Gene therapy by virus-like self-spooling toroidal DNA condensates for revascularization of hindlimb ischemia.

Peripheral arterial diseases (PAD) have been reported to be the leading cause for limb amputations, and the current therapeutic strategies including antiplatelet medication or intervene surgery are reported to not clinically benefit the patients with high-grade PAD. To this respect, revascularization based on angiogenetic vascular endothelial growth factor (VEGF) gene therapy was attempted for the potential treatment of critical PAD. Aiming for transcellular delivery of VEGF-encoding plasmid DNA (pDNA), we proposed to elaborate intriguing virus-like DNA condensates, wherein the supercoiled rigid micrometer-scaled plasmid DNA (pDNA) could be regulated in an orderly fashion into well-defined nano-toroids by following a self-spooling process with the aid of cationic block copolymer poly(ethylene glycol)-polylysine at an extraordinary ionic strength (NaCl: 600 mM). Moreover, reversible disulfide crosslinking was proposed between the polylysine segments with the aim of stabilizing these intriguing toroidal condensates. Pertaining to the critical hindlimb ischemia, our proposed toroidal VEGF-encoding pDNA condensates demonstrated high levels of VEGF expression at the dosage sites, which consequently contributed to the neo-vasculature (the particularly abundant formation of micro-vessels in the injected hindlimb), preventing the hindlimb ischemia from causing necrosis at the extremities. Moreover, excellent safety profiles have been demonstrated by our proposed toroidal condensates, as opposed to the apparent immunogenicity of the naked pDNA. Hence, our proposed virus-like DNA condensates herald potentials as gene therapy platform in persistent expressions of the therapeutic proteins, and might consequently be highlighted in the management of a variety of intractable diseases.

Physical activity during pregnancy and the risk of gestational diabetes mellitus: a systematic review and dose-response meta-analysis.

BACKGROUND: Previous research has indicated the inverse association between physical activity (PA) and gestational diabetes mellitus (GDM). However, the dose-response relationship currently remains undetermined. This study aims to explore the dose-response relationship between PA during the first and second trimesters of pregnancy and GDM risk. METHODS: Studies on the relationship between PA during pregnancy and GDM risk published before April 25, 2023, were searched for in six databases. According to the inclusion and exclusion criteria, all literature was screened for eligibility. The Newcastle-Ottawa Scale (NOS) was used to assess risk of bias. Publication bias was examined using funnel plots, Begg's and Egger's tests, as well as trim-and-fill analysis. We harmonized exposure estimates of PA during pregnancy to the common unit of the metabolic equivalent of task (MET)-h/week. Restricted cubic splines were used to model the dose-response relationship. The criteria from the World Cancer Research Fund were used to assess the certainty of evidence across outcomes. All analyses were performed using Stata 15.1. RESULTS: The results indicated that in contrast with the lowest level of PA, promoting the highest PA level lowers the risk of GDM by 36% (RR = 0.64, 95%CI: 0.53 ~ 0.78). We found a curvilinear dose-response association between PA during the first trimester and incident GDM (Pnonlinearity = 0.012). Compared to inactive pregnant women, for those who achieved the guidelines-suggested minimum level (10 MET-h/week) of PA during the first trimester, the GDM risk was decreased by 13% (RR = 0.87, 95%CI: 0.79 ~ 0.96). A linear relationship was found between PA during the second trimester and the GDM risk (Pnonlinearity = 0.276). The results with a restricted cubic spline model suggested that pregnant women who accumulate 10 MET-h/week have a 1% reduced risk of GDM compared to completely inactive individuals. Twice (20 MET-h/week) or a higher amount of PA (50 MET-h/week) contributed to further reductions in GDM risk. CONCLUSION: There is a dose-response relationship between higher levels of PA in both the first and second trimesters and reduced risk of GDM; the relationship is stronger in the first trimester. Increasing PA during pregnancy can prevent the development of GDM. PROSPERO REGISTRATION NUMBER: CRD42023420564.

Sleeping more than 8 h: a silent factor contributing to decreased muscle mass in Chinese community-dwelling older adults.

BACKGROUND: Muscle mass loss is an age-related process that can be exacerbated by lifestyle, environmental and other factors, but can be mitigated by good sleep. The objective of this study was to investigate the correlation between varying time lags of sleep duration and the decline in muscle mass among individuals aged 60 years or older by using real-world health monitoring data obtained from wearable devices and smart home health monitoring devices. METHODS: This study included 86,037 observations from 2,869 participants in the Mobile Support System database. Missing data were supplemented by multiple imputation. The investigation utilized generalized estimating equations and restricted cubic spline curve to examine the relationship between sleep duration and low muscle mass. Various lag structures, including 0, 1, 2, 0-1, 0-2, and 1-2 months, were fitted, and the interaction effect of observation time with sleep duration was estimated for each lag structure. Additionally, subgroup analyses were conducted. The models were adjusted for various covariates, including gender, age, body mass index, footsteps, smoking status, drinking status, marital status, number of chronic diseases, number of medications, diabetes mellitus, hyperlipidemia, coronary artery disease, respiratory disease, and musculoskeletal disease and an interaction term between time and sleep duration. RESULTS: The results of the generalized estimating equation showed a significant correlation (p < 0.001) between sleep duration of 8 h or more and low muscle mass in older adults, using 6-7 h of sleep as a reference. This effect was seen over time and prolonged sleep accumulated over multiple months had a greater effect on muscle mass loss than a single month. The effect of long sleep duration on muscle mass loss was significantly greater in females than in males and greater in the over-75 than in the under-75 age group. Restricted cubic spline plots showed a non-linear relationship between sleep duration and low muscle mass (p < 0.001). CONCLUSIONS: This study found an association between sustained nighttime sleep of more than eight hours and decreased muscle mass in older adults, especially older women.

Association of HHEX and SLC30A8 Gene Polymorphisms with Gestational Diabetes Mellitus Susceptibility: A Meta-analysis.

Genetics plays a role in the development of gestational diabetes mellitus (GDM), which poses serious risks to pregnant women and their children. Several studies have demonstrated a link between GDM susceptibility and rs13266634 C/T polymorphism in SLC30A8 gene and rs1111875 C/T and rs5015480 C/T, which are located near the linkage disequilibrium block containing the IDE, HHEX, and KIF11 genes. However, the results are conflicting. Therefore, we aimed to investigate the association between susceptibility to GDM and HHEX and SLC30A8 gene polymorphisms. PubMed, Web of Science, EBSCO, CNKI, Wanfang Data, VIP, and SCOPUS were used to search for research articles. The quality of the selected literature was evaluated using the Newcastle-Ottawa scale. A meta-analysis was performed using Stata 15.1. Allelic, dominant, recessive, homozygote, and heterozygote models were used for the analysis. Nine articles with 15 studies were included. (1) Four studies about HHEX rs1111875 showed that the C allele was associated with the susceptibility to GDM; (2) three studies on HHEX rs5015480 indicated that the C allele in rs5015480 was significantly associated with GDM; (3) eight studies about SLC30A8 rs13266634 showed that the C allele was significantly associated with the susceptibility to GDM; and (4) a subgroup analysis showed that the rs5015480 polymorphism in HHEX and rs13266634 polymorphism in SLC30A8 gene were associated with GDM susceptibility in Asians. The meta-analysis provided evidence that the C allele in rs1111875 and rs5015480 in HHEX and rs13266634 in SLC30A8 can increase the risk of GDM.PROSPERO registration number CRD42022342280.

Association of SLC22A1 rs622342 and ATM rs11212617 polymorphisms with metformin efficacy in patients with type 2 diabetes.

Metformin is the first-choice oral anti-hyperglycemic drug for type 2 diabetes mellitus (T2DM) patients. There are controversies about the association of SLC22A1 rs622342, which was not reported in the Chinese population, and ataxia-telangiectasia mutated (ATM) rs11212617 polymorphisms with metformin efficacy in T2DM. Our study was to investigate the effects of the two single nucleotide polymorphisms on the efficacy of metformin in T2DM of Han nationality in Chaoshan China. After enrollment, 82 newly diagnosed T2DM patients went on 2-month metformin monotherapy. According to BMI before treatment, the patients were divided into a normal weight group (≥18.5 and <25 kg/m2) and an overweight group (BMI ≥ 25 and <30 kg/m2). T-test, Pearson χ2 test, and regression analysis, which adjusted for age, BMI, sex, the dose of metformin, education, tea drink, smoking, and sweet, were used to evaluate the effects of rs622342 and rs11212617 on several variables, such as fasting plasma glucose (FPG). Compared with the AA or CC genotype, patients with AC genotype of rs622342 achieved greater reduction in Δ60FPG and Δ(60-30)FPG (P = 0.00820, 0.00089, respectively). For 11212617, the reduction in Δ30FPG and Δ60FPG was significantly different among patients with the AC genotype (P = 0.00026, 0.00820, respectively). Our results indicated that common variants of SLC22A1 rs622342 and ATM rs11212617 were associated with the efficacy of metformin in T2DM of Han nationality in Chaoshan China.

Post-Harvest Processing Methods Have Critical Roles in the Contents of Active Ingredients of Scutellaria baicalensis Georgi.

To find the best post-harvest processing method for Scutellaria baicalensis Georgi, we explored the effects of fresh and traditional processing on the active ingredients in S. baicalensis and evaluated three drying techniques to determine the optimal post-harvest processing technique. We quantified four active ingredients (baicalin, baicalein, wogonoside, and wogonin) in 16 different processed S. baicalensis samples that were harvested from Tongchuan, Shaanxi province, by HPLC (high-performance liquid chromatography). In addition, we performed a similarity analysis (SA), a hierarchical cluster analysis (HCA), and a principal component analysis (PCA) on the common peaks in S. baicalensis that were identified by the HPLC fingerprints. Compared to the traditional processing method, the fresh processing method could better preserve the four active ingredients in S. baicalensis, meanwhile, the similarity analysis (0.997-1.000) showed that the fresh processing was more similar to the traditional processing, and it did not change the type of 18 active ingredients in S. baicalensis. The cluster analysis results showed that the shade drying and sun drying methods results were more similar to each other, while the oven drying (60 °C) method results were clustered into one category. According to the results of the principal component analysis, S9, S7, and S8 had higher scores, and they were relatively well processed under these processing settings. Fresh processing could be an alternative to traditional processing; the moisture content was reduced to 24.38% under the sun drying condition, and it was the optimal post-harvest processing solution for S. baicalensis.

Effects of Different Processing Methods Based on Different Drying Conditions on the Active Ingredients of Salvia miltiorrhiza Bunge.

Compared to the traditional processing method, fresh processing can significantly enhance the preservation of biologically active ingredients and reduce processing time. This study evaluated the influences of fresh and traditional processing based on different drying conditions (sun drying, oven drying and shade drying) on the active ingredients in the roots and rhizomes of S. miltiorrhiza. High-performance liquid chromatography (HPLC) was utilized to determine the contents of six active ingredients in the roots and rhizomes of S. miltiorrhiza. The data were analyzed by fingerprint similarity evaluation, hierarchical cluster analysis (HCA) and principal component analysis (PCA). The results suggest that compared to the traditional processing method, the fresh processing method may significantly increase the preservation of biologically active ingredients. Furthermore, the findings demonstrated that among the three drying methods under fresh processing conditions, the shade-drying (21.02-26.38%) method is most beneficial for retaining the active ingredients in the roots and rhizomes of S. miltiorrhiza. Moreover, the fingerprint analysis identified 17 common peaks, and the similarity of fingerprints among samples processed by different methods ranged from 0.989 to 1.000. Collectively, these results suggest novel processing methods that may improve the yield of active ingredients for S. miltiorrhiza and may be implemented for industrial production.

Bioresorbable Depot for Sustained Release of Immunostimulatory Resiquimod in Suppressing Both Primary Triple-Negative Breast Tumors and Metastatic Occurrence.

In light of immune facilities trafficking toward the pathological sites along upward gradient of immunostimulatory cytokines, a localized resiquimod (Toll-like receptor 7/8 agonist) release depot was manufactured for pursuit of precision immunostimulation toward intractable triple-negative breast carcinoma. In principle, resiquimod/poly(lactic-co-glycolic acid) microspheres were fabricated and embedded into injectable and biodegradable poly(ethylene glycol) (PEG)-based hydrogel. The subsequent investigations approved persistent retention of immunostimulatory resiquimod in tumors upon peritumoral administration, which consequently led to localized and consistent secretion of immunostimulatory cytokines. Initially, not only innate tumor phagocytosis but also adaptive antitumor immunities were successfully cultivated for in situ suppression of the growth of primary solid tumors, more importantly, capable of inhibiting distant pulmonary metastasis, as evidenced by observation of enormous lymphocytes selectively gathering in the pulmonary artery. Hence, our presented study provided an important clinical indication of using immunostimulatory drugs to activate potent innate and adaptive antitumor immunities for precision antitumor therapy. Further immunomodulatory strategies, such as checkpoint blockage and tumor immunogenicity, could also be complementary for development of advanced antitumor immunotherapeutics in treatment of a number of intractable tumors.

Ultrasound-Responsive Nanoparticulate for Selective Amplification of Chemotherapeutic Potency for Ablation of Solid Tumors.

Precision medicine requests preferential transportation of the pharmaceutical substances to the pathological site and impartation of localized therapeutic activities to the targeted cells. To accomplish this goal, we attempted a facile nanoscaled ultrasound-responsive delivery system, characterized by doxorubicin assembled with an amphiphilic copolymer (multiple of hydrophobic stearic segments tethered onto the hydrophilic pullulan backbone through ultrasound-labile oxyl-alkylhydroxylamine linkage). As a consequence of the strategically installed ultrasound-labile oxyl-alkylhydroxylamine linkage to elicit the tailored segregation of the hydrophilic pullulan and the hydrophobic stearic segments upon ultrasound impetus, the constructed nanoscaled self-assembly presented distinctive structural destabilization behaviors and afforded spatiotemporal controlled liberation of the cytotoxic drugs. It is worthy to note that the ultrasound was determined to markedly lower the IC50 of the proposed system from over 10 µg/mL to 2.33 µg/mL (approximate 4-fold), thereby serving as a facile impetus to amplify the cytotoxic potency of the proposed drug delivery vehicles. Furthermore, drastic tumor ablation was validated by dosage of the proposed doxorubicin delivery system to T41 tumor-bearing mice accompanied by the tumor-localized ultrasound impetus, while no observable adverse side effect was confirmed. Therefore, the results advocated our ultrasound-responsive delivery vehicle as a tempting strategy for precise spatiotemporal control of the release of the drug cargo, thus affording selectively amplified cytotoxic potency to the ultrasound-imposed site, which should be highlighted as important progress toward precision medicine.

Association of KCNQ1 polymorphisms with gliclazide efficacy in Chinese type 2 diabetic patients.

OBJECTIVES: To investigate the effects of KCNQ1 polymorphisms on the efficacy of gliclazide in type 2 diabetic patients. MATERIALS AND METHODS: A total of 443 newly diagnosed type 2 diabetic patients were included in this study. After enrollment, patients went on an 8-week gliclazide monotherapy. Fasting plasma glucose (FPG) was measured before and after the treatment. Life-style information was collected by weekly follow-up. Genotyping of the two single-nucleotide polymorphisms was performed using the single base primer extension method. T-test, one-way analysis of variance, and Pearson χ-test were used to evaluate the effects of rs2237892 and rs2237897 on the FPG reduction and treatment success rate. RESULTS: After 8 weeks of gliclazide therapy, the FPG decreased significantly from 10.9±2.8 to 7.4±2.2 mmol/l (P<0.001). Compared with the CC genotype, patients with CT or TT genotypes of rs2237897 achieved greater reduction in FPG (3.9±2.6 vs. 3.2±2.4 mmol/l, P=0.003; 33.9±19.0 vs. 27.7±17.4%, P<0.001) and a higher rate of treatment success (74.1 vs. 65.2%, P=0.042 for criterion 1; 61.1 vs. 44.5%, P<0.001 for criterion 2, respectively), whereas no significant difference was found in the FPG reduction and treatment success rate among different genotypes of rs2237892. CONCLUSION: Our results indicated that a common variant of KCNQ1, rs2237897, was associated with the efficacy of gliclazide after 8-week monotherapy in Chinese newly diagnosed type 2 diabetic patients. The FPG reduction and treatment success rate were significantly higher in carriers of CT and TT genotypes.

Interaction of type 2 diabetes mellitus with chromosome 9p21 rs10757274 polymorphism on the risk of myocardial infarction: a case-control study in Chinese population.

BACKGROUND: Myocardial infarction (MI) is a serious complication of Coronary Artery Disease (CAD). Previous studies have identified genetic variants on chromosome 9p21 and 6p24 that are associated with CAD, but further studies need to be conducted to investigate whether these genetic variants are associated with the pathogenesis of MI. We therefore performed this study to assess the association between the risk of MI and SNP rs10757274 on chromosome 9p21 and SNP rs6903956 on chromosome 6p24, and to explore the gene-environment interactions in a Chinese population. METHODS: A hospital-based case-control study, consisting of 502 MI patients and 308 controls, was conducted in a Chinese population. Demographic, behavioral information and clinical characteristics were collected, and genotyping of the two SNPs was performed using single base primer extension genotyping technology. The unconditional logistic regression (ULR) method was adopted to assess the association of the two SNPs with MI risk. Both generalized multifactor dimensionality reduction (GMDR) and ULR methods were applied to explore the effect of gene-environment interactions on the risk of MI. RESULTS: After adjusting for covariates, it was observed that SNP rs10757274 on chromosome 9p21 was significantly associated with MI. Compared with subjects carrying the AA genotype, subjects carrying the GA or GG genotypes had a higher MI risk (ORa = 1.52, 95% CI:1.06-2.19, pa = 0.0227; ORa = 2.40, 95% CI:1.51-3.81, pa = 0.0002, respectively). Furthermore, a two-factor gene-environment interaction model of CDKN2A/B (rs10757274) and type 2 diabetes mellitus (T2DM) was identified to be the best model by GMDR (p = 0.0107), with a maximum prediction accuracy of 59.18%, and a maximum Cross-validation Consistency of 10/10. By using the ULR method, additive interaction analysis found that the combined effect resulted in T2DM-positive subjects with genotype GG/GA having an MI risk 4.38 times that of T2DM-negative subjects with genotype AA (ORadd = 4.38, 95% CI:2.56-7.47, padd < 0.0001). CONCLUSIONS: These results show that gene polymorphism of CDKN2A/B (rs10757274) is associated with MI risk in a Chinese population. Furthermore, T2DM is likely to have an interaction with CDKN2A/B (rs10757274) that contributes to the risk of MI.

Amphiphilic polymeric nanodrug integrated with superparamagnetic iron oxide nanoparticles for synergistic antibacterial and antitumor therapy of colorectal cancer.

Colorectal cancer (CRC) is one of the most prevalent and deadly malignancies that can be influenced by Fusobacterium nucleatum (Fn), a bacterium that promotes tumor development and chemoresistance, resulting in limited therapeutic efficacy. Traditional antibiotics cannot effectively eliminate Fn at tumor site due to issues like biofilm formation, while chemotherapy alone fails to suppress tumor progression. Therefore, the development of new methods to eliminate Fn and promote antitumor efficacy is of great significance for improving the outcome of CRC treatment. Herein, we developed a nanodrug (OPPL) that integrates oleic acid-modified superparamagnetic iron oxide nanoparticles (O-SPIONs) and an amphiphilic polymer (PPL) to deliver the platinum prodrug and antimicrobial lauric acid (LA) for enhancing the treatment of CRC. We demonstrated that OPPL can synergistically enhance antibacterial and biofilm disruption activities against Fn along with the antimicrobial LA by producing reactive oxygen species (ROS) through its peroxidase-like activity. Furthermore, the OPPL nanodrug can increase intracellular ROS, promote lipid peroxides and deplete glutathione, leading to ferroptosis. By combining chemotherapy and induced ferroptosis, the OPPL nanodrug exhibited high cytotoxicity against CRC cells. In vivo studies showed that the OPPL nanodrug could enhance tumor accumulation, enable magnetic resonance imaging, suppresse tumor growth, and inhibit growth of intratumor Fn. These results suggest that OPPL is an effective and promising candidate for the treatment of Fn-infected CRC. STATEMENT OF SIGNIFICANCE: The enrichment of Fusobacterium nucleatum (Fn) in colorectal cancer is reported to exacerbate tumor malignancy and is particularly responsible for chemoresistance. To this respect, we strategically elaborated multifaceted therapeutics, namely OPPL nanodrug, combining oleic acid-modified superparamagnetic iron oxide nanoparticles (O-SPIONs) with a polymer containing a platinum prodrug and antimicrobial lauric acid. The O-SPION components exert distinctive peroxidase-like activity, capable of stimulating Fenton reactions selectively in the tumor microenvironment, consequently accounting for the progressive production of reactive oxygen species. Hence, O-SPIONs have been demonstrated to not only supplement the antimicrobial activities of lauric acid in overcoming Fn-induced chemoresistance but also stimulate potent tumor ferroptosis. Our proposed dual antimicrobial and chemotherapeutic nanodrug provides an appreciable strategy for managing challenging Fn-infected colorectal cancer.

Triple-transformable dynamic surroundings for programmed transportation of bio-vulnerable mRNA payloads towards systemic treatment of intractable solid tumors.

The surface physiochemical properties of nanomedicine play a crucial role in modulating biointerfacial reactions in sequential biological compartments, accordingly accomplishing the desired programmed delivery scenario to intracellular targets. PEGylation, which involves modifying the surface with a layer of poly(ethylene glycol), has been validated as an effective strategy for minimizing adverse biointerfacial interactions. However, it has also been observed to impede cellular uptake and intracellular trafficking activities. To address this dilemma, we propose a dynamic surface chemistry approach that actively prevents non-specific reactions in systemic circulation, while readily facilitating cellular uptake by converting into a highly cytomembrane-adhesive state. Moreover, the surface becomes more adhesive to endolysosomal membranes, enabling translocation into the cytosol. In this study, PEGylated mRNA delivery nanoparticulates were tethered with charge-reversible polymers to create dynamic surroundings through click chemistry. Importantly, the dynamic surroundings exhibited negative charges under physiological conditions (pH 7.4). This property prevented degradation by anionic nucleases and structural disassembly induced by endogenous charged biological species. Consequently, the nanoparticles exhibited appreciable stealth function, effectively managing the first pass effect, leading to prolonged blood retention and improved bioavailabilities at targeted cells. Furthermore, the dynamic surroundings shifted towards relatively positive charges in the tumor microenvironment (pH 6.8). As a result, the nanoparticles were more likely to be taken up by tumors due to their electrostatic affinities towards polyanionic cytomembranes. Eventually, the internalized mRNA nanomedicine transformed responsive to the surrounding microenvironment into highly positive charges within acidic endolysosomes (pH 5.0), exerting explosive disruptive potencies on the endolysosomal structures, thus facilitating translocation of mRNA from the digestive endolysosomes into the targeted cytosol. Notably, the dynamic surroundings also reduced the immunogenicity of naked mRNA due to their stealthy properties and rapid endolysosomal translocation functions. In summary, our proposed unique triple-transformable dynamic surface chemistry provided an intriguing delivery scenario that overcomes sequential biological barriers, contributing to efficient expression of the encapsulated mRNA at targeted tumors.

pH-responsive organic/inorganic hybrid nanocolloids for transcellular delivery of ribonucleolytic payloads toward targeted anti-glioma therapy.

Proteins have been appreciated to be a superlative modality of therapeutics in view of their direct roles in regulating diverse sets of biological events, nonetheless, the clinical applications of the proteinic therapeutics have been strictly limited to act on the cell surface receptors owing to their inherent cell-impermeable character of the proteins. To this obstacle, we contrived carboxylation reaction upon the proteins (RNase A) into the overall negatively charged pro-RNase, followed by elaboration of intelligent pH-responsive pro-RNase delivery nanocolloids based on co-precipitation of pro-RNase and Arg-Gly-Asp (RGD)-functionalized poly(ethylene glycol) (PEG)-block-polyanion with aids of inorganic calcium phosphate (CaP). The resulting nanocolloids appeared to actively accumulate into glioma due to the specific binding affinities of RGD and glioma-enriched αVß3 and αVß5 integrins. Furthermore, the pH responsiveness to the acidic endolysosomal microenvironment of all compositions of nanocolloids (including: decarboxylation of pro-RNase composition to restore the native RNase A, ionization of CaP composition to elicit osmotic pressure, and charge reversal of PEG-block-polyanion into membrane-disruptive polycation) could stimulate not only efficient endolysosomal escape for translocation into the cytosol but also structural disassembly for ready liberation of the RNase A payloads, eventually exerting non-specific RNA degradation for apoptosis of the affected cells. Systemic dosage of the proposed nanocolloids demonstrated potent anti-tumor efficacies towards xenograft glioma due to massive RNA degradation. Therefore, our proposed RNase A prodrug nanocolloids could represent as a versatile platform for engineering transcellular protein delivery systems, which are expected to spur thriving emergence of a spectrum of proteins in precision intervention of intractable diseases.

Simultaneous Knockdown of Immune Suppressive Markers by Tumor Microenvironment-Responsive Multifaceted Prodrug Nanomedicine.

Tumors managing to exempt from immune clearance are attributable to their overexpressed immune suppressive molecules (CD47, PD-L1, etc.). Leadingly, the checkpoint blockade-based chemoimmunotherapy by means of knockdown of these immunosuppressive checkpoints, together with immunogenetic chemotherapeutics, is perceived to be a valid therapeutic strategy for improving anti-tumor outcomes. Herein, chemotherapeutic camptothecin was covalently introduced into an intriguing multifaceted nanomedicine. Note that the elaborated nanomedicine was chemically engineered to enable targeted transportation to the tumors via systemic administration, possessing intelligent responsiveness to sequential extracellular and intracellular microenvironments in the targeted tumors for prompted transcellular endocytosis owing to enzymolysis by the tumor-enriched matrix metalloproteinases and the selective liberation of cytocidal camptothecin in the cell interiors owing to thiolysis by glutathione. In addition, this chemotherapeutic nanomedicine allowed facile encapsulation of the negatively charged RNA interference payloads. Consequently, aiming for treatment of intractable triple-negative breast tumors, we attempted the small interfering RNA (siRNA) payloads aiming for CD47 and PD-L1 into the aforementioned nanomedicine. The subsequent investigations demonstrated drastic knockdown of these vital immune suppressive checkpoints by this siRNA-encapsulating chemotherapeutic nanomedicine, conducing to the reversal of the immune checkpoint suppressive microenvironment of triple-negative 4T1 tumors. Namely, the inhibited proceedings of the innate and adaptive anti-tumor immunities were revived, as supported by observation of the activated infiltration and retention of CD68+ macrophages and CD4+ and CD8+ lymphocytes into the tumors. Eventually, most potent anti-tumor efficacies were accomplished by systemic administration of this chemoimmunotherapeutic nanomedicine, which verified the amplified contribution from anti-tumor immunities by means of knockdown of the immune suppressive molecules to the ultimate anti-tumor efficacies. Note that the upregulation of the immune suppressive molecules was constantly reported in a variety of clinical therapies; hence, our facile chemoimmunotherapeutic platform should be emphasized in clinical translation for seeking improved therapeutic outcomes.

Activation of pyroptosis by specific organelle-targeting photodynamic therapy to amplify immunogenic cell death for anti-tumor immunotherapy.

Pyroptosis, a unique lytic programmed cell death, inspired tempting implications as potent anti-tumor strategy in pertinent to its potentials in stimulating anti-tumor immunity for eradication of primary tumors and metastasis. Nonetheless, rare therapeutics have been reported to successfully stimulate pyroptosis. In view of the intimate participation of reactive oxygen species (ROS) in stimulating pyroptosis, we attempted to devise a spectrum of well-defined subcellular organelle (including mitochondria, lysosomes and endoplasmic reticulum)-targeting photosensitizers with the aim of precisely localizing ROS (produced from photosensitizers) at the subcellular compartments and explore their potentials in urging pyroptosis and immunogenic cell death (ICD). The subsequent investigations revealed varied degrees of pyroptosis upon photodynamic therapy (PDT) towards cancerous cells, as supported by not only observation of the distinctive morphological and mechanistic characteristics of pyroptosis, but for the first-time explicit validation from comprehensive RNA-Seq analysis. Furthermore, in vivo anti-tumor PDT could exert eradication of the primary tumors, more importantly suppressed the distant tumor and metastatic tumor growth through an abscopal effect, approving the acquirement of specific anti-tumor immunity as a consequence of pyroptosis. Hence, pyroptosis was concluded unprecedently by our proposed organelles-targeting PDT strategy and explicitly delineated with molecular insights into its occurrence and the consequent ICD.

Charge-Reversible Pro-Ribonuclease Enveloped in Virus-like Synthetic Nanocapsules for Systemic Treatment of Intractable Glioma.

We have tailored multifaceted chemistries into the manufacture of artificial virus-like delivery vehicles mimicking viral "intelligent" transportation pathways through sequential biological barriers; these vehicles can acquire the ability to dynamically "program transfer" to their target sites. To accomplish this, we created anionic pro-proteins, which facilitate charge reversal when subject to acidic endosomal pH; in this way, carboxylation reactions are performed on proteins with amine-reactive cis-aconitic anhydride. Electrostatic associations then initiate the envelopment of these pro-proteins into multilayered nanoarchitectural vehicles composed of multiple-segmental block copolycationic cyclic Arg-Gly-Asp (RGD)-poly(ethylene glycol)(PEG)-GPLGVRG-polylysine(thiol). Therefore, upon the pro-proteins' initial binding to the tumors via the protruding RGD ligands, the bio-inert PEG surroundings are detached through the enzymolysis of the intermediate GPLGVRG linkage by tumor-enriched matrix metalloproteinases, unveiling the cationic polylysine palisade and imparting intimate affinities to the anionic cytomembranes of the targeted tumors. Essentially, through their active endocytosis into the subcellular endosomal compartments, the pro-proteins are made capable of retrieving the original amine groups through a charge reversal decarboxylation process, consequently eliciting augmented charge densities (charge nonstoichiometric protein@polylysine(disulfide)) to disrupt the anionic endosomal membranes to facilitate translocation into the cytosol. Eventually, the active protein payloads can be liberated from nonstoichiometric protein@polylysine(thiol) by the disassembly of polylysine palisade upon the cleavage of disulfide crosslinking in response to the very high level of glutathione in the cytosol, thereby contributing toward extreme cytotoxic potency. Hence, our elaborated virus-mimicking platform has demonstrated potent antitumor efficacy through the systemic administration of ribonucleases, which will consequently lead to an innovative new therapeutic method by which proteins could reach intracellular targets.

Spatiotemporal Concurrent Liberation of Cytotoxins from Dual-Prodrug Nanomedicine for Synergistic Antitumor Therapy.

Nanomedicine developed to date by means of directly encapsulating cytotoxins suffers from crucial drawbacks, including premature release and detoxification prior to arrival at pharmaceutics targets. To these respects, redox-responsive polymeric prodrugs of platinum (Pt) and camptothecin (CPT), selectively and concomitantly activated in the cytoplasm, were elaborated in manufacture of dual prodrug nanomedicine. Herein, multiple CPTs were conjugated to poly(lysine) (PLys) segments of block copolymeric poly(ethylene glycol) (PEG)-PLys through the redox responsive disulfide linkage [PEG-PLys(ss-CPT)] followed by reversible conversion of amino groups from PLys into carboxyl groups based on their reaction with cis-aconitic anhydride [PEG-PLys(ss-CPT&CAA)]. On the other hand, Pt(IV) in conjugation with dendritic polyamindoamine [(G3-PAMAM-Pt(IV)] was synthesized for electrostatic complexation with PEG-PLys(ss-CPT&CAA) into dual prodrug nanomedicine. Subsequent investigations proved that the elaborated nanomedicine could sequentially respond to intracellular chemical potentials to overcome a string of predefined biological barriers and facilitate intracellular trafficking. Notably, PEG-PLys(ss-CPT&CAA) capable of responding to the acidic endosomal microenvironment for transformation into endosome-disruptive PEG-PLys(ss-CPT), as well as release of G3-PAMAM-Pt(IV) from nanomedicine, prompted transclocation of therapeutic payloads from endosomes into cytosols. Moreover, concurrent activation and liberation of cytotoxic CPT and Pt(II) owing to their facile responsiveness to the cytoplasmic reducing microenvironment have demonstrated overwhelming cytotoxic potencies. Eventually, systemic administration of the dual prodrug construct exerted potent tumor suppression efficacy in treatment of intractable solid breast adenocarcinoma, as well as an appreciable safety profile. The present study illustrated the first example of nanomedicine with a dual prodrug motif, precisely and concomitantly activated by the same subcellular stimuli before approaching pharmaceutic action targets, thus shedding important implication in development of advanced nanomedicine to seek maximized pharmaceutic outcomes.

Synthetic infrared nano-photosensitizers with hierarchical zoom-in target-delivery functionalities for precision photodynamic therapy.

Surgical assailment at the vulnerable subcellular organelles (e.g. mitochondria) by photodynamic therapy (PDT) is perceived as the most devastating approach to eradicate the tumors. Herein, we programmed a novel near-infrared (NIR) PDT construct illustrating appreciable hierarchical zoom-in targeting scenario, namely, primary cell-level targeting to carcinoma post systemic dosage and subcellular level targeting to mitochondria. Pertaining to tumor-targeting function, charge reversal chemistry selectively responsive to acidic tumoral microenvironments (pH 6.8) was implemented as the external corona of PDT constructs. This charge transformative exterior entitled minimal biointerfacial reactions in systemic retention but intimate affinities to cytomembranes selectively in tumoral microenvironments, thereby resulting in preferential uptake by tumors. Furthermore, the proposed PDT constructs were equipped with mitochondria targeting triphenylphosphonium (TPP) motif, which appeared to propel intriguing 88% colocalization with mitochondria. Therefore, overwhelming cytotoxic potencies were accomplished by our carefully engineered photodynamic constructs. Another noteworthy is the photodynamic constructs characterized to be excited at tissue-penetrating NIR (980 nm) based on energy transfer between their internal components of anti-Stoke upconversion nanoparticles (UCN, donor) and photodynamic chlorin e6 (Ce6, acceptor). Therefore, practical applications for photodynamic treatment of intractable solid carcinoma were greatly facilitated and complete tumor eradication was achieved by systemic administration of the ultimate multifunctional NIR photodynamic constructs.

Chemotherapeutic potency stimulated by SNAI1-knockdown based on multifaceted nanomedicine.

Molecular insights into tumorigenesis have uncovered intimate correlation of SNAI1 with tumor malignancy. Herein, to explore merits of SNAI1-knockdown in tumor therapy, we harnessed RNA interference tool (shSNAI1), together with chemotherapeutic doxorubicin. Owing to abundant hydroxyl groups, pullulan was attempted to be covalently conjugated with a multiple of functional moieties, including positively-charged oligoethylenimine components for electrostatic entrapment of polyanionic shSNAI1 and hydrophobic components for entrapment of lipophilic doxorubicin. Notably, the aforementioned covalent conjugations were tailored to be detachable in response to intracellular reducing microenvironment owing to redox disulfide linkage, thereby accounting for selective intracellular liberation of the therapeutic payloads. Moreover, the surface of nanomedicine was modified with hyaluronic acid, endowing not only excellent biocompatibilities but active tumor-targeting function due to its receptors (CD44) overexpressed on tumor cells. Subsequent investigations approved appreciably targeted co-delivery of shSNAI1 and doxorubicin into solid lung tumors via systemic administration and demonstrated critical contribution of SNAI1-knockdown in amplifying chemotherapeutic potencies.