Ribonuclease A-polymer conjugates via in situ growth for cancer treatment.

Efficient delivery of therapeutic proteins is a critical aspect for protein-based cancer treatment. Herein, an in situ growth approach was employed to prepare ribonuclease A (RNase A)-polymer conjugates by incorporating a cationic polymer, poly(N,N'-dimethylamino-2-ethyl methacrylate) (P(DMAEMA)), and a hydrophobic polymer, poly(N-isopropylacrylamide) (P(NIPAM)), through atom transfer radical polymerization (ATRP). The synthesized RNase A-polymer conjugates (namely R-P(D-b-N)) could preserve the integrity of RNase A and exhibit a unique combination of cationic and hydrophobic properties, leading to enhanced intracellular delivery efficiency. The successful delivery of RNase A by R-P(D-b-N) conjugates effectively triggered the cell apoptosis through the mitochondria-dependent signaling pathway to achieve the anti-proliferative response. Additionally, the conjugates could inhibit cell migration and thus possess the potential for the suppression of tumor metastasis. Overall, our findings highlight that the introduction of cationic and hydrophobic moieties via ATRP provides a versatile platform for the intracellular delivery of therapeutic proteins, offering a new avenue for treating diverse diseases.

Fluorinated polyamidoamine dendrimer-mediated miR-23b delivery for the treatment of experimental rheumatoid arthritis in rats.

In rheumatoid arthritis (RA), insufficient apoptosis of macrophages and excessive generation of pro-inflammatory cytokines are intimately connected, accelerating the development of disease. Here, a fluorinated polyamidoamine dendrimer (FP) is used to deliver miR-23b to reduce inflammation by triggering the apoptosis of as well as inhibiting the inflammatory response in macrophages. Following the intravenous injection of FP/miR-23b nanoparticles in experimental RA models, the nanoparticles show therapeutic efficacy with inhibition of inflammatory response, reduced bone and cartilage erosion, suppression of synoviocyte infiltration and the recovery of mobility. Moreover, the nanoparticles accumulate in the inflamed joint and are non-specifically captured by synoviocytes, leading to the restoration of miR-23b expression in the synovium. The miR-23b nanoparticles target Tab2, Tab3 and Ikka to regulate the activation of NF-κB pathway in the hyperplastic synovium, thereby promoting anti-inflammatory and anti-proliferative responses. Additionally, the intravenous administration of FP/miR-23b nanoparticles do not induce obvious systemic toxicity. Overall, our work demonstrates that the combination of apoptosis induction and inflammatory inhibition could be a promising approach in the treatment of RA and possibly other autoimmune diseases.

Superoxide dismutase-embedded metal-organic frameworks via biomimetic mineralization for the treatment of inflammatory bowel disease.

Inflammatory bowel disease (IBD) is characterized by chronic and spontaneous inflammation in the gastrointestinal tract, and has been associated with an improved level of reactive oxygen species (ROS). Herein, superoxide dismutase (SOD) was encapsulated into zeolitic imidazolate framework-zni (ZIF-zni) to construct a nanocomposite termed SOD@ZIF-zni through biomimetic mineralization, which was then used as a formulation for the IBD treatment. The SOD@ZIF-zni composite could efficiently suppress the level of ROS and pro-inflammatory cytokines, using the colorectal cancer cell line SW480 as a model. Oral administration of the SOD@ZIF-zni composite could relieve the oxidative stress and inhibit the release of pro-inflammatory cytokines in the inflamed colonic tissues, leading to the alleviation of colitis in dextran sulfate sodium-induced colitis mice. Overall, the favorable therapeutic efficacy and biocompatibility of SOD@ZIF-zni gave it potential to be used as a safe and effective formulation for IBD treatment in the future.

Urate oxidase loaded in PCN-222(Fe) with peroxidase-like activity for colorimetric detection of uric acid.

In the past two decades, the number of reports on the construction of uric acid (UA) sensors has increased dramatically, as it is a vital factor in the diagnosis of physiological functions and diseases. Among these sensors, cascade colorimetric detection based on peroxidase mimics has received great attention owing to the advantages of easier operation and more intuitive results. Herein, we report a simple UA detection method via the integration of urate oxidase (UOx) and PCN-222(Fe) with peroxidase-like activity, in which UOx is immobilized in PCN-222(Fe) by physical adsorption. UOx could catalyze the UA oxidation to produce H2O2, and then PCN-222(Fe) catalyzed the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of H2O2 to generate a blue product with a maximum absorbance at 652 nm. Under optimal conditions, the increasing absorbance at 652 nm was proportional to the UA concentration. The linear range for UA detection was 10-800 µM with a limit of detection of 3.5 µM, and the cascade system has been successfully utilised for the detection of UA in human serum samples to demonstrate the accuracy of the proposed method. In conclusion, the cascade catalytic system based on the immobilization of UOx in PCN-222(Fe) has the potential to be used as a rapid and sensitive sensor for UA detection.

Lipoic Acid-Modified Oligoethyleneimine-Mediated miR-34a Delivery to Achieve the Anti-Tumor Efficacy.

MiR-34a, an important tumor suppressor, has been demonstrated to possess great potential in tumor gene therapy. To achieve the upregulation of miR-34a expression level, an oligoethyleneimine (OEI) derivative was constructed and employed as the carrier through the modification with lipoic acid (LA), namely LA-OEI. In contrast to OEI, the derivative LA-OEI exhibited superior transfection efficiency measured by confocal laser scanning microscopy and flow cytometry, owing to rapid cargo release in the disulfide bond-based reduction sensitive pattern. The anti-proliferation and anti-migration effects were tested after the miR-34a transfection to evaluate the anti-tumor response, using human cervical carcinoma cell line HeLa as a model. The delivery of LA-OEI/miR-34a nanoparticles could achieve obvious anti-proliferative effect caused by the induction of cell apoptosis and cell cycle arrest at G1 phase. In addition, it could inhibit the migration of tumor cells via the downregulation of MMP-9 and Notch-1 level. Overall, the LA-OEI-mediated miR-34a delivery was potential to be used as an effective way in the tumor gene therapy.

Artesunate-loaded porous PLGA microsphere as a pulmonary delivery system for the treatment of non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) has emerged to be a significant cause of cancer mortality worldwide. Artesunate (ART) extracted from Chinese herb Artemisia annua L, has been proven to possess desirable anti-cancer efficacy, especially for the metastatic NSCLC treatment. Moreover, the poly(lactic-co-glycolic acid) (PLGA) microsphere has been considered to be a potential pulmonary delivery system for the sustained drug release to enhance the therapeutic efficacy of lung cancer. Herein, the ART-loaded porous PLGA microsphere was prepared through the emulsion solvent evaporation approach. The microsphere was demonstrated to possess highly porous structure and ideal aerodynamic diameter for the pulmonary administration. Meanwhile, sustained ART release was obtained from the porous microsphere within 8 days. The release solution collected from the microsphere could be effectively uptake by the cells and further induce the cell apoptosis and the cell cycle arrest at G2/M phase to execute the anti-proliferative effect, using human lung adenocarcinoma cell line A549 as a model. Additionally, strong inhibitory effect on the cell migration and invasion could be obtained after the treatment with release solution. Taken together, our results demonstrated that the ART-loaded PLGA porous microsphere could achieve excellent anti-cancer efficacy, providing a potential approach for the NSCLC treatment via the pulmonary administration.

Dual ATP/reduction-responsive polyplex to achieve the co-delivery of doxorubicin and miR-23b for the cancer treatment.

Combination therapy based on the co-delivery of therapeutic genes and anti-cancer drugs has emerged as a promising approach in the cancer treatment, and stimuli-responsive delivery systems could further improve the therapeutic efficacy. Herein, an ATP aptamer and its complementary DNA were used to form Duplex into which doxorubicin (DOX) was loaded to construct DOX-Duplex, and then the lipoic acid-modified oligoethyleneimine (LA-OEI) was employed as a carrier to realize the co-delivery of DOX-Duplex and miR-23b. The ternary nanocomplex LA-OEI/miR-23b/DOX-Duplex showed excellent anti-proliferative effect by inducing the cell apoptosis via mitochondrial signaling pathway and arresting the cell cycle at S phase. Meanwhile, the co-delivery of DOX-Duplex and miR-23b could efficiently inhibit the metastasis of cancer cells by reducing the expression level of MMP-9. The favorable anti-tumor efficacy of ternary nanocomplex was attributed to the rapid drug release in response to intracellular ATP concentration and reduction conditions and the synergistic effect between DOX-Duplex and miR-23b. Thus, ATP aptamer and reduction-responsive polymer provided a convenient platform to construct dual stimuli-responsive systems for the co-delivery of gene and drug in the cancer treatment.

Study the lipidoid nanoparticle mediated genome editing protein delivery using 3D intestinal tissue model.

Lipid nanoparticles are promising carriers for oral drug delivery. For bioactive cargos with intracellular targets, e.g. gene-editing proteins, it is essential for the cargo and carrier to remain complexed after crossing the epithelial layer of intestine in order for the delivery system to transport the cargos inside targeted cells. However, limited studies have been conducted to verify the integrity of cargo/carrier nanocomplexes and their capability in facilitating cargo delivery intracellularly after the nanocomplex crossing the epithelial barrier. Herein, we used a traditional 2D transwell system and a recently developed 3D tissue engineered intestine model and demonstrated the synthetic lipid nanoparticle (carrier) and protein (cargo) nanocomplexes are able to cross the epithelial layer and deliver the protein cargo inside the underneath cells. We found that the EC16-63 LNP efficiently encapsulated the GFP-Cre recombinase, penetrated the intestinal monolayer cells in both the 2D cell culture and 3D tissue models through temporarily interrupting the tight junctions between epithelial layer. After transporting across the intestinal epithelia, the EC16-63 and GFP-Cre recombinase nanocomplexes can enter the underneath cells to induce gene recombination. These results suggest that the in vitro 3D intestinal tissue model is useful for identifying effective lipid nanoparticles for potential oral drug delivery.

Nucleolin-Targeting AS1411 Aptamer-Modified Micelle for the Co-Delivery of Doxorubicin and miR-519c to Improve the Therapeutic Efficacy in Hepatocellular Carcinoma Treatment.

BACKGROUND: Multidrug resistance (MDR) has emerged to be a major hindrance in cancer therapy, which contributes to the reduced sensitivity of cancer cells toward chemotherapeutic drugs mainly owing to the over-expression of drug efflux transporters. The combination of gene therapy and chemotherapy has been considered as a potential approach to improve the anti-cancer efficacy by reversing the MDR effect. MATERIALS AND METHODS: The AS1411 aptamer-functionalized micelles were constructed through an emulsion/solvent evaporation strategy for the simultaneous co-delivery of doxorubicin and miR-519c. The therapeutic efficacy and related mechanism of micelles were explored based on the in vitro and in vivo active targeting ability and the suppression of MDR, using hepatocellular carcinoma cell line HepG2 as a model. RESULTS: The micelle was demonstrated to possess favorable cellular uptake and tumor penetration ability by specifically recognizing the nucleolin in an AS1411 aptamer-dependent manner. Further, the intracellular accumulation of doxorubicin was significantly improved due to the suppression of ABCG2-mediated drug efflux by miR-519c, resulting in the efficient inhibition of tumor growth. CONCLUSION: The micelle-mediated co-delivery of doxorubicin and miR-519c provided a promising strategy to obtain ideal anti-cancer efficacy through the active targeting function and the reversion of MDR.

Fluoropolymer-Mediated Intracellular Delivery of miR-23b for the Osteocyte Differentiation in Osteoblasts.

Emerging evidence suggests that microRNAs (miRNAs) play key roles in the regulation of multiple biological processes, including the differentiation of osteoblasts. Although miRNA-based gene therapy holds immense potential in the treatment of a variety of diseases, the intracellular delivery of miRNA remains challenging owing to the lack of efficient and safe gene carriers. In this study, a fluoropolymer (FP) is constructed through the modification of polyamidoamine (PAMAM) using heptafluorobutyric anhydride and then is used as a carrier for miR-23b transfection to induce osteocyte differentiation of osteoblasts. The derivative FP is found to facilitate miR-23b transfection due to its favorable endosomal escape from the "proton sponge" effect. Compared to PAMAM/miR-23b, the FP/miR-23b nanocomplex efficiently promotes the differentiation of osteoblasts and formation of calcified nodules, attributable to enhanced expression of various osteogenesis genes (runt-related transfection factor 2 [RUNX2], alkaline phosphatase [ALP], osteopontin [OPN], and osteocalcin [OCN]). Thus, FP-mediated miR-23b transfection may be used as an effective strategy to facilitate osteogenic differentiation.

In Vitro and In Vivo Study of Amphotericin B Formulation with Quaternized Bioreducible Lipidoids.

Invasive fungal infections are well-known causes of morbidity and mortality in immunocompromised patients. Amphotericin B (AmB) is a polyene fungicidal agent with excellent properties of the broad antifungal spectrum, high activity, and relatively rare drug resistance. However, significant toxicities limit the clinical application of AmB and its conventional formulation AmB deoxycholate (Fungizone). Here we investigated nanoparticle formulations of AmB using synthetic biodegradable lipidoids and evaluated their stability, in vitro antifungal efficacy, and in vivo toxicity and pharmacokinetics. We found that the AmB formulated using a mixture of quaternized lipidoid (Q78-O14B) and DSPE-PEG2000 has the size around 70-100 nm and is stable during storage. The formulation showed no hemotoxicity to red blood cells (RBCs) in vitro. It also possesses the highest antifungal activity (in vitro) and lowest toxicity (both in vitro and in vivo). These metrics are significantly superior to the commercial antifungal product Fungizone. Meanwhile, AmB/Q78-O14B-P exhibited prolonged blood circulation in comparison to Fungizone in vivo. In AmB/Q78-O14B-P formulation, AmB was still detectable in the liver, spleen, and lung tissues with a concentration above the minimum inhibitory concentrations 72 h after low-dose intravenous injection. Based on these results, AmB in lipidoid nanoparticle formulation may produce sustained antifungal activity against blood-borne and systemic organ infections. Moreover, the new AmB formulation showed low nephrotoxicity and hepatotoxicity in rats even at high doses, allowing a dramatically wider and safer therapeutic window than Fungizone. This method provides a means to develop much needed antifungal agents that will be more therapeutically efficacious, more affordable (than AmBisome), and less toxic (than Fungizone) for the treatment of systemic fungal infections.

A genipin-crosslinked protein-polymer hybrid system for the intracellular delivery of ribonuclease A.

BACKGROUND: Therapeutic proteins have been widely used in the treatment of various diseases, and effective carriers are highly required for achieving protein delivery to obtain favorable treatment potency. MATERIALS AND METHODS: A protein-polymer hybrid system was constructed through the genipin-mediated crosslinking of polyethyleneimine with a weight-average molecular weight of 25,000 g/mol (PEI25K) and ribonuclease A (RNase A), namely RGP. RESULTS: The RGP nanoparticles were observed to be easily internationalized in HeLa cells owing to the introduction of positively charged PEI25K, thereby triggering the antiproliferative effects by cleaving RNA molecules in the tumor cells. Moreover, red fluorescence could be obviously visualized in the tumor cells after RGP delivery, which was attributed to the intrinsic characteristics of genipin. CONCLUSION: The protein-polymer hybrid system prepared via the genipin-mediated crosslinking has exhibited potential to be used as a theranostic platform for both in vivo imaging and delivering diverse therapeutic proteins.

Inhibition of proliferation and migration of tumor cells through phenylboronic acid-functionalized polyamidoamine-mediated delivery of a therapeutic DNAzyme Dz13.

BACKGROUND: The phenylboronic acid-functionalized polyamidoamine (PP) was employed as a gene carrier for Dz13 delivery, inducing an obvious anticancer response. MATERIALS AND METHODS: The Dz13 condensation ability of PP was evaluated through gel retardation assay. The cellular uptake mechanism of PP/Dz13 nanoparticles was studied using confocal laser scanning microscope and flow cytometer. The inhibition ability of cell proliferation, migration and invasion was investigated through MTT assay, flow cytometry, wound healing and Transwell migration assays, using hepatocarcinoma cell line HepG2 as a model. Finally, Western blotting analysis was used to detect the signaling pathway associated with the inhibition of cell apoptosis and migration induced by Dz13 delivery. RESULTS: The carrier PP could efficiently condense Dz13 into stable nanoparticles at mass ratios of >1.5. The hydrodynamic diameter and zeta potential of PP/Dz13 nanoparticles were measured to be 204.77 nm and +22.00 mV at a mass ratio of 10.0, respectively. The nanoparticles could realize an efficient cellular uptake in sialic acid-dependent endocytosis manner. Moreover, the nanoparticles exhibited an obvious antiproliferation effect through the induction of cell apoptosis and cell cycle arrest due to the cleavage of c-Jun mRNA. Besides, the suppression of cell migration and invasion could be achieved after the PP/Dz13 transfection, attributing to the decreased expression level of MMP-2 and MMP-9. CONCLUSION: The PP provided a potential delivery system to achieve the tumor-targeting gene therapy.

Phenylboronic acid-modified polyamidoamine-mediated delivery of short GC rich DNA for hepatocarcinoma gene therapy.

Phenylboronic acid was introduced on the surface of polyamidoamine to construct a derivative PP, which was further used as a tumor-targeting carrier for realizing the delivery of short GC rich DNA (GCD). The PP-mediated GCD delivery could disrupt the polymerization of microtubules and thus trigger a strong anti-proliferative effect through the induction of cell apoptosis and cell cycle arrest, using hepatocellular carcinoma cell line HepG2 as a model. In addition, the transfection of PP/GCD nanoparticles could efficiently suppress cell migration and invasion. Moreover, the intravenous injection of PP/GCD nanoparticles could dramatically decrease the tumor growth by inducing the in situ apoptosis of the tumor and meanwhile it exhibited a desirable safety profile. Overall, the development of tumor-targeting carrier PP will provide a promising platform for GCD delivery to obtain an anti-cancer efficacy which is beneficial for facilitating tumor gene therapy in future clinical applications.

Phenylboronic acid-functionalized polyamidoamine-mediated miR-34a delivery for the treatment of gastric cancer.

In the present research, a tumor-targeted gene carrier, PPP, was constructed through the modification of phenylboronic acid onto the surface of a polyamidoamine dendrimer, and then miR-34a delivery was employed as a model to evaluate its anti-tumor efficacy. The carrier PPP was identified to possess favorable miR-34a binding and condensation ability and meanwhile protect miR-34a against nuclease degradation. Through confocal laser scanning microscopy and flow cytometry analysis, PPP-mediated cellular uptake of miR-34a was found to proceed through a sialic acid-dependent endocytosis pathway and the nanoparticles could achieve endosome/lysosome escape within 6 h. Further, an anti-proliferative effect could be obtained after PPP/miR-34a transfection through the induction of cell apoptosis. Meanwhile, the inhibition of migration and invasion could be realized through blocking the Notch-1 signaling pathway after PPP/miR-34a treatment. Finally, PPP possessed acceptable safety and inhibited in vivo tumor growth through the in situ apoptosis of tumor sites, which relied on the specific tumor-targeting ability and long circulation time in the blood. In summary, the derivative PPP could be potentially utilized as an efficient carrier for miR-34a delivery to achieve an anti-tumor response in clinical use.

A comprehensive review on histone-mediated transfection for gene therapy.

Histone has been considered to be an effective carrier in non-viral gene delivery due to its unique properties such as efficient DNA binding ability, direct translocation to cytoplasm and favorable nuclear localization ability. Meanwhile, the rapid development of genetic engineering techniques could facilitate the construction of multifunctional fusion proteins based on histone molecules to further improve the transfection efficiency. Remarkably, histone has been demonstrated to achieve gene transfection in a synergistic manner with cationic polymers, affording to a significant improvement of transfection efficiency. In the review, we highlighted the recent developments and future trends in gene delivery mediated by histones or histone-based fusion proteins/peptides. This review also discussed the mechanism of histone-mediated gene transfection and provided an outlook for future therapeutic opportunities in the viewpoint of transfection efficacy and biosafety.

Inhibition of cell proliferation and migration through nucleobase-modified polyamidoamine-mediated p53 delivery.

INTRODUCTION: The nucleobase 2-amino-6-chloropurine-modified polyamidoamine (AP-PAMAM) was used as a carrier for p53 gene delivery to achieve the antitumor effects. METHODS AND MATERIALS: The condensation of p53 plasmid was studied through gel retardation assay, and the transfection efficiency was evaluated through the transfection assay of pEGFP-N3 and pGL-3 plasmids. Using human cervical carcinoma cell line HeLa as a model, the inhibition of cell proliferation and migration was studied through flow cytometry, wound healing and Transwell migration assays, respectively. The p53 expression level was detected through quantitative polymerase chain reaction and Western blotting analyses. RESULTS: The carrier could condense p53 plasmid into stable nanoparticles at N/P ratios of 2.0, and higher transfection efficiency than polyamidoamine (PAMAM) could be obtained at all the N/P ratios studied. AP-PAMAM-mediated p53 delivery could achieve stronger antiproliferative effect than PAMAM/p53. The antiproliferative effect was identified to be triggered by the induction of cell apoptosis (apoptotic ratio of 26.17%) and cell cycle arrest at S phase. Additionally, AP-PAMAM/p53 transfection has been found to suppress the cell migration and invasion of cancer cells. Finally, the enhanced p53 expression level could be detected after p53 transfection at mRNA and protein levels. CONCLUSION: The PAMAM derivative-mediated p53 delivery could be a promising strategy for achieving tumor gene therapy.

Phenol degradation catalyzed by a peroxidase mimic constructed through the grafting of heme onto metal-organic frameworks.

The aim of this work was to construct a peroxidase mimic for achieving the phenol degradation through Fenton reaction. The enzyme mimic was synthesized through the conjugation of heme with the amino group of 2-amino-1,4-benzene dicarboxylate in UiO-66-NH2 (ZrMOF), namely Heme-ZrMOF. Compared to free heme, the composite Heme-ZrMOF exhibited an obviously enhanced ability for phenol degradation with up to 97.3% of phenol removal after 2h. Meanwhile, it could achieve the easy separation of catalyst from the system and the elimination of iron residues in the process of phenol degradation. Finally, the catalyst Heme-ZrMOF was observed to possess good recyclability in the phenol degradation with still 76.2% of phenol removal after 4 cycles.

Lipase-inorganic hybrid nanoflower constructed through biomimetic mineralization: A new support for biodiesel synthesis.

We reported a facile, economic and green method based on biomimetic mineralization to acquire lipase-inorganic hybrid nanoflower, which was then employed as a biocatalyst for biodiesel production. In the hybrid nanoflower, enzyme molecules and Cu2+ ions were utilized as the organic and inorganic components, respectively. The morphology of nanoflower and the distribution and loading of proteins were systematically characterized by scanning electron microscopy, confocal laser scanning microscopy and ultraviolet-visible spectroscopy, which indicated the successful encapsulation of lipase in the hybrid nanoflower. Using the hydrolysis of p-nitrophenyl caprylate as a model, lipase-inorganic hybrid nanoflower was observed to possess favorable catalytic activity and stability in the ester hydrolysis. Further, the hybrid nanoflower was used as a catalyst for biodiesel production, in which it could convert sunflower oil to biodiesel with 96.5% conversion and remain 72.5% conversion after being used for 5 cycles. Thus, the lipase-inorganic hybrid nanoflower is potential to be used as an economically viable biocatalyst for the production of biofuel as the future petrol-fuel replacement.

Nucleobase-modified polyamidoamine-mediated miR-23b delivery to inhibit the proliferation and migration of lung cancer.

The nucleobase analogue 2-amino-6-chloropurine was modified on the surface of polyamidoamine (PAMAM) to construct a derivative AP-PAMAM, and then it was used as a gene carrier for miR-23b delivery to achieve the anti-tumor effects. The carrier AP-PAMAM could condense miR-23b into stable nanoparticles with a particle size of 97.3 nm (N/P ratio of 50), which was favorable for the cellular uptake of nanoparticles. Compared with PAMAM, AP-PAMAM exhibited an obviously enhanced transfection efficiency through the transfection assay of plasmids pEGFP-N3 and pGL-3. Using the human lung adenocarcinoma cell line A549 as a model, AP-PAMAM-mediated miR-23b delivery could achieve a stronger anti-proliferative effect than PAMAM/miR-23b. The inhibition of cell proliferation was elucidated to be associated with the apoptotic induction (apoptotic ratio of 23.2%) and S phase arrest owing to the decreased expression level of cyclin D1. Meanwhile, the AP-PAMAM-mediated miR-23b delivery could suppress the cell migration and invasion of cancer cells through wound healing and Transwell migration assays. In summary, the PAMAM derivative-mediated miR-23b delivery could be a promising strategy for achieving tumor gene therapy.