The boleveloped ,4,5.lfy-6-null mutants, their expression is not abolished [The ABC model is a scientific model related to flower development comprising three classes. It induces sexual reproduction and floral organ emergence by regulating floral-related gene expression patterns. Recently, the ABC model has been expanded to the ABCDE model and studied for various crops. Class A is composed of floral meristem genes, and class B genes determine the construction of petals and stamens . Althougbolished , indicatAG genes. Previous studies using loss-of-function and gain-of-function analyses have revealed that AGAMOUS-like 19 (AGL19) and AGAMOUS-like 24 (AGL24) are flowering-related genes. Transgenic plants overexpressing the AGL19 gene in the FLC-independent pathway appeared to flower earlier than the wild type [agl19 mutants showed slightly late flowering compared to the wild type on short days. Likewise, the agl24 mutants appeared to be late flowering, and transgenic plants overexpressing the AGL24 gene showed early flowering [AGL19 not only interacts with AGL24 but also indirectly interacts with SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1) in Brassica oleracea [FLOWERING LOCUS C (FLC), VERNALIZATION 1 (VRN1), and SOC1, have been the focus of studies on bolting and flowering in Chinese cabbage [AGAMOUS-like genes involved in bolting and flowering that act independently or are correlated with important regulatory genes.Class C includes ild type . The agloleracea . Several cabbage ,11,12. TMyzus persicae on silencing the acetylcholinesterase 1 (Ace 1) gene [Tetranychus urticae on silencing the coatomer protein complex subunit 2 (COPB2) gene [PpNAC56, a transcription factor involved in numerous plant growth processes, are more resistant to heat stress than are the wild type [mannitol-1-phosphate dehydrogenase (mtlD) gene [Current research is focused on inculcating crops with useful characteristics to overcome the unfavorable external environment using various molecular biological technologies. For example, RNA interference (RNAi) has been used to develop insect-resistant transgenic plants. Transgenic tomato plants expressing artificial microRNA gained improved resistance to  1) gene . TransgeB2) gene . Insect-ild type . Salt tolD) gene . The onllD) gene .OsRR22-edited T1 rice plants [CaERF28-edited T2 chili plants showed improved anthracnose resistance [Previous studies have shown that CRISPR/Cas technology allows T-DNA segregation via next-generation progression after target gene mutation, resulting in the desired traits being displayed in T-DNA-free plants. For example, salinity tolerance has been observed in e plants , and CaEsistance .CRISPR/Cas9 is an acquired immune system inherent in prokaryotes that recognizes and cleaves external viruses . The opeAGAMOUS-like 19 (AGL19) and AGL24 genes [AGL genes, and late bolting was observed in the edited lines.In this study, using the CRISPR/Cas9 system, we confirmed that 24 genes ,26 are rAGL19 (CT001_A03121400 and CT001_A08282630) and AGL24 (CT001_A03122450 and CT001_A01013460) genes of the inbred line ‘CT001.’ The CT001_A03121400 gene, comprising seven exons, was positioned on the A03 chromosome, and the CT001_A08282630 gene, comprising three exons, was identified on the A08 chromosome. CT001_A03122450 was located on the A03 chromosome, and CT001_A01013460 was located on the A01 chromosome. CT001_A03122450 and CT001_A01013460 contained six exons. sgRNAs were designed to target the exonic regions of each gene  sequence targeting the ach gene A. To avo0 AGL-edited lines were generated by transformation of Chinese cabbage. We obtained 14 and 22 tentative E0 lines derived from A1 and A2 editing vectors, respectively. To select the T-DNA-inserted line, two primer sets were designed using a partial sequence of hygR and Cas9hc:NLS:HA (Cas9hc) for PCR analysis  sequence and generation of a stop codon. Similarly, an 8 bp sequence was deleted upstream of the PAM sequence in the CT001_A03122450 gene of the E0 A2-1 line. Five amino acid sequences were mutated, and the stop codon appeared early  analysis . As a result, T-DNA-free E1 AGL-edited lines without PCR amplicon were identified. In the A1-2 and A1-9 lines, four and three T-DNA-free E1 AGL-edited lines were identified, respectively  without a PCR amplicon were identified in the A2-1, A2-11, A2-16, and A2-22 lines  and APETALA1 (AP1), which are floral meristem identity genes, decreased in agl17-1 [LFY directly activates AP1, which plays dual roles: it specifies the floral meristem, and it acts as a class A gene to determine the identity of perianth organs [LFY, AP1, and UNUSUAL FLORAL ORGANS, an F-box gene [ agl17-1 . LFY dirh organs ,29. Actibox gene ,31.LFY also cooperates with a homeobox gene, WUSCHEL (WUS), to activate the AG gene, a type of class C gene that specifies the identity of stamens and carpels [AGL6 is involved in the flowering pathway. The 35S::AGL6 transgenic plants appear to have short vegetative development and early flowering with abnormal phenotype observations in the flower structure [AGL28 gene regulates floral promoters, including FLOWERING CONTROL LOCUS A (FCA) and LUMINIDEPENDENS (LD). Overexpression of AGL28 results in early flowering by upregulation of FCA and LD [AGL24 has been studied in relation to the floral integrator SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) gene. The agl24 mutant showed abnormal phenotypes, such as late flowering and late floral organ development, similar to those of the SOC1 mutant. AGL24 expression is correlated with various flowering pathways. carpels ,33. AGL6tructure ,35,36. TA and LD . In partFLOWERING LOCUS C (FLC), a key regulator gene in flowering, encodes a MADS-box protein that acts as a floral transition repressor, and is downregulated during cold treatment. The FLC gene suppresses floral integrator genes, such as FLOWERING LOCUS T (FT) and SOC1 [Flowering plants are affected by various flowering pathways, including the circadian rhythm, photoperiod, autonomous, vernalization, gibberellin, flowering time, and meristem identity pathways . The traand SOC1 ,40.AGL19 and AGL24 directly or indirectly interact with floral integrators, such as FLC and SOC1. To generate AGL-edited Chinese cabbage lines, AGL19_sgRNA and AGL24_sgRNA were designed for specific regions of AGL genes and cassettes were ligated with the pHAtC vector. Using conserved sequences, one sgRNA can target and knock out multiple homologous genes. For example, sgRNAs were designed to target the conserved sequences among three gene families  in Brassica napus [GmPDS, GmFAD2, and GmALS, which contain two homologous genes and share 90% homology of the amino acid sequence, were modified using one sgRNA targeting the common sequence of each homologous gene in Glycine max [PP2A B regulatory subunit (PR55/B) homologous genes and generate frameshift-triggered self-compatibility in Brassica rapa [AGL-edited lines exhibited a delayed bolting phenotype than did the inbred line ‘CT001′  gene generates an early stop codon, resulting in the translation of an incomplete protein. Therefore, BrVRN1 cannot function to suppress FLC, and the edited plant shows late flowering [PR55/B-edited plants show self-compatibility by producing early stop codons and loss of protein function [This study showed that ca napus . GmPDS, cine max . One sgRica rapa . These r ‘CT001′ . The occproteins . The E0 lowering . PR55/B-function . These gCT001_A01013460 gene of E1 A2-11-6, A2-16-4, and A2-16-12 lines were found to be different from those of the E0 A2-11 and A2-16 lines. Using the CRISPR/Cas9 system, a 1 bp insertion was observed in the A2-11 line, but a 1 bp transversion and a 7 bp deletion appeared in the A2-11-6 line. In addition, a 6 bp deletion was confirmed in the CT001_A01013460 gene of the A2-16-4 and A2-16-12 lines. In previous studies, different InDeL mutations of the target region in next generations have been reported for rice [TaDA2 gene of the T0 plant, but more variations were detected in the T1 generation [AOX gene in T0 rice plants, whereas additional mutations were found in the progeny lines [Occasionally, the mutation patterns of target genes in gene-edited plants change during advancement. In this study, the mutation patterns in the for rice ,45. The ny lines . Mutationy lines .1 AGL-edited lines showing late bolting will be needed. Additionally, it is necessary to identify E2 AGL-edited lines that exhibit a homozygous mutation pattern via next-generation progression. Developing transgenic plants using traditional techniques for inserting external genes has gotten tangled with the issue surrounding genetically modified (GM) crops, namely the retention of T-DNA in the plant genome to obtain a useful transgenic plant phenotype. However, T-DNA removal is possible through generation advancement in gene-edited plants developed using the CRISPR/Cas9 system. Thus, we believe that the CRISPR/Cas9 system is a useful technique for the development of gene-edited transgene-free plants.Finally, transgene-free plants were secured and useful phenotypes were maintained . As the AGL genes were selected to delay the bolting stage of Brassica rapa ssp. pekinensis. sgRNA design and vector construction were performed, as described previously [AGL19 and AGL24 genes after counting the number of each homologous gene in the ‘CT001′ genome using FSTVAL . sgRNAs were designed with 23 nucleotides composed of a 20 bp random guide sequence and a 3 bp NGG sequence  using CRISPR direct . To modulate AGL19 and AGL24 gene expression, candidate sgRNAs were selected from the exonic region, and a low off-targeting frequency was confirmed based on the ‘CT001′ genome using the BioEdit program . To compare similar sequences, sgRNA sequences were blasted to the whole genome sequence using the inbred line ‘CT001′ pseudomolecule sequence as a reference. As a result, it was expected that other genes were not targeted by the designed sgRNAs.Two eviously . sgRNAs AGL19 and AGL24. AGL19_sgRNA was designed to target the fourth exon of A03 and the second exon of A08. AGL24_sgRNA was designed to target the first exons of A01 and A03. The constructed gene-editing vectors (A1 and A2) containing the hygromycin phosphotransferase (hpt) gene as a selection marker were transferred into Agrobacterium tumefaciens LBA4404 using a modified freeze-thaw method [To delay the bolting stage, both sgRNAs were designed to target two homologous genes, w method .AGL-edited Chinese cabbage lines. The seeds of ‘CT001’ were sterilized and sown in MS basal medium in vitro. AGL-edited lines were generated using Agrobacterium-mediated transformation. The infected explants were transferred to the selection medium with hygromycin every 2 weeks until shoots emerged. After root development, the tentative AGL-edited lines were cultivated in a culture room at Kyung Hee University .The inbred line ‘CT001’ was used to develop AGL-edited lines was performed by confirming T-DNA insertion. The genomic DNA (gDNA) of ‘CT001’ and tentative AGL-edited lines was used as a template, and PCR analysis was conducted using the 2× H-Star Taq PCR master mix . Two primer sets were designed to target the partial sequences of the hygR and Cas9hc regions . The purity and concentration of the extracted RNA were checked using a Nanodrop® ND-1000 spectrophotometer , and cDNA was synthesized using the LyoFACT™ RT Pre-Mix . The cDNA synthesis conditions were as follows: reaction at room temperature for 5 min, reverse transcription at 50 °C for 1 h, and RNase inactivation and extension at 95 °C for 10 min. Specific primer sets were designed to target each exonic region of homologous genes . RT-PCR conditions were as follows: initial denaturation at 95 °C for 15 min; 35 cycles of 95 °C for 30 s, 58 °C for 20 s, and 72 °C for 20 s; and a final extension step at 72 °C for 5 min. RT-PCR amplicons were eluted using the P&C Multiple Elution Kit  and sequenced using BTSeq . The obtained sequence data were aligned and translated into amino acid sequences.To confirm the mutations of the us genes . The cDN1 AGL-edited lines were selected using PCR analysis with genomic DNA from 3-week-old E1 seedlings and T-DNA-specific primers sets. Lines without the PCR amplicon were identified as T-DNA-free E1 AGL-edited lines.T-DNA-free EAGL-edited lines, ‘CT001’ and AGL-edited lines were randomly placed and artificially vernalized in a cold room for 7 weeks at 4 °C under a 16 h/8 h light/dark photoperiod. After vernalization, the lines were placed in a greenhouse at 23 °C, and then the bolting date and stem length were recorded. The number of days between the emergence and appearance of the first floral axis of each AGL-edited line was confirmed. The selected E0 AGL-edited lines were self-pollinated and advanced to the E1 generation.To examine the bolting time of 0 AGL-edited lines. gDNA was cut using EcoRI at several recognition sites inside the ‘CT001’ genome [HindIII molecular marker were loaded onto 1% agarose gel, and electrophoresis was conducted at 45 V for 7 h. Denatured DNA was transferred onto a Hybond N+ nylon membrane . The probe was designed using a 709 bp of the hpt gene of the T-DNA, and the eluted PCR amplicon was labeled with 32P-dCTP using the BcaBEST labeling kit . The nylon membrane and labeled probe were hybridized and washed in a shaking incubator at 60 °C. They were then transferred into a cassette for exposure to an X-ray film and visualized.Southern hybridization was conducted to determine the T-DNA copy number in the genome of E’ genome . Digeste0 AGL-edited lines, modified variable-argument–thermal asymmetric interlaced PCR (VA-TAIL PCR) was performed [2O. VA-TAIL PCR analysis was performed using the 2X Taq PCR Master Mix  with a thermocycler  programmed as shown in To confirm the T-DNA insertion site of the Eerformed . To deveAGL-edited Chinese cabbage lines with a late-bolting phenotype using the CRISPR/Cas9 system and targeting the AGL19 and AGL24 genes. To target specific regions, sgRNA sequences were designed with a low off-targeting probability, and gene-editing vectors were constructed. Late-bolting AGL-edited lines were developed, and target sequence mutations were analyzed. Finally, elite lines with one copy of T-DNA inserted into the intergenic region were obtained. Therefore, it was possible to develop late-bolting Chinese cabbage lines by targeting the AGL genes using the CRISPR/Cas9 system. The results of this study are expected to be used as basic data for developing crops with useful traits for responding to climatic changes using the CRISPR/Cas9 system.This is the first study to explore the development of


Herein, we developed a biodegradable theranostic platform (pLMOFePt-TGO) involving composite of dendritic organosilica and FePt alloy, loaded with tamoxifen (TAM) and glucose oxidase (GOx), and encapsulated by platelet-derived growth factor-B (PDGFB)-labeled liposomes, that effectively uses the synergy among chemotherapy, enhanced chemodynamic therapy (CDT), and anti-angiogenesis. The increased concentration of glutathione (GSH) present in the cancer cells induces the disintegration of pLMOFePt-TGO, releasing FePt, GOx, and TAM. The synergistic action of GOx and TAM significantly enhanced the acidity and H2O2 level in the TME by aerobiotic glucose consumption and hypoxic glycolysis pathways, respectively. The combined effect of GSH depletion, acidity enhancement, and H2O2 supplementation dramatically promotes the Fenton-catalytic behavior of FePt alloys, which, in combination with tumor starvation caused by GOx and TAM-mediated chemotherapy, significantly increases the anticancer efficacy of this treatment. In addition, T2-shortening caused by FePt alloys released in TME significantly enhances contrast in the MRI signal of tumor, enabling a more accurate diagnosis. Results of in vitro and in vivo experiments suggest that pLMOFePt-TGO can effectively suppress tumor growth and angiogenesis, thus providing an exciting potential strategy for developing satisfactory tumor theranostics.Chemodynamic therapy of cancer is limited by insufficient endogenous HThe online version contains supplementary material available at 10.1186/s12951-023-01814-5. It can also produce other kinds of ROS, such as the highly toxic hydroxyl radicals (·OH) via Fenton-type reactions [2O2 concentration [2O2 concentration in tumor cells can reach 100 μM, which is four orders of magnitude higher than that in normal cells, around 10 nM [2O2 could be exploited to cause a localized increase in the concentration of ·OH radicals using Fenton catalysts, and form the basis of OH-mediated chemodynamic therapy (CDT) to selectively target and kill tumor cells while avoiding any significant damage to healthy, benign tissues [2O2 overproduction and the relatively weakly acidic conditions present in the TME cannot generate sufficient ·OH to be effective as anti-tumor CDT [2O2 concentration and the acidity in the TME is critical for this strategy to be effective against tumors.Malignant tumors are associated with unique microenvironments that feature weak acidity , overpro2O2) . Althoug2O2) . Althoug2O2) . H2O2 iseactions . The kinntration . Previound 10 nM . This ov tissues . However2O2 in the presence of oxygen [2O2 donor but also an acidity enhancer. Hence, one could logically deduce that the combination of Fenton catalysts and GOx could have significantly higher efficacy and anticancer activity as compared to Fenton catalysts alone. However, the hypoxic nature of the TME could likely limit the catalytic activity of GOx, resulting in less effective anti-cancer efficacy [Ultrasmall iron oxide (USIO), a classical Fenton catalyst, has some inherent limitations such as low catalytic activity and the need for highly acidic conditions (with pH in the range of 2–4), making it unsuitable for achieving effective anti-tumor CDT . Recent f oxygen . It is nefficacy . Recent efficacy , 17. The2O2 levels and acidity. In addition, TAM contributes to chemotherapy and increases cellular acidity by triggering hypoxic glycolysis that results in increased lactate accumulation. The significantly increased acidity and H2O2 levels in the vicinity of the tumor, in turn, boost the Fenton catalytic activity of the FePt alloys, generating more hydroxyl radicals (·OH), thus enhancing anti-cancer CDT efficacy. In addition, the T2 shortening capability of ultrasmall FePt alloys facilitates better contrast in the T2-weighted MRI images of the tumor.We designed a biodegradable theranostic platform (pLMOFePt-TGO) that consists of TAM and GOx-loaded composite nanoparticles consisting of mesoporous organosilica (MON) and FePt alloy, encapsulated by platelet-derived growth factor-B (PDGFB)-labeled liposomes. As shown in, Scheme Ultrasmall FePt alloys were synthesized by a modified thermal decomposition method. Transmission electron microscope (TEM) images showed that the FePt alloys were 2–4 nm in size and had a narrow size distribution Fig. A. High-rSubsequently, the crystal structure of particles was characterized via X-ray diffraction (XRD). The XRD pattern Fig. K shows p2 adsorption-desorption isotherms indicated that the MOFePt alloys had a distinct mesoporous structure and a high specific surface area (272.95 m2/g). These nanoparticles have pore sizes primarily in the range of 2–10 nm and an average pore size of approximately 5.3 nm , and 2850–2930 cm−1   pathway is an important signaling network for the normal development of mesenchymal cells . OverexpWe further explored the biodistribution and tumor-targeted accumulation of pLMOFePt in vivo. A 4T1 tumor-bearing mouse model was constructed by direct subcutaneous injection of 4T1 cells. After about a week, 4T1 tumor-bearing mice were monitored by an animal imaging system after intravenous injection (I.V.) of LMOFePt-Cy7 and pLMOFePt-Cy7. The fluorescence intensity of the tumor in mice injected with pLMOFePt-Cy7 gradually increased with time from 4 to 24 h, and then gradually decreased. It can be seen in Fig. MOFePt alloys were exposed to phosphate buffer solutions with different pH values to simulate normal physiological conditions and TME. As shown in the TEM images, the structure of MOFePt degraded significantly in low pH conditions Fig. A–D. Nume2O2) into highly toxic hydroxyl radicals (·OH) via Fenton catalysis [2O2 solution treated with MOFePt and 10 mM of GSH at 650 nm was significantly higher than that without the addition of GSH. This indicated that the rate of MOFePt catalyzed Fenton reaction is enhanced in the presence of GSH and produced more ·OH as a result. The ability of PLMOFePt-TGO to catalyze the transformation of glucose into gluconic acid and H2O2 was also assessed using an H2O2 assay kit and pH analyzer -5-(and-6)-carboxyfluorescein, an acetoxy-methyl ester (BCECF-AM) whose green fluorescence weakens in acidic conditions. The green fluorescence intensity of MCF-7 cells treated with FePt alloy by themselves as well as that of cells treated with LMOFePt was seen to be similar to that of the MCF-7 cells from the control group. However, the fluorescence intensity of the cells treated with LMOFePt-T and pLMOFePt-T was significantly weaker than the control group, with pLMOFePt-T treated cells showing this effect to a greater degree than the cells treated with LMOFePt-T without PDGFB labeling. These results proved that pLMOFePt-T could effectively enhance cellular acidity via the glycolysis process. In addition, the acidity of MCF-7 cells treated with pLMOFePt-TGO was higher than those treated with pLMOFePt-T, which could be attributed to the synergistic actions of GOx and TAM for enhancing cellular acidity  cells. Notably, while both LMOFePt-T and pLMOFePt-T exhibited a greater degree of inhibition on the viability of MCF-7 and 4T1 cells as compared to TAM, the cytotoxicity of pLMOFePt-T was stronger than that of LMOFePt-T without PDGFB labeling. These results demonstrated that the anti-cancer activity pLMOFePt-T was independent of the presence or absence of estrogen receptors and was strongly associated with glycolysis-enhanced CDT. As expected, pLMOFePt-TGO treatment exhibited the strongest anti-cancer inhibitory effect as compared to the other groups, indicating that the GOx-mediated starvation therapy further enhanced the efficacy of the ant-cancer CDT using pLMOFePt-TGO. In order to confirm the presence of ferroptosis, we explored the viability of MCF-7 cells treated with pLMOFePt-TGO at the absence and presence of iron ion chelate (DFOM) or ferroptotic inhibitor (Fer-1). As shown in Additional file The viability of cells was investigated by MTT assay. As compared to FePt alone, MOFePt did not cause a decrease in cell viability, showing lower cytotoxicity to the THLE-3 cells. This result shows that the MOFePt nanocarriers have excellent biocompatibility Fig. A. In addThe quantitative flow cytometric results shown in Fig. 3 in 13 days. The PBS-treated MCF-7 tumor-bearing mice were euthanized after 13 days, and the tumors were excised. The tumor volume of MCF-7 tumor-bearing mice injected with pLMOFePt-TGO was the smallest among all the groups in this study, indicating it had the strongest anti-cancer activity. After treatment for 19 days, the mice in other groups were also euthanized, and the tumors were excised and photographed  at two-day intervals. As shown in Fig. hed Fig. B. The pLhed Fig. C, D. Thihed Fig. E, F. ThePDGFR-β is known to be overexpressed in most tumor stromal cells and tumor vasculature . Hence, 2) measurements of FePt and pLMOFePt were conducted using a 7.0 T MRI scanner. The r2 values of FePt alloy and pLMOFePt were 179.9 mM−1 s−1 and 91.7 mM−1 s−1 respectively  diagnosis. Transverse relaxivity  The hydrodynamic size of FePt, MOFePt, pLMOFePt-TGO. B) Hydrodynamic size change of pLMOFePt-TGO pLMOFePt-TGO in a different medium. Figure S2.The particle size and polymer dispersity index (PDI) change of pLMOFePt-TGO in (A) distilled water, (B) PBS, and (C) DMEM with standing time. Figure S3. A-F) XPS high-resolution spectra of Fe2p, Pt4f, Si2p, S2p C1s, and full spectra in pLMOFePt-TGO. Figure S4. Zeta potential of FePt, MOFePt,pLMOFePt-TGO. Figure S5. A) Fourier transform infrared spectroscopy of pLMOFePt-TGO. B) Thermogravimetric curve of TAM, MOFePt, and MOFeP-T. C) The UV-vis standard curve of GOx. D) The loading rate of FePt and GOx calculated by ICP-MS analysis and UV-vis standard curve of GOx in E). Figure S6. A) pH value change of different concentration glucose solution treated with pLMOFePt-TGO. B) Cumulative release profile of FePt from pLMOFePt in various pH solutions. The internalization of pLMOFePt in MCF-7 cells: C) low magnification of Bio-TEM observation and D) high magnification of Bio-TEM observation. Figure S7. A) Pharmacokinetic curves of FePt and pLMOFePt analyzed by ICP-MS. B) The standard curve of intracellular acidity obtained by fluorescent probe BCECF-AM. C) The fluorescence spectrum of MCF-7 cells stained with BCECF-AM to assess cellular acidity. Figure S8. The viability of MCF-7 cells treated with pLMOFePt-TGO at the absence and presence of (A) DFOM, (B) Fer-1, (C) NAC, and (D) Z-VAD-FMK. Figure S9. The images of 4T1 cells stained with JC-1 A) and BODIPY B), respectively after treatment with FePt and pLMOFePt-TGO. Figure S10. (A) The tube formation of C166 cells treated with different concentrations of pLMOFePt-TGO; (B) corresponding number of tube formation. Figure S11. H&E staining images of the vital organs in different treatment groups. Figure S12. Ki67 staining images of MCF-7 tumor-bearing mice treated with different samples.


The final outcome of the HLA-E/NKG2A/CD94 interaction on target cells is also highly dependent on the identity of the nonameric peptide incorporated into the HLA-E ligand. To better understand the role played by a nonameric peptide in these immune complexes, we performed a series of multi-microsecond all-atom molecular dynamics simulations. We generated natural and alternative variants of the nonameric peptide bound to the HLA-E ligand alone or in the HLA-E/NKG2A/CD94 complexes. A systematic study of molecular recognition between HLA-E and peptides led to the development of new variants that differ at the strategic 6th position (P6) of the peptide and have favorable in silico properties comparable to those of natural binding peptides. Further examination of a selected subset of peptides in full complexes revealed a new variant that, according to our previously derived atomistic model, can interfere with the signal transduction via HLA-E/NKG2A/CD94 and thus prevent the target cell from evading immune clearance by NK and CD8+ T-cells. These simulations provide an atomistic picture of how a small change in amino acid sequence can lead to a profound effect on binding and molecular recognition. Furthermore, our study also provides new data on the peptide interaction motifs as well as the energetic and conformational properties of the binding interface, laying the structure-based foundation for future development of potential therapeutic peptides, peptidomimetics, or even small molecules that would bind to the HLA-E ligand and abrogate NKG2A/CD94 recognition. Such external intervention would be useful in the emerging field of targeting senescent cells in a variety of age-related diseases, as well as in novel cancer immunotherapies.MHC class I antigen E (HLA-E), a ligand for the inhibitory NKG2A/CD94 receptor of the immune system, is responsible for evading the immune surveillance in several settings, including senescent cell accumulation and tumor persistence. The formation of this ligand-receptor interaction promotes the inhibition of the cytolytic action of immune system natural killer (NK) cells and CD8 Natural killer (NK) cells are part of the innate immune system and belong to a lymphocyte lineage with cytotoxic and cytokine-producing functions. They are involved in the organism’s machinery to combat various types of tumors and microbial infections. In addition, they also have regulatory functions as they can influence many other cell types, limiting or enhancing the immune response. NK cells must effectively recognize the target cells while ensuring tolerance to “self” cells . This is+ T-cells that recognizes and acts via the HLA-E ligand expressed on the target cells. The HLA-E/NKG2A axis plays a role in colorectal cancer ], and the specific nonameric peptide  derived from the signal sequences of other HLA class I molecules  . Remarkamination . Furthermination . In our omplexes .+ immune cells. These are Hsp60sp, R5V mutated leader peptide of HLA-B7  simulations of naturally occurring and alternative designed variants of the nonameric peptides in complex with HLA-E/β2m ligand alone or in the HLA-E/β2m/NKG2A/CD94 complexes to broaden the understanding of the molecular background for their diverse binding affinities which are often associated with only small changes in their structure. Simulations revealed new peptide variants with comparable affinity as natural peptides with highest HLA-E binding affinities. One investigated variant can according to our derived atomistic model , interfeAltogether we built 12 models consisting of HLA-E heavy and HLA-E light chain [called beta-2-microglobulin (β2m)] with various nonameric peptides incorporated into the HLA-E complex along with the apo form of HLA-E. Next, we constructed four models containing the NKG2A/CD94 heterodimer in addition to the HLA-E heavy and light chains and the corresponding nonameric peptide accounting for a total of 17 simulated models. All HLA-E complexes are built using either the crystal structure of the human non-classical major histocompatibility complex molecule HLA-E with bound class Ia MHC leader peptide derived from HLA-B8, solved at 2.85 Å resolution (PDB entry 1MHE) or the crystal structure of human HLA-E, solved at the resolution of 2.50 Å that contains the leader peptide of the HLA class I histocompatibility antigen, alpha chain G (PDB entry 3BZE).HLA-EG_wt consists of HLA-E, β2m, and the G peptide (sequence: VMAPRTLFL), while the remaining models differ in nonameric peptide, incorporated into the HLA-E. The second model (ii) HLA-EB27_wt contains a peptide derived from HLA-B27 . The rest of the models of the HLA-E complex contain a peptide corresponding to the leader sequence of HLA-B8 and HLA-B7 (sequence: VMAPRTVLL). Namely, the third model (iii) HLA-Ewt contains the wild type of the B7 peptide. Then, single mutations of the B7 peptide were introduced in the following models: R5V for model (iv) HLA-ER5V, T6M for (v) HLA-ET6M, T6A for (vi) HLA-ET6A, T6F for (vii) HLA-ET6F, T6Y for (viii) HLA-ET6Y, T6W for (ix) HLA-ET6W, T6L for (x) HLA-ET6L, and T6I for (xi) HLA-ET6I. We also constructed a model with the wild type B7 peptide with capped peptides’ N-terminal residue, (xii) HLA-Ecap. Capped residues are used to neutralize terminals in peptide chains and for neutralization of peptides’ N-terminal in HLA-Ecap model we used the ACE (-C(=O)-CH3) residue. Finally, we prepared the apo model of HLA-E/β2m, (xiii) HLA-Eapo. In the superscript of all model names the name or the variant of the subjected nonameric peptide is listed.The first model (i) NKG2A/HLA-ET6M, T6Y in (xv) NKG2A/HLA-ET6Y, T6W in (xvi) NKG2A/HLA-ET6W, and T6I in (xvii) NKG2A/HLA-ET6I. All x-ray structures used in our simulation contain arginine residue at the position 107 thus corresponding to the HLA-E*01:01 variant.All simulated HLA-E/β2m/peptide/NKG2A/CD94 complexes are based on the crystal structure of CD94/NKG2A in complex with HLA-E with the incorporated leader sequence of HLA-G, solved at 3.40 Å resolution (PDB entry 3CDG). Peptide G derived from HLA-G, originally present in the crystal structure, was replaced with the peptide B7  to which single mutations were introduced, based on the initial computational assessment of known peptides: T6M in model (xiv) 3 × 103.623 Å3 × 99.798 Å3. The assembled systems accommodated approximately 85,000 atoms, including water molecules and 15 Na+ counterions. The dimensions of the HLA-E/β2m/peptide/NKG2A/CD94 complexes are 130.968 Å3 × 123.440 Å3 × 122.079 Å3, including water molecules and 18 Na+ counterions, the systems numbered approximately 193,500 atoms. Tleap module of Ambertools 18 was used to construct disulfide bonds and model topologies -based MD simulation was performed over the next 10 ns, where all restraints were removed and a Berendsen barostat was used to control the pressure of 1 bar  between the peptide and the rest of the protein complexes as well as its per-residue distribution were calculated by the Molecular Mechanics-Generalized Born Surface Area (MM-GBSA) method (The binding free energies (ΔG) method  and the ) method . The cal) method . The int) method .To gain a deeper understanding of the interaction patterns between different peptide variants and HLA-E ligand, we also derived dynamic pharmacophore models . 1,000 equally spaced frames between 10 ns and 1 μs of the equilibrated parts of the trajectories were exported and analyzed using the DynophoreApp . The resHLA-EG_wt, HLA-EB27_wt, HLA-Ewt, HLA-ER5V, HLA-ET6M, HLA-ET6A, HLA-ET6F, HLA-ET6Y, HLA-ET6W, HLA-ET6L, HLA-ET6I, HLA-Ecap, and HLA-Eapo, while the remaining four models also encompass the receptor NKG2A/CD94 counterpart: NKG2A/HLA-ET6M, NKG2A/HLA-ET6Y, NKG2A/HLA-ET6W, and NKG2A/HLA-ET6I, where the superscript indicates the name or the variant of the subjected nonameric peptide.All models simulated in this study were firmly based on the available crystal structures of the extracellular domains of the human CD94/NKG2A proteins in complex with HLA-E (PDB entry: 3CDG) or HLA-E ligand alone . Constructed models include all resolved components consisting of extracellular domains of HLA-E, β2m, and nonameric peptide nested between the α1 and α2 domains of the HLA-E, referred to as ligand part. The full immune complexes consisting of ligand and receptor parts also includes the resolved extracellular domains of the NK receptor counterpart, namely proteins NKG2A and CD94 . TogetheHLA-Ewt, HLA-EG_wt, and HLA-EB27_wt using the MM-GBSA method  with experimentally reported affinities for HLA-E . Per-resA method   capping residue, and perform another MD simulation. Comparison of the per-residue decompositions of the capped peptide of model HLA-Ecap with the wild type peptide HLA-Ewt  revealed an interesting orientation of the side chain of the Arg62 peptide . This ob peptide . Such pog groove . Thus, bHLA-Ecap model revealed that capping of P1 did not improve the binding affinity of the peptide. Both, binding free energy and interaction energy between the capped peptide and the HLA-E/β2m ligand proved to be less favorable than the energies of the HLA-Ewt with bound B7 peptide and closer to that of the HLA-EG _wt model.The MM-GBSA binding free energy as well as Gromacs interaction energy calculations successfully reproduced the experimentally determined order of binding affinities of all three wild-type peptides B7, G, and B27 for HLA-E/β2m  Braud e. This prThese results suggest that the nonameric peptide’s P6 position holds the most potential for further optimization to enhance its binding to the HLA-E ligand. Finally, we analyzed the peptide binding site on the HLA-E with the grid apo-site analysis performed in Ligandscout  as anothBased on the results obtained, the hydrophobic nature seems to be strongest near the P6 site, where the introduction of additional charged structural elements could lead to a desolvation penalty , which c+ NK cells  models, also known as dynamic pharmacophores, for each of the 12 simulated HLA-E:peptide complexes. In them, dynamic nature of the MD simulations and static structure-based pharmacophore models are combined, resulting in a dynamical characterization of the peptides interaction patterns throughout the simulations . In thisondensed . We also peptide .We further substantiated the dynophore geometric analysis with per-residue MM-GBSA decomposition analysis for the generated peptide variants and compared it to the observations obtained for the three wild type peptides with experimentally reported data. All mutations at the peptides 6th residue, with the exception of T6A, resulted in greater contribution of P6 to the binding . HoweverThe per-residue analysis further shows that in all simulated cases the C-terminal site of the peptide is more stably bound, as here two residues (P7 and P9) with large contribution to binding could be observed. On the other hand, only one such residue (P2) could be found at the peptide N-terminal site. The central residue (P5) also makes a large contribution to binding . JoiningHLA-ET6W system resulted in an early detachment of the N-terminal site (residues P1–P4) of the peptide from its binding pocket. Interestingly, the C-terminal site of the peptide (residues P5–P9) remained bound until the end of this simulation  and ligand (HLA-E/β2m/peptide) recognition and outcome.Together, we simulated and analyzed four HLA-E/β2m/peptide/NKG2A/CD94 immune complexes with designed variants of the peptide B7 containing the following mutations: T6M, T6Y, T6W, and T6I. The T6I variant was selected because of its previously discussed favorable HLA-E binding properties. Because the T6W variant partially detached from the HLA-E binding pocket in the In addition to the variants of peptide B7 mutated at position 6, we also present here some new data that we extracted from the simulated HLA-E/β2m/peptide/NKG2A/CD94 complexes with peptides G, B7, B7 with R5V mutation, and its apo model, that we generated and simulated in the scope of our previous work where we studied the molecular recognition events necessary for the inhibitory signal transduction . AccordiCD94-Lys135NKG2A and Ser109CD94-Lys135NKG2A interaction distances as well as their assessment via dynophore models led to the conclusion that T6Y, T6W, and T6I peptide variants are likely to allow the receptor-ligand recognition to take place, thereby preventing the cytotoxic action of the NK cell. On the other hand, the NKG2A/HLA-ET6M model with T6M mutation of the B7 peptide shows the presence of the proposed key interactions at frequencies closer to the negative NKG2A/HLA-ER5V model containing the B7_R5V peptide that abrogates HLA-E-mediated protection. Furthermore, the NKG2A/HLA-ET6M model also has similar average distances between the heavy atoms of the interacting residues as NKG2A/HLA-ER5V. In contrast, the remaining three models with petide variants display comparable features to the models that allow inhibitory signal transduction: NKG2A/HLA-EG_wt and NKG2A/HLA-Ewt  binds to HLA-E with much higher affinity as those with threonine   and thisvia NKG2A/CD94  is the most effective mediator of the inhibitory signalling , despiteG2A/CD94 . Thus, tG2A/CD94  in hematG2A/CD94 .+ T-cell-mediated cytolysis via the NKG2A/CD94 interaction. The data obtained also lay the necessary structure-based foundation for future development of potential therapeutic peptides, peptidomimetics, or even small molecules that would bind to the HLA-E ligand and then abrogate NKG2A/CD94 recognition. The utility of any dynamic model obtained can be further extended to describe the nature of peptide binding to HLA-E/β2m at the atomistic level, offsetting the disadvantage of the lower throughput of MD simulations compared with cell assays  simulations of the HLA-E/β2m/peptide complex enhanced the atomistic understanding of the peptide binding to its binding pocket on HLA-E and the influence of single mutations to binding. Subsequent simulations of HLA-E/β2m/peptide/NKG2A/CD94 immune complex shed a light on the effect of introduced point mutations on the inhibitory signal transduction that protects the cell from the cytotoxic effects of the immune NK and CD8


In this paper, we present a formulation study aimed to develop naked and decorated H2S-DOXO-loaded lipid nanoparticles (NPs). NPs prepared by the “cold dilution of microemulsion” method were decorated with hyaluronic acid (HA) to obtain active targeting and characterized for their physicochemical properties, drug entrapment efficiency, long-term stability, and in vitro drug release. Best formulations were tested in vitro on human-sensitive (MCF7) and human/mouse DOXO-resistant (MDA-MDB -231 and JC) breast cancer cells, on human (U-2OS) osteosarcoma cells and DOXO-resistant human/mouse osteosarcoma cells (U-2OS/DX580/K7M2). HA-decoration by HA-cetyltrimethyl ammonium bromide electrostatic interaction on NPs surface was confirmed by Zeta potential and elemental analysis at TEM. NPs had mean diameters lower than 300 nm, 70% H2S-DOXO entrapment efficiency, and were stable for almost 28 days. HA-decorated NPs accumulated H2S-DOXO in Pgp-expressing cells reducing cell viability. HA-decorated NPs result in the best formulation to increase the inter-cellular H2S-DOXO delivery and kill resistant cells, and therefore, as a future perspective, they will be taken into account for further in vivo experiments on tumor animal model.Several semisynthetic, low-cardiotoxicity doxorubicin (DOXO) conjugated have been extensively described, considering the risk of cytotoxicity loss against resistant tumor cells, which mainly present drug efflux capacity. Doxorubicin 14-[4-]-benzoate (H Streptomyces species, which is used alone or in combination with other anticancer drugs in the treatment of hematologic cancers and solid tumors, lymphomas, and sarcomas. It is presently considered one of the most effective chemotherapeutic agents and has been used to treat cancer for over 40 years -benzoate (H2S-DOXO) was subsequently tested in vitro and in vivo in castration-resistant prostate cancer (CRPC), against which chemotherapy is only transiently effective due to the emergence of chemoresistance, to investigate whether H2S-DOXOs are able to overcome drug resistance and to test their safety. In an androgen-independent and DOXO-resistant DU-145 prostate cancer cell line, good intracellular accumulation and high cytotoxicity were observed [2S-DOXO in nude mice previously injected subcutaneously with DU-145 cells was a reduction in tumor volume by 60% and an increase in the percentage of apoptotic cells. In addition, administration of free DOXO was associated with weight loss and cardiotoxicity, whereas H2S-DOXO was well tolerated and had a better safety profile. Therefore, the authors demonstrated that H2S-DOXO represents novel strategies to reverse chemoresistance in the treatment of CRPC.Of the various Hobserved . The eff2S-DOXO (HALs-H2S-DOXO) were prepared with the aim of exploiting the overexpression in osteosarcomas of the hyaluronic acid (HA) receptor CD44, known to promote tumor growth and metastatic spread [2S-DOXO showed higher in vitro toxicity in mouse DOXO-resistant osteosarcoma cells (K7M2) and human DOXO-sensitive osteosarcoma cells (U-2OS) than DOXO alone or Caelyx®. In addition, they exhibited a favorable drug release profile and showed the same cardiotoxicity as the marketed liposomal DOXO Caelyx® in 6-week-old K7M2 tumor-bearing female Balb/C mice. Moreover, neither DOXO nor Caelyx® reduced the growth of these tumors, which were selected as models for CD44- and Pgp-overexpressing osteosarcomas, nor did they induce apoptosis in the tumor, whereas HALs-H2S-DOXO increased caspase-3 activation in the tumor and decreased Pgp expression in the tumor.Nanotechnology has shown promising advancements in the field of drug development and delivery and its applications for diagnosis, monitoring, control, and treatment of biological systems have been described as nanomedicine . In partc spread ,18 and wc spread . HALs-H22S-DOXO should be carried. The idea was to increase H2S-DOXO chemical stability towards hydrolysis and to overcome the phenomena of self-aggregation, coalescence, flocculation, and precipitation that can occur during liposome formulation or storage, leading to degradation of the vesicle structure and decomposition of the liposomes themselves [On the basis of these stimulatory results and of the previous results we obtained with lipophilic DOXO derivatives ,21, we demselves .2S-DOXO derivatives described in the literature [2S-DOXO: the ester group and the H2S donor substructure, which follow pseudo-first-order kinetics with t1/2 2.8 h.Recently, we developed a NP production technique called “cold dilution of microemulsion” ,24, whicterature  hydrolyz2S-DOXO in a lipid matrix rather than in the aqueous environment of liposomes could improve its stability during storage and after administration.We hypothesize that the inclusion of H2S-DOXO-loaded NPs. After screening the physicochemical properties, drug entrapment efficiency, long-term stability, and in vitro drug release, the best formulations were tested in vitro on human DOXO-sensitive (MCF7) and DOXO-resistant (MDA-MDB-231) breast cancer cells, on DOXO-resistant mouse breast cancer cells (JC), on human (U-2OS) osteosarcoma cells, DOXO-resistant human osteosarcoma cells (U-2OS/DX580), and on DOXO-resistant mouse osteosarcoma cells (K7M2).In this paper, we present a formulation study aimed at selecting the best conditions and the most suitable ingredients for the development of naked and HA-decorated H2S-DOXO in aqueous and organic solvents is reported in The solubility of H2S-DOXO in water/acetonitrile 60/40 v/v  and in methanol (solvent used for extraction from NPs to determining EE%) was tested by RP-HPLC after 30-day storage (t30). In The chemical stability of H2S-DOXO in water/acetonitrile 60/40 v/v in the presence of µE ingredients, at the same ratio (w/w) used in the µE, was tested to select the suitable ingredients and to ensure the integrity of H2S-DOXO into the formulation. In The chemical stability of H2S-DOXO in aqueous and organic solvents was essential for the following selection of the ingredients for the NP set-up.The knowledge of the stability and solubility of Hw/w) are reported in The optimal blank µE formulations (F1 series) without and in the presence of ST or CTAB (2.5% ®200 and NaGC were selected as surfactant/cosurfactant system. Cremophor ®RH60 and NaTC were excluded for H2S-DOXO instability problems. ST is an established cationic molecule employed in drug delivery systems to confer a positive charge to the surface of lipid matrix (NPs) [2S-DOXO, as observed in chemical stability tests  at different concentrations .Epikuron ix (NPs)  or bilayix (NPs) . ST was ty tests . In the After the formulation step, it was crucial to determine mean diameters and Zeta potentials of the different NPs.As the mean diameter in water, determined by DLS, was greater than 500 nm, the mean diameters of the NPs (NP series) were determined in aqueous polymer solutions, to choose the best stabilizing polymer .NPs dispersed in 3% PVP have a mean diameter of around 150 nm, which is the smallest in the whole NP series. A highly negative  Zeta potential was the result for all NP series. The negative surface charge could be due to the presence of NaGC in the lipid matrix, while the disposition of each polymer on NPs, although influencing surface hydrophilicity, does not modify its charge.On the contrary, when µE F1-ST and F1-CTAB were diluted by water , they gaThe presence of a cationic lipid (ST) or a cationic surfactant (CTAB) determined a dramatic change in NP surface charge, as shown by the highly positive value of Zeta potentials, which were in the +18/+20 mV range.w/v). When the HA concentration increased, the negative surface charge also increased.The resulting cationic NPs were decorated with HA via electrostatic interaction between the negative charge of HA and the positive charge of ST or CTAB. The presence of the outer layer of HA dramatically increased particle size and converted the surface charge to a negative value again . To obtaw/v HA, NP-CTAB were also dispersed only in 0.05% w/v HA . H2S-DOXO was also incorporated in µEs F1-ST and F1-CTAB and dispersed in water to facilitate the subsequent ionic bond with HA (used at 0.05% w/v). The obtained NPs were characterized by mean diameter, Zeta potential, and encapsulation efficiency (EE%) measurements  or without HA were used for further characterizations, in terms of microscopic observation and stability studies over time.NPs containing ST, as a positively charged agent, have larger diameters than those containing CTAB, and very low EE%, confirming the results obtained from the chemical stability tests of the DOXO-derivative in presence of ST. Therefore, NP-CTAB-H2S-DOXO 3% PVP and HA-NP-CTAB-H2S-DOXO microphotographs are reported in 2S-DOXO loaded disperse systems, as only particles with a mean diameter higher than 500 nm were visualized, according to optical microscopy resolution power. Anyway, the presence of H2S-DOXO crystals and diffused fluorescence (due to the presence of fluorescent molecule out of the NP matrix) were excluded by observation under polarized light.NP-H2S-DOXO and H2S-DOXO solution over 5 h are reported in Cumulative drug release profiles from HA-NP-CTAB-H2S-DOXO release in 5 h was almost 100% from an aqueous solution with a typical first-order curve (R2 = 0.999). On the other hand, only 40% of the H2S-DOXO incorporated in NP matrix was released after the same time; a typical burst effect was noted in the first hour. These results suggest that most H2S-DOXO is likely be entrapped within NPs.H2S-DOXO 3% PVP and the NPs with the same composition but without H2S-DOXO (NP in 3% PVP) were stable for 28 days (t28) at 4 °C, as reported in 2S-DOXO 3% PVP.Stability studies were performed on those systems which had the best characteristics in term of mean diameter, Zeta potential, and EE%. NP-H2S-DOXO  for 72 h, both being only slightly modified, while EE% dropped to 42% at 25 °C storage  was performed on the 0.05% TEM observation was mainly aimed to assess the presence of HA on NP surface thanks to elemental analysis. In fact, from this analysis, it was also possible to obtain, in addition to an enlarged image of individual NPs, data regarding the elements present on their surface. In fact, from the morphological analysis, it is difficult to distinguish and discriminate between coated and naked NPs. Thanks to the elemental analysis, however, it was possible to obtain information on the presence or absence of HA.As shown in ® 200), while its detection on the surface of NPs decorated with HA may be due to the large amount of glycosaminoglycan.Due to the lightness of this chemical element, detecting the presence of nitrogen by TEM is rather difficult when present in traces, as in the case of naked NPs which possess small quantities of CTAB and lecithin .In a first experimental set, we incubated cells for 6 h with 5 µM free DOXO, free HWe chose two tumor types—osteosarcoma and breast cancer—in which DOXO is used as first-line treatment, but where problems of acquired resistance often occur ,28. DOXO2S-DOXO 3% PVP did not produce a higher intracellular drug delivery than DOXO or H2S-DOXO in sensitive cell lines, characterized by low levels of Pgp [2S-DOXO 3% PVP and particularly HA-NP-CTAB-H2S-DOXO significantly increased the intracellular drug retention in resistant MDA-MB-231 and U-2OS/DX580 cells, both characterized by high levels of Pgp [2S-DOXO is less effluxed by Pgp, because it promotes a sulfhydration followed by degradation of the protein, either as free drug [As shown in s of Pgp ,30, the s of Pgp ,30. The s of Pgp . Collectree drug .2S-DOXO than with NP-H2S-DOXO 3% PVP, likely because HA grants an active targeting of the NPs. Indeed, the HA receptor, CD44, has been detected in U-2OS/DX580 and K7M2 cells [The use of NPs likely offers the further advantage of a better delivery of the drug inside the cells, favoring the accumulation of DOXO. The effect was more pronounced with HA-NP-CTAB-HM2 cells , and is M2 cells . These e2S-DOXO, NP-H2S-DOXO 3% PVP, and HA-NP-CTAB-H2S-DOXO in sensitive MCF-7 and U-2OS cells. By contrast, free DOXO was devoid of any cytotoxic effects against resistant MDA-MB-231, JC, U-2OS/DX580, and K7M2 cells. H2S-DOXO, either as free drug or loaded in NPs, induced a significant release of LDH , polyvinyl alcohol 9000 (PVA 9000), and Kollidon®25 (PVP) were from Merck , Epikuron®200 (phosphatidylcholine 92%) was from Cargill , doxorubicin hydrochloride (DOXO) was purchased from APAC Pharmaceutical , Cremophor®RH60 (PEG-60 hydrogenated castor oil) from BASF , taurocholic acid sodium salt (NaTC) and glycocholic acid sodium salt (NaGC) were from ICN Biomedicals , and trilaurin and tristearin from Alfa-Aesar . Deionized water was obtained by a MilliQ water purification system . Unless specified otherwise, all reagents were purchased from Merck.Hyaluronic acid sodium salt 1600 kDa (HA) was purchased from Farmalabor , stearylamine (ST), cetyltrimethylammonium bromide (CTAB), ethyl acetate (EA), propylene glycol, benzyl alcohol, Pluronic2S-DOXO and the RP-HPLC analysis were performed as described in Chegaev et al. [The synthesis of Hv et al. .2S-DOXO in water/acetonitrile (60/40 v/v ratio), methanol, 0.1 M pH 7.2 phosphate buffer, propylene glycol, and benzyl alcohol was determined as follows: an excess of H2S-DOXO was added to the organic or aqueous solution (1 mL); after 2-h equilibration at room temperature (22–23 °C) in a sealed vial under 600 rpm magnetic stirring, the suspension was centrifuged at 1397× g for 10 min, and the supernatant solution analyzed by RP-HPLC. The time needed to reach equilibrium solubility was determined by analyzing samples of the equilibrating solution at different time points to establish constant drug solubility.The solubility of H2S-DOXO stability in water/acetonitrile (60/40 v/v ratio) and methanol was determined after 30-day (t30) storage at 4 °C following the drug concentration over time by RP-HPLC and comparing it with the concentration at t0, assuming it as 100%.H2S-DOXO stability in water/acetonitrile (60/40 v/v ratio) in the presence of NaTC, NaGC, lecithin (phosphatidylcoline 95–98%), ST, CTAB, and Cremophor®RH60 was determined at t0 and after 24 h (t24) by RP-HPLC comparing the drug concentration with that in water/acetonitrile solution at t0, assuming it as 100%.HNPs were prepared by the method named “cold dilution of microemulsion” ; briefly2S-DOXO was stable. EA, chosen as water miscible solvent, and the aqueous phase (0.1 M pH 7.2 phosphate buffer) were mutually pre-saturated before using them in µE preparation (EAs and PBSs respectively). A total of 1 mL of the formulated µE was then diluted with 5 mL water or a polymeric aqueous solution to precipitate NPs. Various concentrations of aqueous solutions of several polymers, PL F68, PVA 9000, and PVP, were tested to check the best conditions to obtain small NPs and to avoid NPs aggregation [In the present paper, after a preliminary screening on several compositions, µEs were obtained with biocompatible GRAS ingredients  in which Hregation . A very 2S-DOXO were incorporated in 1 mL of the selected µE, prepared with those surfactants/cosurfactants and cosolvents that allow to maintain the structural drug stability.Finally, 2 mg of H2S-DOXO-loaded NPs and free H2S-DOXO were separated by gel filtration using Sepharose CL-B4 column.H2S-DOXO  aqueous dispersion was added into 5 mL of HA solution  dropwise under gentle stirring. After 30 min, electrostatic interactions between HA and cationic NPs led to the formation of HA-NP-CTAB-H2S-DOXO or HA-NP-ST-H2S-DOXO. Blank HA-NP-CTAB and HA-NP-ST were prepared without H2S-DOXO according to the above-described protocol.HA decorated NPs were prepared to obtain active targeting. HA decoration was achieved by the electrostatic interaction between HA and CTAB or ST on the NPs surface ,36. CTAB®G-25 column.Unreacted HA was separated by gel centrifugation using SephadexParticle size and Zeta potential measurement. Particle size and Zeta potential of NPs were determined by dynamic light scattering (DLS) using the Zetasizer Nano ZS90 . Before each measurement, the dispersion of NPs was diluted with Milli-Q water. All measurements were taken at 25 °C and each sample was analyzed in triplicate.Optical microscopy analysis. The possible aggregation of NPs was observed using an optical microscope  equipped with a fluorescent lamp  connected to a digital camera at × 630 magnification. The microscope images were analyzed using the Motic Images 2000 software.Transmission electron microscopy (TEM). The shape and morphology of naked and HA-decorated NPs were studied by transmission electron microscopy (TEM) via JEOL JEM 3010-UHR  microscope operating at 300 kV and equipped with an Oxford Inca Energy TEM 200 EDS X-rays analyzer. For the measurements, samples were prepared by dripping on a carbon-coated copper grid and water was evaporated in air.Determination of drug entrapment efficiency (EE%). H2S-DOXO concentration in NP suspension, after elution in gel filtration (GF) and gel centrifugation (GC) column, was determined by the HPLC method used for in vitro quantification of drug. The drug amount resulting after GF and GC was considered as totally entrapped in the NP matrix. EE% is calculated as the ratio between the amount of NP-entrapped drug (post-column drug concentration) and the total amount used in NP preparation (pre-column drug concentration) × 100.In vitro drug release. Release of H2S-DOXO from NPs was evaluated using the dialysis method [2S-DOXO was placed in a dialysis bag, and the dialysis bag, sealed at the ends, was immersed into 40 mL of phosphate buffered saline  at 37 °C stirring at 100 rpm. At predetermined time intervals, 1 mL PBS was withdrawn and replaced with an equal volume of fresh PBS. Release was monitored for 5 h. The overtime released H2S-DOXO was analyzed by RP-HPLC. As a reference, a 20% propylene glycol aqueous solution of H2S-DOXO was used.s method . An aliqStability studies of NP formulations. The stability of NP systems was studied in terms of mean diameter, Zeta potential, and EE% after 28-day storage at 4 °C and after 72-h storage at 25 °C.Cell lines. Human breast cancer MCF-7 and MDA-MB-231 cells, murine mammary cancer JC cells, human osteosarcoma U-2OS cells, and murine osteosarcoma K7M2 cells were purchased by the American Type Cell Collection (ATCC). U-2OS/DX580 cells were generated by parental U-2OS cells with a stepwise selection in a medium containing increasing concentration of DOXO and after 25 passages maintaining in the presence of 580 ng/mL of DOXO in the culture medium [v/v penicillin-streptomycin and 10% fetal bovine serum, at 37 °C and 5% CO2.e medium . Cells w2S-DOXO, or with 5 μM DOXO or H2S-DOXO, as reference. Samples under study were: free DOXO, free H2S-DOXO, NP-H2S-DOXO 3% PVP, and HA-NP-CTAB-H2S-DOXO.In each of the following experimental sets, cells were incubated with specific dilutions of NPs to reach a final concentration of 5 μM HIntracellular DOXO accumulation. 2 × 106 cells were rinsed with fresh medium, detached with trypsin/EDTA (0.05/0.02% v/v), rinsed in 0.4 mL of a 1:1 solution ethanol/HCl 0.3 N, and sonicated in ice with 10 s . Moreover, 5 μL of cell lysate were used to measure the intracellular protein content . The remaining sample was used to measure the intracellular content of DOXO fluorimetrically, using a Synergy HTX 96-well reader , using λem = 475 nm, λexc = 553 nm. The fluorescence units were transformed in nmoles DOXO/mL, according to a titraton curve prepared with scalar dilutions of DOXO (range 1 μM–1 nM). The results were expressed as nmol DOXO/mg cell proteins.Acute cytotoxicity. Culture medium from 2 × 106 cellule was centrifuged at 12,000 g for 15 min to pellet cell debris, while cells were washed with PBS twice, detached with trypsin/EDTA (0.05/0.02% v/v), rinsed in 0.2 mL of 82.3 mM triethanolamine phosphate-HCl (pH 7.6), and sonicated in ice with 10 s ; 5 μL of cell lysate was used to measure the intracellular protein content (BCA1 kit). The activity of extracellular lactate dehydrogenase (LDH), considered as an index of DOXO acute cell damage [Viability. 1 × 105 cells were seeded in 96 well plates and incubated for 72 h with DOXO, H2S-DOXO or the different NPs. Cell viability was evaluated using the ATPLite kit , as per manufacturer’s instructions, using a Synergy-HTX 96-well reader . The relative luminescence units (RLUs) of the untreated cells were considered 100%; the RLUs of the other experimental conditions were expressed as percentage versus untreated cells.Statistical analysis. All data in the text and figures are provided as means ± SEM. The results were analyzed by a one-way analysis of variance (ANOVA) and Tukey’s test. p < 0.05 was considered significant.2S-DOXO in phosphate buffer at pH 7.2, at which the charge on the amino sugar portion is neutralized, H2S-DOXO can be more easily internalized into the oily phase of the O/W µE. Its good solubility in the solvents used in the µE formulation improves its loading. µE was formulated using those ingredients in which H2S-DOXO was more stable for at least 24 h. Although CTAB was found to be the positively charged agent in which H2S-DOXO was most stable, ST was also tested. Unfortunately, subsequently, it was revealed to be not optimal to H2S-DOXO loading in NPs. For stability purposes, NPs need to be dispersed in a polymer solution and the 3% w/v PVP solution was the best in terms of resulting NPs average diameter. Positively charged NPs, stabilized by cationic agents, had optimal diameters when dispersed in water. The successful conjugation of positive NPs with HA was confirmed by the inversion of the Zeta potential charge and by the elemental analysis at TEM. All the developed systems, blank and H2S-DOXO-loaded, naked or HA-decorated were stable for at least 28 days in terms of diameters and Zeta potential. Samples must be stored at 4 °C. In an in vitro cell study, H2S-DOXO loaded in NPs (more evident in HA-NP-CTAB-H2S-DOXO than in NP-H2S-DOXO 3% PVP) accumulates in resistant cells with high levels of Pgp more than DOXO and free H2S-DOXO, although no increase in acute damage (24 h) was noted, as H2S-DOXO was probably released slowly from NPs. Instead, after 72 h, a reduced cell viability was observed in resistant breast and osteosarcoma of both human and murin origin. HA-NP-CTAB-H2S-DOXO resulted the best formulation to increase the inter-cellular drug delivery and kill resistant cells.Given the poor solubility of the H


Sleeping Beauty (SB) is the first DNA transposon employed for efficient transposition in vertebrate cells, opening new applications for genetic engineering and gene therapies. A transposon-based gene delivery system holds the favourable features of non-viral vectors and an attractive safety profile. Here, we employed SB to engineer HEK293 cells for optimizing the production of a chimpanzee Adenovector (chAd) belonging to the Human Mastadenovirus C species. To date, chAd vectors are employed in several clinical settings for infectious diseases, last but not least COVID-19. A robust, efficient and quick viral vector production could advance the clinical application of chAd vectors. To this aim, we firstly swapped the hAd5 E1 with chAd-C E1 gene by using the CRISPR/Cas9 system. We demonstrated that in the absence of human Ad5 E1, chimp Ad-C E1 gene did not support HEK293 survival. To improve chAd-C vector production, we engineered HEK293 cells to stably express the chAd-C precursor terminal protein (ch.pTP), which plays a crucial role in chimpanzee Adenoviral DNA replication. The results indicate that exogenous ch.pTP expression significantly ameliorate the packaging and amplification of recombinant chAd-C vectors thus, the engineered HEK293ch.pTP cells could represent a superior packaging cell line for the production of these vectors. These features highlighted the advantage of chAd versus hAd5 as vaccine carrier in humans. To date, a number of clinical trials aimed at preventing the infection of Ebola, Malaria, HIV, Hepatitis C Virus, Respiratory Syncytial Virus [Human Adenoviral vectors, in particular those belonging to serotype 5 (Ad5), have been extensively used in clinical trials with an excellent safety profile, mainly as vectors for Human Immunodeficiency Virus (HIV) vaccines ,2. Howeval Virus  and receal Virus . HoweverS. pyogenes (SpCas9) driven by a guide RNA (gRNA) to the target DNA, where it induces double-strand breaks (DSBs) [A possible strategy to establish an engineered HEK293 cell line for chAd viral vector production exploits CRISPR-mediated genome editing. The CRISPR/Cas9 system is an effective genome editing tool relying on an engineered nuclease from s (DSBs) ,11. The s (DSBs) ,13,14,15s (DSBs) , and couSleeping Beauty system [Sleeping Beauty (SB) belongs to the Tc1/mariner superfamily of DNA transposons [Sleeping Beauty could be delivered as a non-viral system through nucleofection [As alternative strategy, adenoviral factors could be modulated to improve vector production. Indeed, it has been shown that human Ad5 pre-Terminal Protein (pTP) overexpression enhanced viral replication of oncolytic Adenovirus in cancer cells . Moreovey system  to stablnsposons  comprisinsposons  and follnsposons ,24,25. Ansposons  and is bnsposons , the T2 nsposons ,29 and tofection  or lipofofection , Adeno-aofection , Baculovofection  and inteofection  have beeTo achieve our aim, we transposed  the ch.pOur results demonstrate that ch.pTP-overexpressing HEK293 cells improve yield and quality of recombinant chAd-C vectors. Moreover, clones #13 and #25 isolated from HEK293ch.pTP bulk represent good candidates for new packaging cells tailored to the production of chAd-C vectors, already employed in clinical trial for vaccine therapy.Several simian Adenovirus have been employed as E1-deleted Ad vectors for gene delivery or vaccine vectors exploited in human therapies ,37,38,39Sleeping Beauty system, which allows a genuine and safe integration profile [To establish a stable HEK293 cell line able to boost the production and amplification of chAd vectors, we sought to permanently express the pTP from chAd-C (ch.pTP) belonging to Human Mastadenovirus C species  into HEK profile  of the gPmeI-linearized plasmid DNA into engineered HEK293ch.pTP or parental HEK293 cells and measured the viral titers  rescued at P0 and propagated at passage 1 (P1). No further amplification steps were performed since the effect of the ch.pTP expression in the packaging cells would benefit preferentially the P0 of vector production which, in HEK293 cells, occurs in the absence of available initiator of Adenoviral replication. We measured, by Real Time PCR, the amount of ch.pTP mRNA produced during the packaging of a chAd-C vector at P0 from 3 dpt up to full cytopathic effect (15 dpt), in HEK293ch.pTP cells compared to HEK293 cells (n = 4) harvested at the same time frame (15 dpt) indicated that chAd-C Venus-rescue (P0) was significantly more efficient in engineered HEK293ch.pTP cells compared to the parental HEK293 (n = 3), as measured by VP/mL and TU/mL , measured by TU/mL, was approximately 4-fold more efficient in HEK293ch.pTP cells and the P1 in these cells produced 6-fold more transducing particles compared to HEK293 cells . Again, Once demonstrated the stable packaging advantages of the engineered HEK293ch.pTP cells, we performed cell cloning to isolate HEK293ch.pTP clones producing high-titer chAd-C vector. The expression of ch.pTP in 34 isolated clones was measured by RTqPCR A. Nine cThe selected nine clones were also tested, in two independent experiments, for the efficiency to package the chAd-C Venus vector compared to HEK293ch.pTP and HEK293 cells. Compared to HEK293 cells, all the selected clones showed a similar or higher production of chAd-C at P0 A,C, and Overall, these data indicate that exogenous expression of ch.pTP can significantly improve the rescue and propagation of chAd-C vectors and new packaging clones for chAd-C vector production were isolated.2–103 VP/cell [The adenoviral E1-deleted vectors isolated from chimpanzees used so far, i.e., chAd-C show reduced yields upon propagation on HEK293 cells (10 VP/cell ) indicatThereby, we replaced the human Ad5 E1 with chAd-C E1 in HEK293 cells with a CRISPR-based strategy. However, although chAd-C belongs to the highly immunogenic virus of C species, its E1 gene does not have the potential to stably transform human cells such as the hAd5 E1 gene. It is accepted that transformation of primary human cell lines with Adenovirus-derived sequences is a highly inefficient process, and it is possible that HEK293 cells have adapted to, and depend on the specific hAd5 E1 sequences present in their genome for survival. Indeed, in hAd5-E1-transformed human amniotic fluid cells (a mix of at least two different populations), only mesenchymal stem cell lines  could be derived, and in these cells the expression pattern of the different E1-derived proteins is different from the one observed in HEK293 cells , raisingOverall hAd5 and chAd-C E1 share 73.4% amino acid homology: 66.7% for E1A-13S, 65.8% for E1A-12S, 69.31% for E1B-19K and 82.64% for E1B-55K. It is possible that chAd E1-derived proteins interact less efficiently than their hAd5 counterpart with host cell factors necessary for transformation, including the Retinoblastoma protein (pRb), p53, and components of the DNA repair machinery like Mre11 . This neSleeping Beauty system to stably integrate the ch.pTP in HEK293 cells. Although the transposition efficiency was remarkable, the hygromycin selection was required for the enrichment of cells expressing a level of ch.pTP compatible with HEK293 cell survival. Moreover, hygromycin selection without transposition events resulted in lower ch.pTP expression in the HEK293 bulk population, suggesting that a safe and genuine integration profile provided by the SB system benefits the expression of the ch.pTP.To pursue the aim of chAd-C growth optimization, we took advantage of the previously reported data claiming that overexpression of hAd5 pTP in HEK293 cells significantly improves the packaging and amplification of human recombinant Adenoviruses . We emplThe HEK293ch.pTP bulk and parental HEK293 were compared in the rescue and propagation of chAd-C Venus belonging to Human Mastadenovirus C species. HEK293ch.pTP cells significantly ameliorated the yield and the quality of chAd-C vector rescued at P0, underlining the key role of the pTP, the initiator of adenoviral replication. Indeed, the amount of chAd-C vector particles produced at P1 in HEK293ch.pTP and HEK293 cells were comparable, but the viral particles propagated in HEK293ch.pTP cells had a significantly higher infectious titer measured as TU/mL. Noteworthy, the ch.pTP integrated in the HEK293ch.pTP cell line shares over 99% homology at protein level with the pTP of the chimpanzee-derived Mastadenoviruses SAdV31.1, SAdV31.2, SAdV34, SAdV40.1 and SAdV40.2  reported by Roy et al.  hinting Not surprisingly, the chAd-E vector belonging Human Mastadenovirus E species benefits to a minor extent from HEK293ch.pTP cells, probably due to an uncomplete homology between the ch.pTP expressed in the HEK293ch.pTP and the pTP from chAd-E, which share an overall 79.05% identity.During viral DNA replication, pTP interacts with two additional viral E2 gene products, namely the ssDNA binding protein DBP and the viral DNA polymerase, and two host cell factors, the nuclear factor I (NFI) and Oct-1. We reasoned that if ch.pTP in HEK293ch.pTP cells is able to promote viral growth of chAd-C and, to a lesser extent, of chAd-E, the problem might lie in the interaction with viral factors, rather than with cellular factors. The crucial amino acid for pTP interaction with Ad DNA polymerase in hAd5 is Ser580 . This SeThe SB-mediated transposition of the ch.pTP in the HEK293 bulk confers a reproducible and stable production of chAd vectors over time allowing the isolation of new packaging clones for chAd vectors. Although no linear correlation between ch.pTP expression and chAd-C viral production was observed, clones #13 and #25 provided robust growth of the chAd-C Venus vector in two independent experiments. To conclude, we established a HEK293ch.pTP packaging cell line and we isolated new packaging clones to improve the generation and production of recombinant chAd-C vectors that would be beneficial for most applications in vaccine therapy.BbsI site, and then the U6-gRNA.E1B cassette into the pX330.gRNA.E1A. For the HDR donor plasmid pBS.chAd-C E1.HA, the chAd-C E1 sequence from the last 46 nt of the 5′-UTR of E1A to 20 nt-downstream E1B STOP codon was amplified by PCR from pchAd-C E1. The promoterless chAd-C E1 sequence was cloned between the 5′ and 3′ homology arms , amplified by PCR from hAd5 in HEK293 genomic DNA, and cloned in pBSIISK(−) plasmid. To generate the pBS.PGK.chAd-C E1.HA donor plasmid, the PGK sequence was cloned downstream of the 5′-HA, just before the chAd-C E1 sequence in the pBS.chAd-C E1.HA plasmid by digestion of the NheI site. The pCMVSB100X and pT2VENUS [To generate the effector plasmid pX330gRNA.E1A.gRNA.E1B, gRNA.E1A or gRNA.E1B were firstly cloned in the pX330-U6-Chimeric_BB-CBh-hSpCas9 plasmid, by oligo annealing in the pT2VENUS  plasmids5 cells was performed using Lipofectamine 2000  with 4 μg of total plasmid DNA. In transposition experiments, the transposon/transposase amounts of plasmid DNA was calculated accordingly to the stoichiometric ratio of 2:1 in a total quantity of 4 μg. In control samples, pCDNA3.1 plasmid  was used instead of pCMVSB100X. The percentage of Venus+ cells was determined by flow cytometry at indicated time points post-transfection. The transposition efficiency was calculated as %Venus+ cells 20 dpt/%Venus+ cells 2 dpt × 100. For Hygromycin selection, starting from the day after transfection, cells were treated with 0.2 mg/mL of Hygromycin to select antibiotic-resistant cells. To isolate single-cell clones, 36 days post-transfection Hygromycin-resistant bulk were limiting-diluted to obtain a concentration of 0.3 cells/well and seeded in a 96-well plate.HeLa and HEK293 cells were grown in cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% foetal calf serum (FCS), 100 U/mL penicillin and 100 mg/mL streptomycin . Transfection of 8 × 10The amount of viral particles produced during the rescue (P0) and propagation (P1), obtained by infecting HEK293s.pTP bulk or clones at MOI 150 (VP/mL), was measured using digital droplet PCR. In short, cells infected with the test vector were harvested at the indicated time point and subjected to three consecutive cycles of freeze/thawing at −80 °C/37 °C, followed by centrifugation at 2000 rpm at room temperature for 10 min to pellet debris.Ten- or hundred-fold-diluted virus-containing supernatants were treated with DNAse  at 37 °C for 30 min to digest unpackaged DNA, according to the manufacturers’ protocol. Following EDTA treatment to inactivate the DNAse, DNAse-resistant viral genomes were released by incubation of the mix with SDS at final concentration of 0.1%, and following another ten- to hundred-fold dilution, DNA was subjected to digital droplet PCR-based quantitation using promoter-specific oligonucleotides .4 HeLa cells per well. Forty-eight hours after infection with serially diluted chAd vector preparations, the percentage of EGFP or Venus positive cells was measured by acquiring on a FACS CANTO cytofluorimeter .The infectious titers of stocks of chAd vectors were determined by endpoint dilution in 96-well plates containing 10Genomic DNA was extracted from HEK293 cells treated for CRISPR-mediated replacement or pTP transposition, using QIAamp DNA mini or micro kits  following the manufacturer’s instructions, at indicated time points post-transfection or transduction. To analyse the replacement of hAd5E1 with chAd-C E1, genomic DNA of HEK293 cells co-transfected with effector plasmid and HDR donor plasmid was amplified by PCR with primers ChAdE1-3HA-F2 and PSG4-RC  to analyGAPDH assay  was used as housekeeping control. Reactions were performed at 50 °C for 2 min and 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. The relative expression of the target genes was normalized to the level of GAPDH housekeeping gene in the same cDNA by using the 2−ΔΔCT quantification. The replicated Relative Quantity (RQ) values for each biological sample were averaged. Total RNA was isolated with the RNeasy Mini and Micro kit  according to the manufacturer’s instructions. cDNA was synthesized in a 20 μL reaction by Superscript III Reverse Transcription kit . To evaluate the expression of ch.pTP mRNA from the integrated transgene and during the viral packaging, SYBR green Real Time PCR was performed with ABI Prism 7900 Sequence Detection System  with Power SYBR Green PCR Master Mix . Primers were designed to amplify both transcripts from the transgene and viral vector . Data were analysed for statistical significance using Student’s


Circular RNAs (circRNAs) play a critical role in colorectal cancer (CRC) progression, including metastasis. However, the detailed molecular mechanism is not fully understood.Differentially expressed circRNAs between primary KM12C and liver metastatic KM12L4 colon cancer cells were identified by microarray. The expression of circRNAs was measured by semi-quantitative (semi-qPCR) and real time-quantitative PCR (RT-qPCR). Metastatic potential including invasive and migratory abilities, and liver metastasis were examined by transwell assays and intrasplenic injection, respectively. CircPPFIA1-associated microRNA (miRNA) and RNA-binding protein (RBP) were screened by an antisense oligonucleotide (ASO) pulldown experiment. The effects of circPPFIA1 on target gene expression were evaluated by RT-qPCR and western blot analyses.PPFIA1 with different length, which named circPPFIA1-L (long) and -S (short). They were significantly downregulated in liver metastatic KM12L4 cells compared to primary KM12C cells. The knockdown of circPPFIA1s in KM12C enhanced metastatic potential and increased liver metastasis. Conversely, overexpression of circPPFIA1s weakened metastatic potential and inhibited liver metastasis. circPPFIA1s were found to function as sponges of oncogenic miR-155-5p and Hu antigen R (HuR) by an ASO pulldown experiment. circPPFIA1s upregulated tumor-suppressing CDX1 expression and conversely downregulated oncogenic RAB36 by decoying miR-155-5p and by sequestering HuR, respectively.By analyzing circRNA microarray data, we identified two anti-metastatic circRNAs generated from Our findings demonstrate that circPPFIA1s inhibit the liver metastasis of CRC via the miR-155-5p/CDX1 and HuR/RAB36 pathways.The online version contains supplementary material available at 10.1186/s12943-022-01667-w. Colorectal cancer (CRC) is the third most common type of malignant tumor and the second leading cause of cancer-related death . DespiteRecent CRC research on the development of diagnostic and therapeutic targets has gradually expanded from protein-coding genes to non-coding RNAs, such as microRNAs (miRNAs), antisense transcripts, long intergenic non-coding RNAs, and circular RNAs (circRNAs) , 7. AlthZEB1-targeting miR-382/587/616 [Several circRNAs are reported to influence metastatic potential by acting as sponges of microRNA (miRNA) and RNA-binding protein (RBP) in CRC –15. For /587/616 . Convers/587/616 .Here, we aimed to identify novel circRNAs that can control the liver metastasis of CRC. Through a circRNA microarray, two circPPFIA1s were found to be downregulated in liver metastatic CRC. Transwell assays and intrasplenic injection mouse experiments revealed that circPPFIA1s negatively regulated metastatic potential and liver metastasis of CRC. Furthermore, we found that circPPFIA1s exhibited anti-metastatic effects by sponging oncogenic miR-155-5p, thereby increasing caudal type homeobox 1 (CDX1) expression, and decreasing the expression of RAB36 via the sequestration of an oncogenic RBP, Hu antigen R (HuR). Taken together, we demonstrated that circPPFIA1s are promising therapeutic targets for treatment for liver metastatic CRC.All tissues were collected from CRC patients who had undergone surgery at Samsung Medical Center . Six pairs of primary CRC tumor tissues and corresponding liver metastatic tumor tissues were obtained from surgical resections of CRC patients without any radiotherapy or chemotherapy before surgery. The samples were pathologically confirmed and stored in liquid nitrogen after surgery until use. All human specimens were approved by the Institutional Review Board of the Samsung Medical Center . Written informed consent was obtained from all patients.The expression levels of circPPFIA1-L and -S in tissues were assessed by BaseScope Assay . Basescope probes for circPPFIA1-L and -S were designed to target the junction sequences of circPPFIA1-L and -S. BaseScope assays were performed using BaseScope Detection Reagent Kit-RED  in accord with the manufacturer’s protocol. Fast RED followed by counterstaining with hematoxylin . The images were visualized using ScanScope AT turbo  and analyzed by ImageScope .Primary and liver metastatic CRC cells  and colorectal cancer cells (DLD1 and RKO) were cultured with Dulbecco’s modified Eagle’s medium . All cell lines were free of mycoplasma contamination and verified by STR analysis  as described by the manufacturer’s protocol. The circRNA microarray was performed by Arraystar . The differentially expressed circRNAs were analyzed with the criteria of The circular structure of circPPFIA1-L and -S was confirmed by testing the stability via RNase R resistance and actinomycin D treatment. To verify the divergent region of circPPFIA1-L and -S, Sanger sequencing was carried out. Detailed information on experimental procedures was shown in ® Protein G (Thermo Fisher Scientific) as described in a previous report [The RIP assay was performed using Dynabeadss report . Briefly™ (Invitrogen) were added for 4 h at 4 °C. LacZ ASO was used as a negative pulldown control  (10 mg/kg) via intraperitoneal injection. A small left abdominal flank incision was made, and the spleen was exteriorized for intrasplenic injection. For the preparation of the cells to be injected, KM12C and KM12L4 cells were transfected with circPPFIA1 siRNA or the overexpression vector, respectively. An equal number of transfected cells (2.0 × 106 cells) were suspended in 50 µl of Hanks’ Balanced Salt Solution (Gibco) and injected into mouse spleens with a 30-gauge needle. After 4 weeks, we examined the liver metastasis with magnetic resonance imaging (MRI) and sacrificed the mice to obtain liver tissues. The animal experiments were performed in a specific pathogen-free animal experiment center at the Samsung Medical Center. Ethics approval for animal use was obtained from the Samsung Medical Center Laboratory Animals Committee .Six- to seven-week-old female BALB/c nude mice  were anesthetized with a mixture of ketamine  (30 mg/kg) and xylazine  , was found in the list of differentially expressed circRNA , 37 circRNAs are possibly generated from PPFIA1  and cDNA were used for the PCR analysis with convergent and divergent primers. Whereas PCR products of the convergent primers were observed for gDNA and cDNA templates, the divergent primers generated PCR products only from cDNA  were used to search for circPPFIA1-interacting miRNAs. The only common prediction in all algorithms was miR-155-5p  experiment was performed. The introduction of pre-miR-155-5p into KM12C cells increased the enrichment of circPPFIA1-L and -S in Ago2 IP material compared to the control IgG . To verify the direct interaction between circPPFIA1s and predicted RBPs, ASO pulldown was followed by western blot analysis , HuR was identified as a putative sponging RBP of circPPFIA1s Fig. A. MoreovNext, the expression of HuR was compared in KM12C and KM12L4 cells. Interestingly, the western blot results revealed that the expression level of HuR in both cells was almost similar Fig. D. MoreovTo test whether HuR can regulate metastatic potential, the invasive and migratory abilities of KM12L4 cells were examined by transwell assays. The knockdown of HuR dramatically decreased the number of invaded and migrated cells Fig. I. We alsRAB36 was selected as a HuR target gene using the reference investigation  were found to be putative HuR target genes  are derived from exons , 23. MosPPFIA1, interacts with the leukocyte common antigen-related family of tyrosine phosphatases and plays an important role in axon guidance and mammary gland development [www.circbase.org), 37 circRNAs are generated from PPFIA1 , circRNAs regulate miRNA function by inhibiting binding of miRNAs to 3’UTR of their targets. For example, circHIPK3 promotes CRC progression and metastasis by sponging miR-7 . CircSAPARID2 . AdditioARID2 –39. In od in CRC , 41 and d in CRC . CDX1 inogenesis . As miR-There has been controversy over whether circRNA can regulate the expression of miRNA. However, emerging evidences have revealed that circRNA as a ceRNA could not affect the level of miRNA since they do not degrade their sponging miRNA. CircTLK1 did not influence the expression level of miR-136-5p but inhibited its inhibitory effect on CBX4 expression as a miRNA sponge . MoreovePTBP1) mRNA [MMP1) mRNA [In addition to miRNA sponge, circRNAs also affect the function of RBP as a RBP sponge. For example, circPTPRA suppresses bladder cancer progression by blocking the interaction between IGF2BP1 and its target mRNAs . For expP1) mRNA . CircDLCP1) mRNA . By a siP1) mRNA . AccordiPPFIA1, were significantly downregulated in liver metastatic cells. The circular structure of circPPFIA1s was verified by RNase R resistance and Sanger sequencing. Using in vitro transwell assays and in vivo intrasplenic injection mouse experiments, we found that circPPFIA1s negatively regulated the liver metastasis of CRC. Mechanistically, an ASO pulldown assay revealed that both circPPFIA1-L and -S function as sponges for oncogenic miR-155-5p and HuR. circPPFIA1s upregulate tumor-suppressing CDX1 by decoying CDX1-targeting miR-155-5p and downregulate oncogenic RAB36 by sequestering HuR. Taken together, circPPFIA1s, as a metastasis suppressor, are a promising therapeutic target for the treatment of the liver metastasis of CRC.We searched for metastasis-associated circRNAs using KM12C CRC cells and its liver metastatic derivative KM12L4. Using a circRNA microarray, we identified several circRNAs downregulated in KM12L4 cells compared to KM12C cells. Two circPPFIA1s, generated from Additional file 1: Supplementary Table S1. Verification of cell lines used in this study by STR analysis. Supplementary Table S2. PCR primers and siRNAs used in this study. Supplementary Table S3. List of antibodies used in this study. Supplementary Table S4. Composition of buffers used in this study. Supplementary methods. Supplementary Figure S1. Comparison of metastatic potential of primary (KM12C) and liver metastatic colorectal cancer cells (KM12L4). A Schematic illustration of the establishment of the liver metastatic colorectal cancer cell model. KM12C was originally obtained from a human specimen and KM12L4 was generated through the fourth selection-isolation of intrasplenic injection. B Liver metastases were examined using in vivo intra-splenic injection. At four weeks post-injection, optical and MRI images were obtained. The degree of liver metastasis (n = 5) was calculated by giving scores in arbitrary units (0–3). Supplementary Figure S2. Analyses of circRNA microarray data. A A heat map analysis of circRNA microarray. B A volcano plot of circRNA microarray data showing a list of the upregulated and downregulated circRNAs. C Detailed information on circPPFIA1-L and -S. Supplementary Figure S3. List of PPFIA1-originated circRNAs. circRNAs that are generated from the PPFIA1 gene are listed in a public circRNA database . Supplementary Figure S4. Primer sequences used in this study. A Schematic illustration and sequences of RT-qPCR primers detecting circPPFIA1-L, circPPFIA1-S, and linear PPFIA1. B Validation of the expression of circPPFIA1-L and -S by RT-qPCR using the above primer sets. C Schematic illustration and sequences of semi-qPCR primers detecting circPPFIA1-L, circPPFIA1-S, and linear PPFIA1. D Schematic illustration and sequences of primers for Sanger sequencing. Supplementary Figure S5. Comparison of circPPFIA1-L and -S expression between adjacent normal and tumor tissues of CRC patients. Tumor tissues and their matched normal tissues were obtained from 14 CRC patients at the Samsung Medical Center. Among the 14 patients, seven did not exhibit metastasis while the other seven patients showed liver metastasis. All samples were collected with the informed consent of patients under institutional review board-approved protocols and stored at -80°C until use. The expression level of circPPFIA1-L and -S was determined by RT-qPCR . Supplementary Figure S6. Characterization of circPPFIA1-L and -S. A The stability of circPPFIA1s and linear PPFIA1 was examined by RT-qPCR using total RNA isolated from actinomycin D-treated KM12C cells. B Schematic illustration of divergent and convergent semi-qPCR primers for circPPFIA1-L and -S. Semi-qPCR analysis was conducted using genomic (gDNA) and complementary (cDNA) DNA. GAPDH was used as a negative control. C Sanger sequencing results of circPPFIA1-L and -S. Supplementary Figure S7. Design and validation of siRNAs targeting circPPFIA1-L and -S.  Schematic illustration and sequences of circPPFIA1-L siRNAs (A), and -S (C).  Each siRNA efficiently decreases the expression level of circPPFIA1-L (B) and -S (D). (E) For a cotransfection experiment, KM12C cells were simultaneously transfected with siRNAs targeting circPPFIA1-L and -S. The expression levels of circPPFIA1s and GAPDH were determined by semi-qPCR. Supplementary Figure S8. Potentiation of metastatic properties by knockdown of circPPFIA1-L and -S in KM12C cells.  KM12C cells were transfected with the indicated siRNA , and transwell assays were performed to determine invasive (A) and migratory (B) abilities.  Cell proliferation of KM12C cells transfected with individual or a mixture of siRNAs targeting circPPFIA1-L (C) or -S (D) was determined by counting the number of viable cells. Supplementary Figure S9. Increase in liver metastasis in vivo by knockdown of circPPFIA1s. The effect of circPPFIA1-L and -S on liver metastasis in vivo was examined through intrasplenic injection of KM12C cellstransfected with circPPFIA1s siRNAs.  The expression level of circPPFIA1-L (A) and -S (C) was determined by semi-qPCR.  Liver metastases were examined using in vivo intrasplenic injection. At four weeks post-injection, optical and MRI images were obtained. The degree of liver metastasis (n = 7) was calculated by giving scores in arbitrary units (0–3). Supplementary Figure S10. Suppression of metastatic potential by overexpression of circPPFIA1-L and -S in KM12L4 cells. (A-C) By introducing the circPPFIA1-L overexpression vector into KM12L4 cells, three independent clones (#1–#3) were generated and were used to investigate the effect of circPPFIA1-L on metastatic potential. Invasive ability was examined by transwell invasion assays, and the expression levels of circPPFIA1-L and PPFIA1 mRNA were measured by RT-qPCR. (D) In the same way as above, two clones (#1 and #2) in which circPPFIA1-S was overexpressed were generated, and invasive and migratory abilities were determined by transwell invasion and migration assays. (E) The effect of circPPFIA1 overexpression on cell proliferation was examined by counting the number of viable cells. Supplementary Figure S11. Decrease in liver metastasis in vivo by overexpression of circPPFIA1s. The effect of circPPFIA1-L and -S on liver metastasis in vivo was examined via intrasplenic injection of KM12L4 cells, wherein circPPFIA1-L or -S was overexpressed.  The expression levels of circPPFIA1-L (A) and -S (C) were determined by semi-qPCR.  Liver metastases were examined via in vivo intrasplenic injection of circPPFIA1-L (B) or -S (D) overexpressing KM12L4 cells. At four weeks post-injection, optical and MRI images were obtained. The degree of liver metastasis (n = 7) was calculated by giving scores in arbitrary units (0–3). Supplementary Figure S12. Negative regulation of invasive and migratory abilities via circPPFIA1-L and -S in DLD1 and RKO cells.  Following transfection of DLD1 (A) and RKO (B) cells with indicated siRNA, transwell assays were conducted to measure invasive and migratory abilities. The expression levels of circPPFIA1-L, -S, and PPFIA1 mRNA were determined by semi-qPCR. GAPDH was used as a loading control.  For overexpression of circPPFIA1-L and -S, DLD1 (C) and RKO (D) cells were transfected with indicated vector. The number of invaded and migrated cells was assessed using transwell assays. The expression levels of circPPFIA1-L, -S, and PPFIA1 mRNA were determined by semi-qPCR. GAPDH was used as a loading control. Supplementary Figure S13. Venn diagram for selecting putative interacting miRNAs with circPPFIA1. By prediction of circPPFIA1-interacting miRNAs using four prediction algorithms , miR-155-5p was selected for further studies. Supplementary Figure S14. Schematic illustration and design of antisense oligonucleotide (ASO) for the pulldown experiments.  ASOs for pulldown were designed to bind to the divergent region of circPPFIA1-L (A) or -S (B). circPPFIA1-L and -S were captured using three and two ASOs, respectively. The enrichment of the corresponding circRNA in pulldown materials was assessed by semi-qPCR. (C) The sequences of ASOs for pulldown experiments. LacZ was used as a control ASO. Supplementary Figure S15. Construction of luciferase vectors harboring wild-type (WT) or mutant (MT) sequences of miR-155-5p miRNA recognition element (MRE) in circPPFIA1-L and -S. (A) Two miR-155-5p MREs (#1 and #2) were predicted by bioinformatics approaches in the sequence of exon 18 of PPFIA1. (B) Dual-luciferase vectors harboring wild-type (WT) or mutant (MT) sequences of each miR-155-5p MRE were manufactured. Supplementary Figure S16. Regulation of metastatic potential by miR-155-5p in DLD1 and RKO cells. DLD1 (A) and RKO (B) cells were transfected with pre-miR-155-5p or anti-miR-155-5p. Invasive and migratory abilities were assessed using transwell invasion and migration assays. Supplementary Figure S17. Rescue experiment for proving that miR-155-5p is required for the increase in metastatic potential by knockdown of circPPFIA1. KM12C cells were simultaneously transfected with circPPFIA1 siRNA and anti-miR-155-5p. Invasive and migratory abilities were examined using transwell invasion (A) and migration (B) assays. Supplementary Figure S18. Venn diagram for screening common target genes that are regulated via circPPIFIA1s/miR-155-5p. By comparing the lists of downregulated genes obtained from RNA sequencing data, and miR-155-5p target genes predicted by miRDB and TargetScan, six genes  were identified as putative targets. Supplementary Figure S19. Schematic of dual-luciferase reporter vectors harboring wild-type or mutant sequences of miR-155-5p MRE in CDX1 mRNA. Supplementary Figure S20. Prediction and validation of circPPFIA1-interacting RNA-binding proteins. (A) Venn diagram for selecting circPPFIA1-interacting RBPs using three prediction algorithms . (B) The levels of predicted RBPS in ASO pulldown materials were assessed by western blot analyses using the indicated antibodies. (C) ASO pulldown followed by western blot analysis was conducted to verify the interaction of HuR with circPPFIA1s . Supplementary Figure S21. High proportion of common target genes harbor the HuR-binding motif.Upregulated common target genes were screened based on the following three criteria: (i) genes upregulated genes by knockdown of circPPFIA1-L, (ii) genes upregulated genes by knockdown of circPPFIA1-S, (iii) genes upregulated in KM12L4 compared to KM12C. By comparing and analyzing the public data of HuR CLIP-seq with the selected common target genes, we observed that 77% (48 genes out of 62 genes) of selected target genes have HuR-binding motifs in their 3’-UTRs.