Dual-responsive nanocarriers for efficient cytosolic protein delivery and CRISPR-Cas9 gene therapy of inflammatory skin disorders.

Developing protein drugs that can target intracellular sites remains a challenge due to their inadequate membrane permeability. Efficient carriers for cytosolic protein delivery are required for protein-based drugs, cancer vaccines, and CRISPR-Cas9 gene therapies. Here, we report a screening process to identify highly efficient materials for cytosolic protein delivery from a library of dual-functionalized polymers bearing both boronate and lipoic acid moieties. Both ligands were found to be crucial for protein binding, endosomal escape, and intracellular protein release. Polymers with higher grafting ratios exhibit remarkable efficacies in cytosolic protein delivery including enzymes, monoclonal antibodies, and Cas9 ribonucleoprotein while preserving their activity. Optimal polymer successfully delivered Cas9 ribonucleoprotein targeting NLRP3 to disrupt NLRP3 inflammasomes in vivo and ameliorate inflammation in a mouse model of psoriasis. Our study presents a promising option for the discovery of highly efficient materials tailored for cytosolic delivery of specific proteins and complexes such as Cas9 ribonucleoprotein.

Radiation reduction for interventional radiology imaging: a video frame interpolation solution.

PURPOSE: The aim of this study was to diminish radiation exposure in interventional radiology (IR) imaging while maintaining image quality. This was achieved by decreasing the acquisition frame rate and employing a deep neural network to interpolate the reduced frames. METHODS: This retrospective study involved the analysis of 1634 IR sequences from 167 pediatric patients (March 2014 to January 2022). The dataset underwent a random split into training and validation subsets (at a 9:1 ratio) for model training and evaluation. Our approach proficiently synthesized absent frames in simulated low-frame-rate sequences by excluding intermediate frames from the validation subset. Accuracy assessments encompassed both objective experiments and subjective evaluations conducted by nine radiologists. RESULTS: The deep learning model adeptly interpolated the eliminated frames within IR sequences, demonstrating encouraging peak signal-to-noise ratio (PSNR) and structural similarity index (SSIM) results. The average PSNR values for angiographic, subtraction, and fluoroscopic modes were 44.94 dB, 34.84 dB, and 33.82 dB, respectively, while the corresponding SSIM values were 0.9840, 0.9194, and 0.7752. Subjective experiments conducted with experienced interventional radiologists revealed minimal discernible differences between interpolated and authentic sequences. CONCLUSION: Our method, which interpolates low-frame-rate IR sequences, has shown the capability to produce high-quality IR images. Additionally, the model exhibits potential for reducing the frame rate during IR image acquisition, consequently mitigating radiation exposure. CRITICAL RELEVANCE STATEMENT: This study presents a critical advancement in clinical radiology by demonstrating the effectiveness of a deep neural network in reducing radiation exposure during pediatric interventional radiology while maintaining image quality, offering a potential solution to enhance patient safety. KEY POINTS: • Reducing radiation: cutting IR image to reduce radiation. • Accurate frame interpolation: our model effectively interpolates missing frames. • High visual quality in terms of PSNR and SSIM, making IR procedures safer without sacrificing quality.

Piperazine-modified dendrimer achieves efficient intracellular protein delivery via caveolar endocytosis bypassing the endo-lysosomal pathway.

Intracellular protein delivery has been a major challenge due to various physiological barriers including low proteolytic stability and poor membrane permeability of the biologics. Nanoparticles were widely proposed to deliver cargo proteins into cells by endocytosis, however, the materials and complexes with proteins are often entrapped in endosomes and subject to lysosome degradation. In this study, we report a piperazine modified dendrimer for stabilizing the complexes via a combination of electrostatic interaction and hydrophobic interactions. The complexes show rapid cell internalization and the loaded proteins are released into the cytosols as early as half an hour post incubation. Mechanism study suggests that the complexes are endocytosed into cells via caveolae-based pathways, which could be inhibited by inhibitors such as genistein, filipin III, brefeldin A and nystatin. The phenylpiperazine-modified polymer enables the delivery of cargo proteins with reserved bioactivity and show high permeability in three-dimensional cell spheroids. The results prove the beneficial roles of phenylpiperazine ligands in polymer-mediated cytosolic protein delivery systems. STATEMENT OF SIGNIFICANCE: We synthesized a list of piperazine and derivatives modified dendrimers as cytosolic protein delivery vectors via facile reactions. Phenylpiperazine modification enables the efficient protein binding through the combination of electrostatic, hydrogen bonding and hydrophobic interactions. Phenylpiperazine modified dendrimers were internalized into the cells via a caveolae-based endo/lysosome-independent path and could release the cargo proteins into the cytosols as early as half an hour post incubation. Phenylpiperazine modified dendrimers delivered cargo proteins with reserved bioactivity and showed high permeability in three-dimensional cell spheroids.

Lead exposure as a causative factor for metabolic associated fatty liver disease (MAFLD) and a lead exposure related nomogram for MAFLD prevalence.

The relationship between lead exposure and neurological disorders has been extensively studied, but the effects of lead exposure on hepatotoxicity are unknown. Metabolically related fatty liver disease (MAFLD) is an update of previous non-alcoholic fatty liver disease (NAFLD). It redefines the diagnostic conditions and emphasizes metabolic factors while considering non-alcoholic factors. Lead can affect the endocrine system and metabolism, so we believe that lead exposure may contribute to MAFLD. 41,723 individuals who had undergone blood lead testing from 2005 to 2018 in the National Health and Nutrition Examination Survey (NHANES) database were selected for this study. The characteristics of population lead exposure in the last decade or so, the effect of lead exposure on liver function and whether lead exposure can cause MAFLD were analyzed. Co-variates were adjusted according to age, ethnicity, body mass index (BMI), waist circumference, visceral adiposity index (VAI), poverty indices (PIR), diabetes, hypertension, and hyperlipidemia. The results showed that blood lead concentrations stabilized at a low level after a decreasing trend from year to year. The differences in blood lead concentrations were associated with differences in age, sex, race, education level, and PIR. Lead exposure was an independent risk factor for MAFLD, and lead and nine other factors were used as independent risk factors for MAFLD, so a nomogram was established to predict the prevalence probability of MAFLD.

Robust Reversible Cross-Linking Strategy for Intracellular Protein Delivery with Excellent Serum Tolerance.

Intracellular protein delivery has attracted increasing attentions in biomedical applications. However, current delivery systems usually have poor serum stability due to the competitive binding of serum proteins to the polymers during delivery. Here, we report a reversible cross-linking strategy to improve the serum stability of polymers for robust intracellular protein delivery. In the proposed delivery system, nanoparticles are assembled by cargo proteins and cationic polymers and further stabilized by a glutathione-cleavable and traceless cross-linker. The cross-linked nanoparticles show high stability and efficient cell internalization in serum containing medium and can release the cargo proteins in response to intracellular glutathione and acidic pH in a traceless manner. The generality and versatility of the proposed strategy were demonstrated on different types of cationic polymers, cargo proteins, as well as cell lines. The study provides a facile and efficient method for improving the serum tolerance of cationic polymers in intracellular protein delivery.

Super-Selective Partial Splenic Embolization for Hereditary Spherocytosis in Children: A Single-Center Retrospective Study.

Background: Hereditary spherocytosis (HS) is the most common hemolytic anemia due to erythrocyte membrane defects. Total splenectomy is the most effective treatment for moderate or severe HS. As a conservative alternative, partial splenic embolization (PSE) can preserve part of the spleen's function, thus reducing the risk of overwhelming post-splenectomy infection (OPSI) or sepsis, especially for pediatric patients. However, it is not easy to precisely control the scope of interventional embolization, limiting PSE applications. The present study aims to optimize the PSE procedure on smaller, which is named super-selective PSE (SPSE), to improve the controllability and assess the feasibility and effectiveness of SPSE. Results: This study was conducted by retrospectively reviewing clinical data from HS patients treated by surgical treatments, which were diagnosed at the children's hospital of Chongqing medical university from January 2015 to December 2019. Patients were divided into two groups according to their treatment preference: SPSE (16 patients) group and total splenectomy (41 patients) group. The mean proportion range of splenic embolism by SPSE was 82.4%, close to the expected value (70-85%). The average hemoglobin value was increased significantly from 6.85 (5.6-8.0) g/dl before SPSE to 12.4 (10.4-13.3) g/dl after SPSE (p < 0.001). All children after SPSE suffered mild post-embolization syndrome, such as pain, fever, and vomiting, which could easily be controlled with appropriate supportive therapy. Conclusions: Super-selective partial splenic embolization is a safe and effective treatment for moderate or severe HS in children. However, with a longer follow-up, more patients further assess the value of SPSE.

Study Protocol: Design and Implementation of the Pediatric Liver Transplantation Biobank.

Background: Early treatment of neonatal biliary atresia (BA) and other end-stage liver diseases can delay or prevent the necessity of liver transplantation (LT). The establishment of a standardized clinical pediatric liver transplantation (PLT) biobank is the prerequisite for scientific research, which helps to provide a qualified sample resource platform for research. Methods: Following standardized procedures to establish biobanks, the operational processes and quality control system were formulated. Liver tissue, blood, and stool samples undergoing LT were regularly collected, managed, and stored. Systematic management was conducted in collected specimens and corresponding clinical information. Results: Since implementation in August 2018, we have enrolled 49 unique subjects (0-18 years of age); the biobank contains nearly 3000 biospecimen aliquots. The most common LT diagnosis is BA (61.23%). Conclusion: The establishment of this biobank is a valuable resource that incorporates detailed clinical and biological information. It will help accelerate the pace of PLT discovery research. ClinicalTrials.gov ID: NCT04477967.

Therapeutic Protein PEPylation: The Helix of Nonfouling Synthetic Polypeptides Minimizes Antidrug Antibody Generation.

Polymer conjugation is a clinically proven approach to generate long acting protein drugs with decreased immune responses. Although poly(ethylene glycol) (PEG) is one of the most commonly used conjugation partners due to its unstructured conformation, its therapeutic application is limited by its poor biodegradability, propensity to induce an anti-PEG immune response, and the resultant accelerated blood clearance (ABC) effect. Moreover, the prevailing preference of unstructured polymers for protein conjugation still lacks strong animal data support with appropriate control reagents. By using two biodegradable synthetic polypeptides with similar structural compositions (l-P(EG3Glu) and dl-P(EG3Glu)) for site-specific protein modification, in the current study, we systematically investigate the effect of the polymer conformation on the in vivo pharmacological performances of the resulting conjugates. Our results reveal that the conjugate l20K-IFN, interferon (IFN) modified with the helical polypeptide l-P(EG3Glu) shows improved binding affinity, in vitro antiproliferative activity, and in vivo efficacy compared to those modified with the unstructured polypeptide analogue dl-P(EG3Glu) or PEG. Moreover, l20K-IFN triggered significantly less antidrug and antipolymer antibodies than the other two. Importantly, the unusual findings observed in the IFN series are reproduced in a human growth hormone (GH) conjugate series. Subcutaneously infused l20K-GH, GH modified with l-P(EG3Glu), evokes considerably less anti-GH and antipolymer antibodies compared to those modified with dl-P(EG3Glu) or PEG (dl20K-GH or PEG20K-GH). As a result, repeated injections of dl20K-GH or PEG20K-GH, but not l20K-GH, result in a clear ABC effect and significantly diminished drug availability in the blood. Meanwhile, immature mouse bone marrow cells incubated with the helical l20K-GH exhibit decreased drug uptake and secretion of proinflammatory cytokines compared to those treated with one of the other two GH conjugates bearing unstructured polymers. Taken together, the current study highlights an urgent necessity to systematically reassess the pros and cons of choosing unstructured polymers for protein conjugation. Furthermore, our results also lay the foundation for the development of next-generation biohybrid drugs based on helical synthetic polypeptides.

Macrocyclization of Interferon-Poly(α-amino acid) Conjugates Significantly Improves the Tumor Retention, Penetration, and Antitumor Efficacy.

Cyclization and polymer conjugation are two commonly used approaches for enhancing the pharmacological properties of protein drugs. However, cyclization of parental proteins often only affords a modest improvement in biochemical or cell-based in vitro assays. Moreover, very few studies have included a systematic pharmacological evaluation of cyclized protein-based therapeutics in live animals. On the other hand, polymer-conjugated proteins have longer circulation half-lives but usually show poor tumor penetration and suboptimal pharmacodynamics due to increased steric hindrance. We herein report the generation of a head-to-tail interferon-poly(α-amino acid) macrocycle conjugate circ-P(EG3Glu)20-IFN by combining the aforementioned two approaches. We then compared the antitumor pharmacological activity of this macrocycle conjugate against its linear counterparts, N-P(EG3Glu)20-IFN, C-IFN-P(EG3Glu)20, and C-IFN-PEG. Our results found circ-P(EG3Glu)20-IFN to show considerably greater stability, binding affinity, and in vitro antiproliferative activity toward OVCAR3 cells than the three linear conjugates. More importantly, circ-P(EG3Glu)20-IFN exhibited longer circulation half-life, remarkably higher tumor retention, and deeper tumor penetration in vivo. As a result, administration of the macrocyclic conjugate could effectively inhibit tumor progression and extend survival in mice bearing established xenograft human OVCAR3 or SKOV3 tumors without causing severe paraneoplastic syndromes. Taken together, our study provided until now the most relevant experimental evidence in strong support of the in vivo benefit of macrocyclization of protein-polymer conjugates and for its application in next-generation therapeutics.

Harnessing Phosphato-Platinum Bonding Induced Supramolecular Assembly for Systemic Cisplatin Delivery.

To improve the therapeutic index of cisplatin (CDDP), we present here a new paradigm of drug-induced self-assembly by harnessing phosphato-platinum complexation. Specifically, we show that a phosphato-platinum cross-linked micelle (PpY/Pt) can be generated by using a block copolymer methoxy-poly(ethylene glycol)-block-poly(l-phosphotyrosine) (mPEG-b-PpY). Coating of PpY/Pt with a R9-iRGD peptide by simple mixing affords a targeting micelle with near neutral-charged surface (iPpY/Pt). The micelles feature in well-controlled sizes below 50 nm and high stability under physiological conditions, and can withstand various environmental stresses. Importantly, the micelles demonstrate on-demand drug release profiles in response to pathological cues such as high ATP concentration and acidic pH. In vitro, the micelles are efficiently internalized and almost equally potent compared to CDDP. Moreover, iPpY/Pt induce greater cytotoxicity than PpY/Pt in a 3D tumor spheroid model likely due to its deeper tumor penetration. In vivo, the micelles exhibit prolonged circulation half-lives, enhanced tumor accumulation, excellent tumor growth inhibition in a xenograft HeLa model and an orthotropic mammary 4T1 model, and improved safety profiles evidenced by the reduced nephrotoxicity. Together, this work demonstrates for the first time that phosphato-platinum complexation can be exploited for effective delivery of CDDP, and suggests a paradigm shift of constructing nanosystems for other anticancer metallodrugs.

Facile hydrothermal preparation of recyclable S-doped graphene sponge for Cu2+ adsorption.

Graphene sponge (GS) has been widely employed for water purification, but adsorption capacity loss frequently occurs during the formation of spongy structure. In this study, we reported the hydrothermal preparation of S-doped GS for the removal of Cu(2+) with a huge adsorption capacity of 228 mg/g, 40 times higher than that of active carbon. The adsorption isotherm could be well fitted into the Freundlich model with a KF value of 36.309(L/mg)(1/n). The equilibrium adsorption could be fully achieved in the first 5 min. In the thermodynamics study, the negative ΔG indicated that the adsorption was spontaneous and physisorption in nature. The positive ΔH implied that the adsorption was endothermic. The changes of both pH and ionic strength had no apparent influence on the adsorption. S-doped GS could be easily regenerated by washing with acidic thiourea. Moreover, S-doped GS could be used for the adsorption of other heavy metal ions, too. The implication to the applications of S-doped GS in water treatment is discussed.

On the status and comparison of glucose intolerance in female breast cancer patients at initial diagnosis and during chemotherapy through an oral glucose tolerance test.

AIMS: This study is to estimate the status and comparison of glucose intolerance in female breast cancer patients at initial diagnosis and during chemotherapy through an oral glucose tolerance test (OGTT), as well as to learn the effect of chemotherapy on the glucose metabolism of breast cancer patients. METHODS: All the 79 breast cancer patients at initial diagnosis, with the mean age of 53.2 years, and 96 breast cancer patients before the 5th or 6th cycle of chemotherapy, with the mean age of 51.5 years, participated in the study from December 2012 to October 2013. After an overnight fast, participants underwent OGTT test, and fasting and 2-hour glucose levels were measured to identify undiagnosed diabetes and prediabetes (i.e., impaired fasting glucose or impaired glucose tolerance) in them. Previously diagnosed diabetes among the female breast cancer patients was determined on the self-report and the medical record. RESULTS: The overall incidences of total normal glucose tolerance, prediabetes, diabetes in female breast cancer patients at initial diagnosis and during chemotherapy were 24.1% and 38.5% (p<0.05), 50.6% and 28.1% (p<0.05), and 25.3% and 33.3% (p>0.05), respectively, and the differences of normal glucose tolerance and prediabetes instead of diabetes between the two groups were statistically significant. About 84% of the total diabetes and prediabetes in the female breast cancer patients at initial diagnosis and 79.7% of those during chemotherapy need to be diagnosed with OGTT. CONCLUSIONS: Breast cancer patients have high incidences of diabetes and prediabetes. After chemotherapy even with steroids, some breast cancer patients with abnormal glucose metabolism may even become normal. Isolated hyperglycemia 2 hours after glucose loading is common, and OGTT should be made for breast cancer patients at initial diagnosis and during chemotherapy.

On the status of ß-cell dysfunction and insulin resistance of breast cancer patient without history of diabetes after systemic treatment.

To estimate the status of ß-cell dysfunction and insulin resistance of breast cancer (BC) patient without history of diabetes mellitus (DM) after systemic treatment through an oral glucose tolerance test (OGTT) and insulin releasing test (IRT). All the 128 BC patients without history of DM after systemic treatment underwent OGTT and IRT test. Fasting and 2-h glucose levels were measured to confirm undiagnosed DM and prediabetes. Insulin sensitivity was estimated by homeostasis model assessment of insulin resistance (HOMA-IR) and Matsuda index and disposition index (IGI/HOMA-IR). Insulin secretion was estimated by the insulinogenic index (IGI) [Δ insulin/Δ glucose (30-0 min)]. Insulin concentrations during the OGTT and IRT at baseline were used to derive the patterns of insulin secretion curve (pattern 1, pattern 2, pattern 3, pattern 4 and pattern 5), which were used to estimate the risk of developing DM. Of 128 BC patients without history of DM after systemic treatment, there were 46 cases (35.9%) of NGT, 60 cases (46.9%) of prediabetes and 22 cases (17.2%) of DM. The BMI of prediabetes and DM were higher than NGT groups with statistical significance. After adjusted for BMI, IGI was significantly lower in DM group but not significantly different between NGT group and prediabetes group. HOMA-IR, Matsuda index and disposition index were significantly different in DM group compared with NGT group and prediabetes and also significantly different between NGT and prediabetes groups. The total rates of patterns 4 and 5 in NGT and prediabetes groups were 15.3% (10.9 and 4.4%) and 48.3% (31.6 and 16.7%), respectively. ß-Cell dysfunction and insulin resistance may appear in BC patients after systemic treatment. BC patients have high risk in development of DM even in NGT and prediabetes groups confirmed by OGTT.

Discordance and clinical significance of ER, PR, and HER2 status between primary breast cancer and synchronous axillary lymph node metastasis.

Discordance of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) status between primary breast cancer, metastatic lesion and synchronous axillary lymph node metastasis has been reported in the series studies. Systemic treatment of primary invasive breast cancer patients with synchronous axillary metastasis is currently based on the biomarker characteristics of the primary tumor; however, hormone receptors and HER2 status may change throughout tumor progression from the primary tumor to the synchronous axillary metastasis. As local metastasis, the synchronous axillary lymph node metastasis may represent the potentially metastatic breast cancer cells much better than the primary tumor. Hence, the determination of hormone receptors and HER2 status should be routinely performed in synchronous axillary nodal metastasis, together with primary tumor, to guide therapy management and evaluate the recurrent risk of primary invasive breast cancer patients with synchronous axillary nodal metastasis, which may even change the postoperative risk categories (St. Gallen consensus) of breast cancer in these patients. This article will review the studies on the discordance and clinical significance of ER, PR, and HER2 receptor status between primary breast cancer and synchronous axillary lymph node metastasis.

Clinicopathologic features of breast cancer patients with type 2 diabetes mellitus in southwest of China.

The aim of this study was to study the prevalence and clinicopathologic features of breast cancer patients with type 2 diabetes mellitus in southwest of China for providing clinical guidance and prognosis appreciation for these patients. Through a case-control study of 3,381 primary breast cancer patients initially diagnosed from January 2007 to May 2013, one case group (164 female breast cancer patients with type 2 diabetes) and two control groups (first control group consists of 328 randomly selected nondiabetic breast cancer patients and second control group consists of 279 nondiabetic breast cancer patients without diabetes-related diseases such as cardiovascular or cerebrovascular diseases) were selected. The clinicopathological features between them were statistically analyzed. (1) Of 3,381 primary breast cancer patients with the average age of 50.5, ranging from 21 to 97 years of age, 164 (4.9 %) cases (with the average age of 60.7) suffered diabetes (previously diagnosed diabetes). (2) The differences of clinicopathologic features between the case group and first control group (with the average age of 61.5) were the ratio of hypertension (41.5 vs 26.1 %, P = 0.001) and axillary lymph node metastasis (51.1 vs 38.1 %, P = 0.046); and the differences of clinicopathologic features between the case group and second control group (with the average age of 64.3) were axillary lymph node metastasis (51.1 vs 35.8 %, P = 0.017), tumor size (≥ T2: 62.3 vs 53.1 %, P = 0.019) and p53 expression (51.0 vs 62.7 %, P = 0.018). No statistical significances (P > 0.05) of histological type, histological grade, or the expressions of estrogen receptor (ER), progesterone receptor, human epidermal growth factor 2 (HER2) and Ki67 were found between them. (3) The clinicopathologic features of ER-positive and ER-negative patients in each group were as follows: (1) In the case group, the ER-negative patients have more advanced tumor histological grade (G3, 19.0 vs 2.8 %, P = 0.012), more positive expression of Her-2 (16.9 vs 8.1 %, P = 0.029) and more axillary lymph node metastasis (63.3 vs 44.4 %, P = 0.048). (2) In the first control group, the same results with tumor histological grade (G3, 15.6 vs 6.2 %, P = 0.025) and positive expression of Her-2 (16.7 vs 4.3 %, P = 0.001), and more positive expression of Ki67 (65.1 vs 52.0 %, P < 0.001) were found. (3) In the second control group, the ER-negative patients have more positive expression of Ki67 (70.5 vs 55.7 %, P = 0.009) and fewer family history of malignancy (1.9 vs 10.0 %, P = 0.013). Diabetes has a high incidence in breast cancer patients and is more common with postmenopausal patients. It is suggested that initially diagnosed breast cancer patients should undertake oral glucose tolerance test screening for occult diabetes and prediabetes. More concerns should be put onto diabetic patients with breast cancer.

Incidences of diabetes and prediabetes among female adult breast cancer patients after systemic treatment.

Patients with cancer frequently show glucose intolerance. This study is to estimate the status of total diabetes and prediabetes in breast cancer patients after systemic treatment through an oral glucose tolerance test (OGTT) in China. All the 119 breast cancer patients more than 3 months after systemic treatment with surgery and chemotherapy participated in the study. All the patients without the diagnosis of diabetes underwent OGTT, and fasting and 2-h glucose levels were measured to identify undiagnosed diabetes and prediabetes. Previously diagnosed diabetes were determined on the self-report and the medical record. Of the 119 breast cancer patients, with the median age of 50.1 years and the mean age of about 48 years when they were initially diagnosed with breast cancer, which showed the similar characters of China and Asia breast cancer patients, the overall incidences of total diabetes and prediabetes were 21.8 and 43.7 %, respectively. About 80 % of the diabetes were previously undiagnosed. About 80.0 % of the cases of undiagnosed diabetes and prediabetes met the criteria for elevated 2-h plasma glucose levels through OGTT but not the criteria for elevated fasting glucose levels. Our study firstly documents high incidences of previously undiagnosed diabetes and prediabetes in breast cancer patients during follow-up after systemic treatment through OGTT, indicating that greater diabetes screening, especially through OGTT, prevention, and treatment strategies among breast cancer patients, after systemic treatment for these patients is needed.