Fabrication of Bacterial Cellulose-Based Dressings for Promoting Infected Wound Healing.

Bacterial cellulose (BC) holds several unique properties such as high water retention capability, flexibility, biocompatibility, and high absorption capacity. All these features make it a potential material for wound healing applications. However, it lacks antibacterial properties, which hampers its applications for infectious wound healings. This study reported BC-based dressings containing ε-polylysine (ε-PL), cross-linked by a biocompatible and mussel-inspired polydopamine (PDA) for promoting infectious wound healing. BC membranes were coated with PDA by a simple self-polymerization process, followed by treating with different contents of ε-PL. The resulted membranes showed strong antibacterial properties against tested bacteria by both in vitro and in vivo evaluations. The membranes also exhibited hemocompatibility and cytocompatibility by in vitro investigations. Moreover, the functionalized membranes promoted infected wound healing using Sprague-Dawley rats as a model animal. A complete wound healing was observed in the group treated with functionalized membranes, while wounds were still open for control and pure BC groups in the same duration. Histological investigations indicated that the thickness of newborn skin was greater and smoother in the groups treated with modified membranes in comparison to neat BC or control groups. These results revealed that the functionalized membranes have great potential as a dressing material for infected wounds in future clinical applications.

Transplantation of encapsulated human Leydig-like cells: A novel option for the treatment of testosterone deficiency.

Previous studies have demonstrated that the transplantation of alginate-poly-ʟ-lysine-alginate (APA)-encapsulated rat Leydig cells (LCs) provides a promising approach for treating testosterone deficiency (TD). Nevertheless, LCs have a limited capacity to proliferate, limiting the efficacy of LC transplantation therapy. Here, we established an efficient differentiation system to obtain functional Leydig-like cells (LLCs) from human stem Leydig cells (hSLCs). Then we injected APA-encapsulated LLCs into the abdominal cavities of castrated mice without an immunosuppressor. The APA-encapsulated cells survived and partially restored testosterone production for 90 days in vivo. More importantly, the transplantation of encapsulated LLCs ameliorated the symptoms of TD, such as fat accumulation, muscle atrophy and adipocyte accumulation in bone marrow. Overall, these results suggest that the transplantation of encapsulated LLCs is a promising new method for testosterone supplementation with potential clinical applications in TD.

Rational Design of IR820- and Ce6-Based Versatile Micelle for Single NIR Laser-Induced Imaging and Dual-Modal Phototherapy.

Phototherapy as a promising cancer diagnostic and therapeutic strategy has aroused extensive attention. However, single-wavelength near-infrared (NIR) light-triggered combinational treatment of photothermal therapy (PTT) and photodynamic therapy (PDT) is still a great challenge. Herein, a multifunctional micelle activated by a single-wavelength laser for simultaneous PTT and PDT as well as fluorescence imaging is developed. Briefly, new indocyanine green (IR820) is conjugated to d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) via the linker 6-aminocaproic acid, and then, chlorin e6 (Ce6) is encapsulated into the micelles formed by TPGS-IR820 conjugates to fabricate TPGS-IR820/Ce6 micelles. As the well-designed TPGS-IR820 conjugate shares a similar peak absorption wavelength with Ce6, this micelle can be applied with a single NIR laser (660 nm). The stable micelles exhibit excellent photothermal conversion efficiency in vitro and in vivo as well as high singlet oxygen generation capacity in tumor cells. After efficient cellular internalization, the as-prepared micelles display outstanding anticancer activity upon single NIR laser irradiation in vitro and in vivo. Furthermore, TPGS-IR820/Ce6 micelles show negligible systemic toxicity. The highly safe and effective TPGS-IR820/Ce6 micelles can offer an innovative strategy to construct single NIR light-induced PTT and PDT combined phototherapy nanoplatforms via suitable modification of organic phototherapeutic agents.

Lumbrokinase/paclitaxel nanoparticle complex: potential therapeutic applications in bladder cancer.

BACKGROUND: Lumbrokinase (LK) is an enzyme complex with antithrombotic, antioxidant, antitumor, and immunomodulatory effects. It has been extensively studied and used in clinical anti-tumor therapy. However, its half-life is short, its bioavailability is low, and its toxicity and side effects are great, which greatly limit its clinical application. Therefore, LK is often combined with other drugs (such as immune agents, hormones, or Chinese herbal medicine) to reduce its dosage and side effects and to improve its anti-tumor effects. METHODS AND RESULTS: Here, we described an LK/paclitaxel (PTX) nanocarrier based on poly(ethylene glycol)-b-(poly(ethylenediamine l-glutamate)-g-poly(ε-benzyoxycarbonyl-l-lysine)-r-poly(l-lysine)) (PEG-b-(PELG-g-(PZLL-r-PLL))). In the present study, LK and PTX were loaded by electrostatic and/or hydrophobic effects under mild conditions, thereby increasing the half-life and bioavailability of the drugs via the sustained release and enhancement of tumor site enrichment by the LK/PTX/PEG-b-(PELG-g-(PZLL-r-PLL)) complex through passive targeting. In this study, using bladder cancer cells (J82 cells) and rat bladder cancer model as the object, the structure of the nanocarrier, the relationship between drugs composition and antitumor properties were systematically studied. CONCLUSION: We propose that the block copolymer PEG-b-(PELG-g-(PZLL-r-PLL)) may function as a potent nanocarrier for augmenting anti-bladder cancer pharmacotherapy, with unprecedented clinical benefits.

Development of non-pyrogenic magnetosome minerals coated with poly-l-lysine leading to full disappearance of intracranial U87-Luc glioblastoma in 100% of treated mice using magnetic hyperthermia.

Magnetic hyperthermia was reported to increase the survival of patients with recurrent glioblastoma by 7 months. This promising result may potentially be further improved by using iron oxide nanoparticles, called magnetosomes, which are synthesized by magnetotactic bacteria, extracted from these bacteria, purified to remove most endotoxins and organic material, and then coated with poly-l-lysine to yield a stable and non-pyrogenic nanoparticle suspension. Due to their ferrimagnetic behavior, high crystallinity and chain arrangement, these magnetosomes coated with poly-l-lysine (M-PLL) are characterized by a higher heating power than their chemically synthesized counterparts currently used in clinical trials. M-PLL-enhanced antitumor efficacy was demonstrated by administering 500-700 µg in iron of M-PLL to intracranial U87-Luc tumors of 1.5 mm3 and by exposing mice to 27 magnetic sessions each lasting 30 min, during which an alternating magnetic field of 202 kHz and 27 mT was applied. Treatment conditions were adjusted to reach a typical hyperthermia temperature of 42 °C during the first magnetic session. In 100% of treated mice, bioluminescence due to living glioblastoma cells fully disappeared 68 days following tumor cell implantation (D68). These mice were all still alive at D350. Histological analysis of their brain tissues revealed an absence of tumor cells, suggesting that they were fully cured. In comparison, antitumor efficacy was less pronounced in mice treated by the administration of IONP followed by 23 magnetic sessions, leading to full tumor bioluminescence disappearance in only 20% of the treated mice.

TLR3 agonist and Sorafenib combinatorial therapy promotes immune activation and controls hepatocellular carcinoma progression.

Hepatocellular carcinoma (HCC) is associated with high mortality and the current therapy for advanced HCC, Sorafenib, offers limited survival benefits. Here we assessed whether combining the TLR3 agonist: lysine-stabilized polyinosinic-polycytidylic-acid (poly-ICLC) with Sorafenib could enhance tumor control in HCC. Combinatorial therapy with poly-ICLC and Sorafenib increased apoptosis and reduced proliferation of HCC cell lines in vitro, in association with impaired phosphorylation of AKT, MEK and ERK. In vivo, the combinatorial treatment enhanced control of tumor growth in two mouse models: one transplanted with Hepa 1-6 cells, and the other with liver tumors induced using the Sleeping beauty transposon. Tumor cell apoptosis and host immune responses in the tumor microenvironment were enhanced. Particularly, the activation of local NK cells, T cells, macrophages and dendritic cells was enhanced. Decreased expression of the inhibitory signaling molecules PD-1 and PD-L1 was observed in tumor-infiltrating CD8+ T cells and tumor cells, respectively. Tumor infiltration by monocytic-myeloid derived suppressor cells (Mo-MDSC) was also reduced indicating the reversion of the immunosuppressive tumor microenvironment. Our data demonstrated that the combinatorial therapy with poly-ICLC and Sorafenib enhances tumor control and local immune response hence providing a rationale for future clinical studies.

Gold nanoparticles stabilized with MPEG-grafted poly(l-lysine): in vitro and in vivo evaluation of a potential theranostic agent.

As the number of diagnostic and therapeutic applications utilizing gold nanoparticles (AuNPs) increases, so does the need for AuNPs that are stable in vivo, biocompatible, and suitable for bioconjugation. We investigated a strategy for AuNP stabilization that uses methoxypolyethylene glycol-graft-poly(l-lysine) copolymer (MPEG-gPLL) bearing free amino groups as a stabilizing molecule. MPEG-gPLL injected into water solutions of HAuCl4 with or without trisodium citrate resulted in spherical (Zav = 36 nm), monodisperse (PDI = 0.27), weakly positively charged nanoparticles (AuNP3) with electron-dense cores (diameter: 10.4 ± 2.5 nm) and surface amino groups that were amenable to covalent modification. The AuNP3 were stable against aggregation in the presence of phosphate and serum proteins and remained dispersed after their uptake into endosomes. MPEG-gPLL-stabilized AuNP3 exhibited high uptake and very low toxicity in human endothelial cells, but showed a high dose-dependent toxicity in epithelioid cancer cells. Highly stable radioactive labeling of AuNP3 with (99m)Tc allowed imaging of AuNP3 biodistribution and revealed dose-dependent long circulation in the blood. The minor fraction of AuGNP3 was found in major organs and at sites of experimentally induced inflammation. Gold analysis showed evidence of a partial degradation of the MPEG-gPLL layer in AuNP3 particles accumulated in major organs. Radiofrequency-mediated heating of AuNP3 solutions showed that AuNP3 exhibited heating behavior consistent with 10 nm core nanoparticles. We conclude that PEG-pPLL coating of AuNPs confers "stealth" properties that enable these particles to exist in vivo in a nonaggregating, biocompatible state making them suitable for potential use in biomedical applications such as noninvasive radiofrequency cancer therapy.

Targeted delivery of tanshinone IIA-conjugated mPEG-PLGA-PLL-cRGD nanoparticles to hepatocellular carcinoma.

Tanshinone IIA (TSIIA) is an active constituent of the traditional Chinese medicinal plant Salvia miltiorrhiza that is known to have anti-tumor properties. In order to increase the selectivity of TSIIA's anti-tumor activity, the current study evaluated the tumor-targeting efficacy of TSIIA incorporated into nanoparticles (NPs). TSIIA was loaded into mPEG-PLGA-PLL-cRGD (methoxy polyethylene glycol, polylactic-co-glycolic acid, poly-L-lysine, cyclic arginine-glycine-aspartic acid) nanoparticles (TNPs) and used to treat hepatocellular carcinoma in vitro and in vivo. Our data demonstrate that TNPs were stable and had an even size distribution as well as an extended TSIIA releasing time, and improved tumor-targeting activity. As a novel drug carrier system, TNPs significantly inhibited the development of liver cancer both in vitro and in vivo, proving to be a novel promising targeted treatment for liver cancer.

Electrically tunable organic bioelectronics for spatial and temporal manipulation of neuron-like pheochromocytoma (PC-12) cells.

BACKGROUND: Organic bioelectronic devices consisting of alternating poly(3,4-ethylenedioxythiophene) (PEDOT) and reduced graphite oxide (rGO) striped microelectrode arrays were fabricated by lithography technology. It has been demonstrated that the organic bioelectronic devices can be used to spatially and temporally manipulate the location and proliferation of the neuron-like pheochromocytoma cells (PC-12 cells). METHODS: By coating an electrically labile contact repulsion layer of poly(l-lysine-graft-ethylene glycol) (PLL-g-PEG) on the PEDOT electrode, the location and polarity of the PC-12 cells were confined to the rGO electrodes. RESULTS: The outgrowth of spatially confined bipolar neurites was found to align along the direction of the 20µm wide electrode. The location of the PC-12 cells can also be manipulated temporally by applying electrical stimulation during the neurite differentiation of PC-12 cells, allowing the PC-12 cells to cross over the boundary between the PEDOT and the rGO regions and construct neurite networks in an unconfined manner where the contact repulsive coating of PLL-g-PEG was removed. CONCLUSIONS: This adsorption and desorption of the PLL-g-PEG without and with electrical stimulation can be attributed to the tunable surface properties of the PEDOT microelectrodes, whose surface charge can switch from being negative to positive under electrical stimulation. GENERAL SIGNIFICANCE: The electrically tunable organic bioelectronics reported here could potentially be applied to tissue engineering related to the development and regeneration of mammalian nervous systems. The spatial and temporal control in this device would also be used to study the synapse junctions of neuron-neuron contacts in both time and space domains. This article is part of a Special Issue entitled Organic Bioelectronics - Novel Applications in Biomedicine.

Novel antiviral therapeutics to control foot-and-mouth disease.

Foot-and-mouth disease virus (FMDV) causes a highly contagious disease of cloven-hoofed animals. Vaccines require ∼7 days to induce protection; thus, before this time, vaccinated animals are still susceptible to the disease. Our group has previously shown that swine inoculated with 1×10(11) focus forming units (FFU) of a replication-defective human adenovirus containing the gene for porcine interferon alpha (Adt-pIFN-α) are sterilely protected from FMDV serotypes A24, O1 Manisa, or Asia 1 when the animals are challenged 1 day postadministration, and protection can last for 3-5 days. Polyriboinosinic-polyribocytidylic acid stabilized with poly-l-lysine and carboxymethyl cellulose (poly ICLC) is a synthetic double-stranded RNA that is a viral mimic and activates multiple innate immune pathways through interaction with toll-like receptor 3 and MDA-5. It is a potent inducer of IFNs. In this study, we initially examined the effect of poly IC and IFN-α on FMDV replication and gene induction in cell culture. Poly ICLC alone or combined with Adt-pIFN-α was then evaluated for its therapeutic efficacy in swine against intradermal challenge with FMDV A24, 1 day post-treatment. Groups of swine were subcutaneously inoculated either with poly ICLC alone (4 or 8 mg) or in combination with different doses of Adt-pIFN-α (2.5×10(9), 1×10(9), or 2.5×10(8) FFU). While different degrees of protection were achieved in all the treated animals, a dose of 8 mg of poly ICLC alone or combined with 1×10(9) FFU of Adt-pIFN-α was sufficient to sterilely protect swine when challenged 24 h later with FMDV A24. IFN-stimulated gene (ISG) expression in peripheral blood mononuclear cells at 1 day post-treatment was broader and higher in protected animals than in nonprotected animals. These data indicate that poly ICLC is a potent stimulator of IFN and ISGs in swine and at an adequate dose is sufficient to induce complete protection against FMD.

Evaluation of the effects of a new drug candidate (GEMSP) in a chronic EAE model.

Chronic Experimental Autoimmune Encephalomyelitis (EAE) was induced in rats to evaluate a new drug candidate (GEMSP) for the treatment of multiple sclerosis. This work is a part of preclinical studies on GEMSP, which is made up of fatty acids, vitamins and amino acids or their derivatives; all these compounds were linked to Poly-L-Lysine. In order to evaluate the effects of GEMSP, animals were divided into three experimental groups: 1) EAE rats treated with GEMSP; 2) EAE rats treated with NaCl; and 3) non-EAE rats. Using immunocytochemical techniques with a pan-leukocyte marker (anti-CD 45), differential leukocyte infiltration was compared in the central nervous systems of the different experimental groups. Antibodies directed against a component of GEMSP, the conjugated methionine, were used in all three groups. We found that: 1) GEMSP was effective in abolishing EAE. The crises and clinical scores were completely abolished in the animals of the first group, but not in the animals belonging to the second group; 2) the degree of leukocyte infiltration varied, depending on the different EAE stages, but was not related to the clinical score; and 3) after using anti-conjugated methionine antibodies, we observed immunoreactivity only in the motoneurons of the ventral horn of the spinal cord in the animals of the first group. This immunoreactivity was not found in the animals of the second or third groups. No methionine immunoreactivity was found in the brain. Our results suggest that GEMSP may be a potential drug candidate against the pathogenic processes involved in multiple sclerosis, inhibiting EAE episodes and brain leukocyte infiltration. Our results also show that one component of GEMSP, the methionine compound, is stored inside motoneurons. The possible physiological actions of GEMSP on spinal cord motoneurons are discussed.

Coencapsulation of hepatocytes and bone marrow cells: in vitro and in vivo studies.

Bioencapsulation of cells is one of the many areas of artificial cells being extensively investigated by centers around the world. This includes the bioencapsulation of hepatocytes. A number of methods have been developed to maintain the specific function and phenotype of the bioencapsulated hepatocytes for in vitro and in vivo applications. These include supplementation of factors in the culture medium; use of appropriate substrates and the co-cultivation of hepatocytes with other type of cells, the so called "feeder cells". These feeder cells can be of liver origin or non-liver origin. We have recently studied the role of bone marrow cells in the maintenance of hepatocytes viability and phenotype by using the coculture of hepatocytes with bone marrow cells (nucleated cells including stem cells), and the coencapsulation of hepatocytes with bone marrow stem cells. This way, the hepatocytes viability and specific function can be maintained significantly longer. In vivo studies of both syngeneic and xenogeneic transplantation show that the hepatocytes viability can be maintained longer when coencapsulated with bone marrow cells. Transplantation of coencapsulated hepatocytes and bone marrow cells enhances the ability of the hepatocytes in correcting congenital hyperbilirubinmia in Gunn rats. Both in vitro and in vivo studies show that bone marrow cells can enhance the viability and phenotype maintenance of hepatocytes. Thus, bone marrow cells play an important role as a new type of feeder cells for bioencapsulated hepatocytes for the cellular therapy of liver diseases.

Microencapsulation of Leydig cells: a system for testosterone supplementation.

The use of testosterone supplementation for elderly men has increased markedly over the last decade due to a recognized gradual decline in serum testosterone, which may lead to decreased bone mass, muscle strength, and libido. Testosterone supplementation is also used widely to treat some forms of erectile dysfunction, androgen deficiency, and infertility. However, long-term exogenous testosterone therapy has been associated with several complications, such as fluid retention, nitrogen retention, and hypertension. Due to these problems, alternate treatment modalities, involving more physiological and longer-acting systems for androgen delivery, have been pursued. Alginate-poly-L-lysine-encapsulated Leydig cell microspheres were used as a novel method for the delivery of testosterone in vivo. Encapsulated Leydig cells, which were stimulated with human chorionic gonadotropin, secreted high levels of testosterone in culture. Unencapsulated cells injected i.p. or s.c. failed to produce any testosterone levels, even with human chorionic gonadotropin stimulation. Castrated rats that were administered encapsulated Leydig cells i.p. or s.c. maintained a serum testosterone level between 0.23 and 0.51 ng/ml. Similar levels of testosterone were obtained for 43 d when the encapsulated Leydig cells were injected s.c. (0.28-0.48 ng/ml). Approximately 10% of a normal adult rat Leydig cell population was injected into each castrated animal; however, this resulted in serum testosterone levels of up to 40% of normal. Clinically, testosterone is usually delivered for supplementation and not for full replacement therapy. Therefore, the findings of this study suggest that microencapsulated Leydig cells may be a viable option as a therapeutic modality involving testosterone supplementation.

Killing of Leishmania donovani amastigotes by poly ICLC in hamsters.

In vitro as well as in vivo studies suggest that cytokine-induced synthesis of nitric oxide (NO) from L-arginine is a major effector mechanism against intracellular pathogens. In this study, we demonstrate that golden hamsters infected with Leishmania donovani amastigotes upon treatment with polyinosinic-polycytidylic acid stabilized with polylysine and carboxymethylcellulose (poly ICLC), a potent interferon inducer and immune enhancer, in combination with L-arginine, develop the capacity to eliminate intracellular pathogens. This antileishmanial activity of poly ICLC was suppressed by N w nitro-L-arginine (N w NLA), an inhibitor of inducible NO synthase. Furthermore, prolonged treatment of infected animals with L-arginine alone for 5 days more after 5 day treatment with poly ICLC plus L-arginine increased the antileishmanial activity compared with 5 day treatment with poly ICLC plus L-arginine, suggesting that inducible NO synthase, once activated, produces NO for 5 days more. Our results suggest that an L-arginine-dependent, NO-mediated mechanism is probably responsible for the antileishmanial action of poly ICLC.

Delivery of a secretable adenosine deaminase through microcapsules--a novel approach to somatic gene therapy.

Many current gene therapy protocols require genetic modification of autologous cells. An alternate approach is to use universal recombinant cell lines engineered to secrete in vivo the desired gene products. Enclosing these cells within immunoprotective devices before implantation would prevent rejection of the nonautologous donor cells. To overcome the limitation that not all therapeutic gene products are secreted, we now propose to fuse a signal sequence to the amino terminus of a nonsecreted protein such as human adenosine deaminase (ADA), thus directing the product into a secretory pathway for release from the cells. A fusion gene constructed between the cDNA of the beta-lactamase signal sequence and human ADA expressed a product after in vitro transcription and translation that was immunologically similar to the human protein. Mouse fibroblasts transfected with the fusion gene demonstrated secreted ADA activity that resembled the human cytosolic enzyme in its heat stability, pH optimum, KM, electrophoretic mobility, and immunologic reactivity. Hence, the secreted enzyme expressed from the fusion gene is antigenically and enzymatically similar to the authentic human form. When transfected mouse fibroblasts or myoblasts were enclosed in permselective alginate-poly-L-lysine alginate microcapsules, ADA activity was secreted from the microcapsules and the cells remained viable for over 5 months. Hence, a secretable and functional human ADA has been constructed that can be delivered from recombinant cells within immunoprotective capsules. The success of this strategy provides the prototype for engineering nonsecreted gene products for therapy via this novel method of somatic gene therapy.

A conjugate of lactosaminated poly-L-lysine with adenine arabinoside monophosphate, administered to mice by intramuscular route, accomplishes a selective delivery of the drug to the liver.

A conjugate of the antiviral agent adenine arabinoside monophosphate (ara-AMP) with a low molecular mass lactosaminated poly-L-lysine, administered to mice by i.m. route, selectively delivers the drug to the liver. In mice the conjugate is devoid of acute toxicity even at high dose (1.3 mg/g) and injected i.m. for 20 days does not induce antibodies. Moreover it is highly soluble in water; this means that a pharmacologically active dose may be administered in a small volume compatible with the i.m. route. Compared to the similar ara-AMP complex with lactosaminated albumin which must be injected intravenously, the present conjugate might assure a better compliance of patients with hepatitis B virus infection for a long lasting, liver targeted antiviral treatment.

Oligonucleotide-poly(L-lysine)-heparin complexes: potent sequence-specific inhibitors of HIV-1 infection.

Poly(L-lysine)-conjugated oligonucleotides complementary to the translation initiation region of the tat protein were tested for their capacity to inhibit HIV-1 replication in de novo infected cells. Sequence-specific antiviral effects were observed with these conjugates at 0.5 microM; their activity was transient, and the viral production was only delayed for a few days. Interestingly, their efficiency was significantly increased by the addition of heparin, a sulfated polyanion that also presents antiviral properties against HIV-1. A single addition, at the time of virus exposure, of the ternary complex formed between oligonucleotide-poly(L-lysine) (75 nM) and heparin (50 micrograms/mL) totally protects cells from HIV-1 infection. Primary interference with virus adsorption is essential for the strong antiviral effect. However, this protection remains strictly sequence specific as demonstrated in experiments performed with different HIV-1 isolates. As comparison, treatments that combine AZT and heparin at the same concentrations did not promote such a complete protection.