3D-printing magnesium-polycaprolactone loaded with melatonin inhibits the development of osteosarcoma by regulating cell-in-cell structures.

Melatonin has been proposed as a potent anticarcinogen presents a short half-life for osteosarcoma (OS). Cell-in-cell (CIC) structures play a role in the development of malignant tumors by changing the tumor cell energy metabolism. This study developed a melatonin-loaded 3D printed magnesium-polycaprolactone (Mg-PCL) scaffold and investigated its effect and molecular mechanism on CIC in OS. Mg-PCL scaffold was prepared by 3D-printing and its characteristic was determined. The effect and molecular mechanism of Mg-PCL scaffold as well as melatonin-loaded Mg-PCL on OS growth and progression were investigated in vivo and in vitro. We found that melatonin receptor 1 (MT1) and CIC expressions were increased in OS tissues and cells. Melatonin treatment inhibit the key CIC pathway, Rho/ROCK, through the cAMP/PKA signaling pathway, interfering with the mitochondrial physiology of OS cells, and thus playing an anti-invasion and anti-metastasis role in OS. The Mg-PCL-MT could significantly inhibit distant organ metastasis of OS in the in vivo model. Our results showed that melatonin-loaded Mg-PCL scaffolds inhibited the proliferation, invasion and metastasis of OS cells through the CIC pathway. The Mg-PCL-MT could be a potential therapeutics for OS.

Tensile properties of synthetic, absorbable monofilament suture materials before and after incubation in phosphate-buffered saline.

OBJECTIVE: To compare tensile properties of synthetic, absorbable, monofilament suture material before and after incubation in phosphate-buffered saline (PBS). STUDY DESIGN: Two sizes (2-0 and 3-0) of Biosyn, Maxon, Monocryl, PDS II, Securocryl, and Securodox were tested. SAMPLE POPULATION: Ten suture loops per group. METHODS: Tensile strength, elongation, and modulus of suture loops were measured at baseline and after 7, 14, 21, or 28 days of incubation in PBS. RESULTS: Size, suture material, and size × suture material interaction influenced maximum breaking load, maximum elongation, and modulus of elasticity. At baseline, 2-0 and 3-0 Maxon had the highest breaking loads (111.67 N and 79.71 N, respectively) for their size, and 2-0 PDSII and 3-0 Securodox had the lowest (68.71 N and 48.73 N, respectively). Maxon 2-0 and 3-0 had the greatest elongations (9.68 mm and 8.45 mm, respectively) for their size, and 2-0 Biosyn and 3-0 Securocryl had the least (7.21 mm and 6.58 mm, respectively). Biosyn 2-0 and 3-0 had the highest modulus. With incubation, Maxon (2-0), PDS II (2-0, 3-0), and Securodox (2-0, 3-0) maintained or gained strength over 4 weeks. Strengths of 2-0 and 3-0 Biosyn and 3-0 Maxon were maintained for 2 weeks, while Monocryl and Securocryl lost 20% to 44% of baseline strength within 1 week and 60% to 72% within 2 weeks. Day 7 strengths of 2-0 Biosyn and 2-0 Monocryl were greater than baseline strength of 2-0 PDS II. Strength of 3-0 Biosyn at day 14 was greater than strength of Monocryl at days 7 and 14 and greater than strength of 3-0 PDS II, 3-0 Securodox, and 3-0 Securocryl at baseline and days 7 and 14. CONCLUSION: Suture tensile properties varied with suture size, composition, and brand. At baseline, Maxon suture had the greatest strength and elongation, and Biosyn had the greatest stiffness. CLINICAL SIGNIFICANCE: Tensile strength retention, when reported as a percentage of original strength, provides insufficient information for comparison of suture tensile properties.

Examination of laryngeal function of healthy dogs by using sedation protocols with dexmedetomidine.

OBJECTIVE: To determine the ability to evaluate laryngeal function under sedation with dexmedetomidine alone or in combination with opioids. STUDY DESIGN: Randomized, crossover, blinded study. ANIMALS: Eight adult research hounds weighing 8 to 22.5 kg. METHODS: Dogs were sedated with propofol, dexmedetomidine, dexmedetomidine and butorphanol, or dexmedetomidine and hydromorphone. Digital images were collected with video laryngoscopy before and after doxapram administration. Maximal inspiratory normalized glottal gap (GGAn ) and laryngeal motion were compared between and within protocols before and after doxapram by using a difference of least squares mean. RESULTS: Normal laryngeal function was confirmed in all dogs with all protocols except propofol, which resulted in two false positive results. No difference between protocols was detected for predoxapram GGAn . Postdoxapram GGAn was greater than predoxapram GGAn for all four sedation protocols (P ≤ .0030). Compared with propofol, postdoxapram GGAn was greater for all three dexmedetomidine protocols (P ≤ .0420). CONCLUSION: Dexmedetomidine alone or in combination with opioids was an effective sedation protocol for laryngeal examination, producing sufficient immobilization to prevent jaw motion and without affecting arytenoid abduction. CLINICAL SIGNIFICANCE: Dexmedetomidine sedation does not inhibit normal laryngeal motion. Laryngeal examination with propofol alone can produce false positive results.

Polylysine-modified polyethylenimine polymer can generate genetically engineered mesenchymal stem cells for combinational suicidal gene therapy in glioblastoma.

Glioblastoma remains the most resistant malignant brain tumor owing to the lack of an efficient delivery system for therapeutic genes or drugs, especially in outgrowing tumor islands. Cell-based delivery systems such as mesenchymal stem cells (MSCs) are a potential candidate in this regard. Conventionally, MSCs have been genetically modified for cancer therapy by using viral vectors that can illicit oncogenicity and limit their use in clinical trials. In this study, we have used nonviral agents such as the polylysine-modified polyethylenimine (PEI-PLL) copolymer to generate genetically engineered MSCs with suicidal genes, namely, HSV-TK and TRAIL. Our results demonstrated that an intratumoral injection of polymer-double-transfected MSCs along with prodrug ganciclovir injections can induce a significant synergistic therapeutic response both in vitro and in vivo compared to single plasmid transfections or untransfected MSCs. The proliferation marker Ki67 and the angiogenesis marker VEGF were also significantly reduced in treatment groups, whereas the TUNEL assay demonstrated that apoptosis is significantly increased after treatment. Our findings suggest that the PEI-PLL copolymer can successfully modify MSCs with therapeutic genes and can produce a pronounced impact during glioblastoma therapy. This study proposes a potential nonviral approach to develop a cell-based therapy for the treatment of glioma. STATEMENT OF SIGNIFICANCE: In this study, we have used a polylysine-modified polyethylenimine polymer (PEI-PLL) copolymer, a non viral transfection agent, for gene delivery in mesenchymal stem cells. These PEI-PLL-transfected mesenchymal stem cells with HSV-TK and TRAIL genes have the potential to treat glioma both in vitro and in vivo. This combinational therapy through PEI-PLL-transfected mesenchymal stem cells can provide cost-effective, low immunogenic, and tumor-targeted delivery of suicideal genes (HSV-TK and TRAIL) for promising glioblastoma treatment.

Pyroptosis induced by enterovirus A71 infection in cultured human neuroblastoma cells.

Enterovirus A71 (EV-A71) infection can cause hand, foot and mouth disease (HFMD), and even fatal meningoencephalitis. Unfortunately, there is currently no effective treatment for EV-A71 infection due to the lack of understanding of the mechanism of neurological diseases. In this study, we employed SH-SY5Y human neuroblastoma cells to explore the roles of caspase-1 in neuropathogenesis. The expression and activity of caspase-1 were analyzed. The potential immuneconsequences mediated by caspase-1 including cell death, lysis, DNA degradation, and secretion of pro-inflammatory were also examined. We found the gene expression levels of caspase-1, IL-1ß, IL-18 and active caspase-1 were markedly increased in the SH-SY5Y cells at 48 h post EV-A71 infection. The cell death, lysis, and DNA degradation were also increased during infection, which could be significantly alleviated by caspase-1 inhibition. These observations provided additional experimental evidence supporting caspase-1-mediated pyroptosis as a novel pathway of inflammatory programmed cell death.

Polylysine-modified polyethylenimine (PEI-PLL) mediated VEGF gene delivery protects dopaminergic neurons in cell culture and in rat models of Parkinson's Disease (PD).

Parkinson's Disease (PD) is a chronic neurodegenerative disorder characterized by motor deficits which result from the progressive loss of dopaminergic neurons. Gene therapy using growth factors such as VEGF seems to be a viable approach for potential therapeutic treatment of PD. In this study, we utilized a novel non-viral gene carrier designated as PEI-PLL synthesized by our laboratory to deliver VEGF gene to study its effect by using both cell culture as well as animal models of PD. For cell culture experiments, we utilized 6-hydroxydopamine (6-OHDA) mediated cell death model of MN9D cells following transfection with either a control plasmid or VEGF expressing plasmid. As compared to control transfected cells, PEI-PLL mediated VEGF gene delivery to MN9D cells resulted in increased cell viability, increase in the number of Tyrosine hydroxylase (TH) positive cells and decreased apoptosis following 6-OHDA insult. Next, we studied the therapeutic potential of PEI-PLL mediated VEGF gene delivery in SNPc by using unilateral 6-OHDA Medial forebrain bundle (MFB) lesion model of PD in rats. VEGF administration prevented the loss of motor functions induced by 6-OHDA as determined by behavior analysis. Similarly, VEGF inhibited the 6-OHDA mediated loss of DA neurons in Substantia Nigra Pars Compacta (SNPc) as well as DA nerve fibers in striatum as determined by TH immunostaining. In addition, PEI-PLL mediated VEGF gene delivery also prevented apoptosis and microglial activation in PD rat models. Together, these results clearly demonstrated the beneficial effects of PEI-PLL mediated VEGF gene delivery on dopaminergic system in both cell culture and animal models of PD. STATEMENT OF SIGNIFICANCE: In this report, we exploited the potential of PEI-PLL to deliver VEGF gene for the potential therapeutic treatment of PD by using both cell culture and animal models of PD. To the best of our knowledge, this is the first report describing the use of novel polymeric gene carriers for the delivery of VEGF gene to DA neurons with improved transfection efficiency. Finally, the study will lead to a significant advancement in the field of non-viral PD gene therapy treatment.

A non-viral suicide gene delivery system traversing the blood brain barrier for non-invasive glioma targeting treatment.

Herpes simplex virus type I thymidine kinase gene (HSV-TK) in viral vector is a promising strategy against glioblastoma multiforme (GBM). However, the biosafety risk restricts its application in clinic. In this work, poly (l-lysine)-grafted polyethylenimine (PEI-PLL), which combines the high transfection efficiency of polyethylenimine and the good biodegradability of poly (l-lysine), was adopted as the non-viral vector backbone. Angiopep-2, a blood brain barrier (BBB) crossing and glioma targeting bifunctional peptide was conjugated on PEI-PLL via polyethyleneglycol (PEG) and designated as PPA. The optimal transfection ratio of PPA/DNA complexes nanoparticles (PPA NPs) was firstly characterized. Next, the glioma targeting of the PPA NPs was confirmed through cellular uptake and transfection analysis. The in vivo imaging studies demonstrated that the PPA NPs could not only penetrate BBB but also accumulate in striatum and cortex via systemic administration. Moreover, the PPA/HSV-TK NPs showed remarkably anti-glioma effect and survival benefit in an invasive orthotopic human GBM mouse model through inhibiting proliferation and inducing apoptosis (p<0.05 vs control). This study firstly illustrated that the cationic polymer PPA could be exploited as an efficient gene vector to cross the BBB, and innovatively provided a potential non-viral nanomedicine for noninvasive suicide gene therapy in the glioma treatment.

Presence of Porphyromonas gingivalis in esophagus and its association with the clinicopathological characteristics and survival in patients with esophageal cancer.

BACKGROUND: Mounting evidence suggests a causal relationship between specific bacterial infections and the development of certain malignancies. However, the possible role of the keystone periodontal pathogen, Porphyromonas gingivalis, in esophageal squamous cell carcinoma (ESCC) remains unknown. Therefore, we examined the presence of P. gingivalis in esophageal mucosa, and the relationship between P. gingivalis infection and the diagnosis and prognosis of ESCC. METHODS: The presence of P. gingivalis in the esophageal tissues from ESCC patients and normal controls was examined by immunohistochemistry using antibodies targeting whole bacteria and its unique secreted protease, the gingipain Kgp. qRT-PCR was used as a confirmatory approach to detect P. gingivalis 16S rDNA. Clinicopathologic characteristics were collected to analyze the relationship between P. gingivalis infection and development of ESCC. RESULTS: P. gingivalis was detected immunohistochemically in 61 % of cancerous tissues, 12 % of adjacent tissues and was undetected in normal esophageal mucosa. A similar distribution of lysine-specific gingipain, a catalytic endoprotease uniquely secreted by P. gingivalis, and P. gingivalis 16S rDNA was also observed. Moreover, statistic correlations showed P. gingivalis infection was positively associated with multiple clinicopathologic characteristics, including differentiation status, metastasis, and overall survival rate. CONCLUSION: These findings demonstrate for the first time that P. gingivalis infects the epithelium of the esophagus of ESCC patients, establish an association between infection with P. gingivalis and the progression of ESCC, and suggest P. gingivalis infection could be a biomarker for this disease. More importantly, these data, if confirmed, indicate that eradication of a common oral pathogen could potentially contribute to a reduction in the overall ESCC burden.

miRNA oligonucleotide and sponge for miRNA-21 inhibition mediated by PEI-PLL in breast cancer therapy.

MicroRNA-21 (miR-21) inhibition is a promising biological strategy for breast cancer therapy. However its application is limited by the lack of efficient miRNA inhibitor delivery systems. As a cationic polymer transfection material for nucleic acids, the poly (l-lysine)-modified polyethylenimine (PEI-PLL) copolymer combines the high transfection efficiency of polyethylenimine (PEI) and the good biodegradability of polyllysine (PLL). In this work, PEI-PLL was successfully synthesized and confirmed to transfect plasmid and oligonucleotide more effectively than PEI in MCF-7 cells (human breast cancer cells). In this regard, two kinds of miR-21 inhibitors, miR-21 sponge plasmid DNA (Sponge) and anti-miR-21 oligonucleotide (AMO), were transported into MCF-7 cells by PEI-PLL respectively. The miR-21 expression and the cellular physiology were determined post transfection. Compared with the negative control, PEI-PLL/Sponge or PEI-PLL/AMO groups exhibited lower miR-21 expression and cell viability. The anti-tumor mechanism of PEI-PLL/miR-21 inhibitors was further studied by cell cycle and western blot analyses. The results indicated that the miR-21 inhibition could induce the cell cycle arrest in G1 phase, upregulate the expression of Programmed Cell Death Protein 4 (PDCD4) and thus active the caspase-3 apoptosis pathway. Interestingly, the PEI-PLL/Sponge and PEI-PLL/AMO also sensitized the MCF-7 cells to anti-tumor drugs, doxorubicin (DOX) and cisplatin (CDDP). These results demonstrated that PEI-PLL/Sponge and PEI-PLL/AMO complexes would be two novel and promising gene delivery systems for breast cancer gene therapy based on miR-21 inhibition. STATEMENT OF SIGNIFICANCE: This work was a combination of the high transfection efficiency of polyethylenimine (PEI), the good biodegradability of polyllysine (PLL) and the breast cancer-killing effect of miR-21 inhibitors. The poly (l-lysine)-modified polyethylenimine (PEI-PLL) copolymer was employed as the vector of miR-21 sponge plasmid DNA (Sponge) or anti-miR-21 oligonucleotide (AMO). PEI-PLL showed more transfection efficiency and lower cytotoxicity in human breast cancer cells than PEI. Moreover, the breast cancer cells exhibited significantly lower miR-21 expression and cell viability post transfection with sponge or AMO. Interestingly, the PEI-PLL/miR-21 inhibitor complexes also sensitized the cancer cells to anti-cancer chemotherapy drugs, doxorubicin (DOX) and cisplatin (CDDP). This synergistic effect provides a good application prospect of co-delivery miR-21 inhibitors and chemical drugs in breast cancer therapy.

Hydrophobic poly (amino acid)-modified PEI-mediated delivery of single-chain antibody scFv1C9 inhibits HepG2 cell cycle process and xenograft growth in nude mice.

The safe and effective gene delivery vector remains the key step for gene therapy. Hydrophobic-modified Phe-PEI (PP80) was exhibited in advantage with biocompatibility and gene delivery with smaller size and easier penetration into cells and tissues. PP80 delivery of rev-casp-3 gene was demonstrated effectively to inhibit HeLa xenograft growth in our previous work. However, it was necessary to evaluate its applicability in other cells or tissues as gene carrier. Here, we quantitatively optimized the complex ratio of PP80 and plasmid DNA (pDNA) and evaluated the potential pyrogenicity by rabbit pyrogen test. In addition, PP80-mediated expression of scFv1C9 gene blocked HepG2 cell cycle progress in vitro. Subsequently, PP80-scFv1C9 was injected into HepG2 xenograft and significantly inhibited the xenograft growth in nude mice. Further investigation indicated that PP80 was an effective gene carrier and possible for entering hepatic xenograft. These features of PP80 made it attractive as a potential gene carrier for cancer therapy.

pH-responsive zwitterionic copolypeptides as charge conversional shielding system for gene carriers.

A novel rapid pH-responssive polymer polyethylenimine-poly(l-lysine)-poly(l-glutamic acid) (PELG) was designed as the shielding system. The zwitterionic copolypeptide PELG with negatively charged at physical pH can act as the shielding system to shield positively charged polyplexes. PELG was used to shield PEI25k/DNA to form ternary polyplex, the polyplex surface zeta potential can change from a negative to positive nearly pH value of 6.9. Because the pH value of tumor extracellular environment is about 6.5, the positive charges on the polyplexes could be restored in tumors, which is beneficial to the electrostatic interactions between positive polyplexes and negative tumor cells, leading to high cell uptake efficiency and high transfection efficiency.

Polylysine-modified polyethylenimine inducing tumor apoptosis as an efficient gene carrier.

Polyethylenimine (PEI) is receiving increasing attention as a gene carrier with high transfection efficiency. However, its high charge density and cytotoxic effects limit its application. Polylysine (PLL) is another polymeric gene carrier with good biodegradability and biocompatibility, although its lack of endosomal escape ability strongly impairs its transfection efficiency. In this study, PLL was introduced to PEI by ring-opening polymerization of ε-benzyloxycarbonyl-l-lysine N-carboxyanhydride, followed by deprotection of carbobenzyloxy groups. As-prepared PEI-PLL multiarm hyperbranched copolymers were characterized as gene carriers in vitro by measuring their particle size, zeta potential, cytotoxicity, transfection efficiency, and cell internalization. The optimum transfected efficiency of PEI-PLL was nearly seven times higher than that of PEI with a molecular weight of 25kDa. Furthermore, pKH3-rev-casp-3 plasmid DNA was used as a gene for anti-tumor treatment in a xenograft model using nude mice. Compared with 25kDa PEI, PEI-PLL exhibited better tumor inhibition effects in 23days. In addition, terminal deoxynucleotidyl transferase dUTP nick end labeling, immunohistochemistry, and western blot analysis were used to determine the anti-tumor mechanism of PEI-PLL. The results showed that tumor cell apoptosis led to tumor inhibition, which could be attributed to pKH3-rev-casp-3 inducing poly(ADP-ribose) polymerase-1 cleavage. PEI-PLL is a promising gene carrier candidate for further application in vivo.

Self-reinforced endocytoses of smart polypeptide nanogels for "on-demand" drug delivery.

The pH and reduction dual-responsive polypeptide nanogels with self-reinforced endocytoses were prepared through ring-opening polymerization of l-glutamate N-carboxyanhydrides, deprotection of benzyl group and subsequent quaternization reaction between γ-2-chloroethyl-l-glutamate unit in polypeptide block and 2,2'-dithiobis(N,N-dimethylethylamine). The nanogels were revealed to exhibit smart pH and reduction dual-responsiveness, and excellent biocompatibilities, which expressed great potential as antitumor drug nanocarriers. Doxorubicin (DOX) as a model antitumor drug was loaded into nanogels through dispersion. DOX-loaded nanogels displayed a stable core-cross-linked structure under normal physiological condition (pH7.4), while rapidly releasing the payloads in the mimicking endosomal (pH5.3), tumor tissular (pH6.8) or intracellular reductive microenvironments (10.0mM glutathione). Confocal fluorescence microscopy demonstrated that DOX-loaded nanogels could deliver DOX into HepG2 cells (a human hepatoma cell line) more efficiently than the parent DOX-loaded micelle and free DOX. The enhanced cellular internalizations of DOX-loaded nanogels were more significant under tumor tissular acidic condition (pH6.8) ascribed to the quaternary ammonium groups in the cores. In addition, DOX-loaded nanogels exhibited improved in vitro and in vivo antitumor activities, and in vivo securities compared with DOX-loaded micelle and free DOX. These excellent features of the smart nanogels with quaternary ammonium groups were endowed with a bright prospect for intracellular targeting antitumor drug delivery.

Hydrophobic poly (amino acid) modified PEI mediated delivery of rev-casp-3 for cancer therapy.

Recent studies in amphiphilic cationic polymers have demonstrated their potential as gene carriers with high transfection efficiency and low cytotoxicity in the in vitro settings to deliver drug, siRNA and plasmid DNA. Yet their safety and efficacy in vivo remain to be a challenge, and require further investigation. In our previous work, PP80 was synthesized as a novel amphiphilic cationic polymer by grafting hydrophobic polyphenylalanine segment on PEI, which displayed higher transfection efficiency than PEI in a number of cell lines in vitro. Here, we reported the favorable biocompatibility displayed by PP80/pDNA complex both in vitro and in vivo. Furthermore, when therapeutic gene rev-casp-3 was conjugated to PP80 and administered intratumorally to a HeLa xenograft model, significant tumor apoptosis was induced with concurrent tumor growth inhibition, indicating that PP80 mediated expression of rev-casp-3 gene in solid tumors with not detectable side effects on the tumor-bearing mice. These data demonstrated that PP80 warrants further investigation as a promising cancer gene delivery vehicle.

Hydrophobic polyphenylalanine-grafted hyperbranched polyethylenimine and its in vitro gene transfection.

A series of amphiphilic multi-armed PPn copolymers were prepared by ROP of Phe-NCA with PEI-25k as a macroinitiator. The particle size of the PPn/DNA complexes was about 100 nm and the zeta potentials were below 20 mV. An MTT assay demonstrated that all the PPn copolymers had lower cytotoxicity compared to PEI-25k. In vitro gene transfection studies were also conducted in HeLa, 293 and CT 26 cells. The optimal quantity of hydrophobic phenylalanine segments in PP80 led to higher transfection efficiency in various cell lines based on this study. The results indicate that PP80 was the best candidate for gene delivery among these PPn copolymers.