Adhesive anti-fibrotic interfaces on diverse organs.

Implanted biomaterials and devices face compromised functionality and efficacy in the long term owing to foreign body reactions and subsequent formation of fibrous capsules at the implant-tissue interfaces1-4. Here we demonstrate that an adhesive implant-tissue interface can mitigate fibrous capsule formation in diverse animal models, including rats, mice, humanized mice and pigs, by reducing the level of infiltration of inflammatory cells into the adhesive implant-tissue interface compared to the non-adhesive implant-tissue interface. Histological analysis shows that the adhesive implant-tissue interface does not form observable fibrous capsules on diverse organs, including the abdominal wall, colon, stomach, lung and heart, over 12 weeks in vivo. In vitro protein adsorption, multiplex Luminex assays, quantitative PCR, immunofluorescence analysis and RNA sequencing are additionally carried out to validate the hypothesis. We further demonstrate long-term bidirectional electrical communication enabled by implantable electrodes with an adhesive interface over 12 weeks in a rat model in vivo. These findings may offer a promising strategy for long-term anti-fibrotic implant-tissue interfaces.

Why do some immobilized N-alkylated polyethylenimines far surpass others in inactivating influenza viruses?

A number of N-alkylated polyethylenimines (PEIs) were covalently attached to glass-slide surfaces, and their virucidal efficacies against three different strains of influenza viruses were examined quantitatively. The anti-influenza activities of the modified surfaces varied widely, with the most potent, immobilized N,N-hexyl,methyl-PEI and N,N-dodecyl,methyl-PEI, reducing the viral titer by over three logs (i.e., >99.9%). While the virucidal activities of the glass surfaces derivatized with N-alkylated PEIs displayed no discernible correlation with such surface properties as hydrophobicity, charge, protein affinity, roughness, adhesive interactions, and polymer-chain extension lengths, they exhibited a marginal correlation with the surface density of the quaternary ammonium group, as titrated by means of fluorescein binding. However, this correlation markedly improved (to the correlation coefficient of 0.97 with a two-tailed p value of 0.044) when the titration was instead carried out using a macromolecular conjugate, the dye coupled to the protein lysozyme, suggesting that the critical determinant of the virucidal activity is the density of the immobilized quaternary ammonium groups accessible to influenza virions.

Mechanism of inactivation of influenza viruses by immobilized hydrophobic polycations.

N,N-dodecyl,methyl-polyethylenimine coatings applied to solid surfaces have been shown by us to disinfect aqueous solutions of influenza viruses. Herein we elucidate the mechanism of this phenomenon. Infectivity-, protein-, RNA-, and scanning electron microscopy-based experiments reveal that, upon contact with the hydrophobic polycationic coating, influenza viruses (including pathogenic human and avian, both wild-type and drug-resistant, strains) irreversibly adhere to it, followed by structural damage and inactivation; subsequently, viral RNA is released into solution, while proteins remain adsorbed.

In Vitro Engineering Chimeric Antigen Receptor Macrophages and T Cells by Lipid Nanoparticle-Mediated mRNA Delivery.

Chimeric antigen receptor (CAR)-engineered adoptive cell therapy marks a revolution in cancer treatment based on the highly successful responses to CAR T cell therapy in the treatment of blood cancers. Due to the versatile structure of CARs, this technology can be easily adapted to other immune cell types, including macrophages and NKs, and applied in the treatment of many other cancers. However, high costs and fatal adverse effects represent significant concerns for future development. In vitro transcribed (IVT) mRNA therapeutics, which possess a high safety profile and straightforward production methods, could provide a useful alternative for CAR cell construction. However, the low stability and transfection efficiency of IVT-mRNA in immune cells limit further applications. In this work, we successfully engineered CAR macrophages (CAR-Ms) and CAR T cells with CAR mRNA using lipid nanoparticles (LNPs). Both the LNP formulations and mRNA modifications were optimized for in vitro mRNA transfection. More importantly, the CAR macrophages and CAR T cells both demonstrated significant cytotoxic effects on B lymphoma in vitro, underscoring the great potential of mRNA-engineered adoptive cell therapy.

Hydrophobic polycationic coatings disinfect poliovirus and rotavirus solutions.

Coating surfaces with N-alkylated polyethylenimines (PEIs), namely branched N,N-hexyl,methyl-PEI via covalent attachment to glass or linear N,N-dodecyl,methyl-PEI by physical deposition ("painting") onto polyethylene, enables the resultant materials to quickly and efficiently disinfect aqueous solutions of (non-enveloped) poliovirus and rotavirus.

Bifunctional polymeric inhibitors of human influenza A viruses.

PURPOSE: New antiviral agents were prepared by attaching derivatives of sialic acid (1) and of the drug zanamivir (2) to poly(isobutylene-alt-maleic anhydride) (poly-(1 + 2)) or by mixing poly-1 and poly-2, followed by assaying them against wild-type and drug-resistant influenza A Wuhan viruses. METHODS: Individually or together, 1 and 2 were covalently bonded to the polymer. The antiviral potencies of the resultant poly-1, poly-2, poly-(1 + 2), and poly-1 + poly-2, as well as 1 and 2, were assessed using plaque reduction assay. RESULTS: Attaching 1 to the polymer improved at best millimolar IC(50) values over three orders of magnitude. While 2 exhibited micromolar IC(50) values, poly-2 was >100-fold even more potent. The IC(50) of poly-(1 + 2) against the wild-type strain was >300-fold and approximately 17-fold better than of poly-1 and poly-2, respectively. In contrast, the potency of poly-(1 + 2) vs. poly-2 against the mutant strain merely doubled. The mixture of poly-1 + poly-2 inhibited both viral strains similarly to poly-2. CONCLUSIONS: The bifunctional poly-(1 + 2) acts synergistically against the wild-type influenza virus, but not against its drug-resistant mutant, as compared to a physical mixture of the monofunctional poly-1 and poly-2.

Temporal release of a three-component protein subunit vaccine from polymer multilayers.

Sustained antigen and adjuvant availability have been shown to improve antiviral immune responses following vaccination. Transcutaneous delivery of vaccines using microneedles has also shown promise and may be particularly relevant for mosquito-borne viruses. We aim to combine these traits to create a three-component Protein Subunit vaccine on Microneedle Arrays (PSMNs) for transcutaneous delivery using layer-by-layer (LbL) assembly. Polymer multilayer thin films were generated to co-deliver a model combination of three chemically distinct vaccine components, a dengue virus Envelope protein Domain III (EDIII) subunit antigen and two adjuvants, a double-stranded RNA (Poly (inosinic:cytidylic acid) (PolyI:C)) and an amphiphilic hexapeptide, Pam3CSK4. Following application of PSMNs to the skin, implanted thin films facilitated sustained and temporal release of individual vaccine components from polymer multilayers. By modulating LbL composition and architecture, component release profiles in the skin could be independently tuned to allow release of adjuvants and antigen from days up to two weeks. Uptake of antigen and adjuvant from implanted vaccine films by antigen-presenting cells was demonstrated using in vivo mouse and ex vivo human skin models. Overall, we believe that such modular vaccine strategies offer design principles for enhancing the immunogenicity of protein subunit vaccines.

Non-viral siRNA delivery to the lung.

SiRNAs exert their biological effect by guiding the degradation of their cognate mRNA sequence, thereby shutting down the corresponding protein production (gene silencing by RNA interference or RNAi). Due to this property, siRNAs are emerging as promising therapeutic agents for the treatment of inherited and acquired diseases, as well as research tools for the elucidation of gene function in both health and disease. Because of their lethality and prevalence, lung diseases have attracted particular attention as targets of siRNA-mediated cures. In addition, lung is accessible to therapeutic agents via multiple routes, e.g., through the nose and the mouth, thus obviating the need for targeting and making it an appealing target for RNAi-based therapeutic strategies. The clinical success of siRNA-mediated interventions critically depends upon the safety and efficacy of the delivery methods and agents. Delivery of siRNAs relevant to lung diseases has been attempted through multiple routes and using various carriers in animal models. This review focuses on the recent progress in non-viral delivery of siRNAs for the treatment of lung diseases, particularly infectious diseases. The rapid progress will put siRNA-based therapeutics on fast track to the clinic.

Conjugation to polymeric chains of influenza drugs targeting M2 ion channels partially restores inhibition of drug-resistant mutants.

By attaching multiple copies of the influenza M2 ion channel inhibitors amantadine (1) and rimantadine (2) to polymeric chains, we endeavored to recover their potency in inhibiting drug-resistant influenza viruses. Depending on loading densities, as well as the nature of the drug, the polymer, and the spacer arm, polymer-conjugated drugs were up to 30-fold more potent inhibitors of drug-resistant strains than their monomeric parents. In particular, a 20% loading density and a short linker group on the negatively charged poly-l-glutamate resulted in one of the most potent inhibitors for 2's conjugates against drug-resistant influenza strains. Although full recovery of the inhibitory action against drug-resistant strains was not achieved, this study may be a step toward salvaging anti-influenza drugs that are no longer effective.

Conjugating drug candidates to polymeric chains does not necessarily enhance anti-influenza activity.

Using the plaque reduction assay, relatively simple bicyclic quinone molecules, as well as multiple copies thereof covalently attached to a long polyglutamate-based polymeric chain, were examined as new inhibitors of various naturally occurring strains of influenza A virus. The polymer-conjugated inhibitors were found to have a far greater potency (for some as high as two orders of magnitude when a long spacer arm was employed) than their corresponding parent molecules against the human Wuhan influenza strain. However, such polymeric inhibitors failed to exhibit higher potency compared with their small molecule predecessors against the human Puerto Rico and avian turkey influenza strains. These observations, further explored by means of molecular modeling, reveal the previously unrecognized unpredictability of the benefits of multivalency, possibly because of poor accessibility of the viral targets to polymeric agents.

Attaching zanamivir to a polymer markedly enhances its activity against drug-resistant strains of influenza a virus.

Effects of the commercial drug zanamivir (Relenza) covalently attached to poly-l-glutamine on the infectivity of influenza A viruses are examined using the plaque reduction assay and binding affinity to viral neuraminidase (NA). These multivalent drug conjugates exhibit (i) up to a 20,000-fold improvement in anti-influenza potency compared with the zanamivir parent against human and avian viral strains, including both wild-type and drug-resistant mutants, and (ii) superior neuraminidase (NA) inhibition constants, especially for the mutants. These findings provide a basis for exploring polymer-attached inhibitors as more efficacious therapeutics, particularly against drug-resistant influenza strains.

Hydrophobic polycationic coatings inactivate wild-type and zanamivir- and/or oseltamivir-resistant human and avian influenza viruses.

Glass slides painted with the hydrophobic long-chained polycation N,N-dodecyl,methyl-polyethylenimine (N,N-dodecyl,methyl-PEI) are highly lethal to waterborne influenza A viruses, including not only wild-type human and avian strains but also their neuraminidase mutants resistant to currently used anti-influenza drugs.

Identification of novel superior polycationic vectors for gene delivery by high-throughput synthesis and screening of a combinatorial library.

PURPOSE: Low efficiency and toxicity are two major drawbacks of current non-viral gene delivery vectors. Since DNA delivery to mammalian cells is a multi-step process, generating and searching combinatorial libraries of vectors employing high-throughput synthesis and screening methods is an attractive strategy for the development of new improved vectors because it increases the chance of identifying the most overall optimized vectors. MATERIALS AND METHODS: Based on the rationale that increasing the effective molecular weight of small PEIs, which are poor vectors compared to the higher molecular weight homologues but less toxic, raises their transfection efficiency due to better DNA binding, we synthesized a library of 144 biodegradable derivatives from two small PEIs and 24 bi- and oligo-acrylate esters. A 423-Da linear PEI and its 1:1 (w/w) mixture with a 1.8-kDa branched PEI were cross-linked with the acrylates at three molar ratios in DMSO. The resulting polymers were screened for their efficiency in delivering a beta-galactosidase expressing plasmid to COS-7 monkey kidney cells. Selected most potent polymers from the initial screen were tested for toxicity in A549 human lung cancer cells, and in vivo in a systemic gene delivery model in mice employing a firefly luciferase expressing plasmid. RESULTS: Several polycations that exhibited high potency and low toxicity in vitro were identified from the library. The most potent derivative of the linear 423-Da PEI was that cross-linked with tricycle-[5.2.1.0]-decane-dimethanol diacrylate (diacrylate 14), which exhibited an over 3,600-fold enhancement in efficiency over the parent. The most potent mixed PEI was that cross-linked with ethylene glycol diacrylate (diacrylate 4) which was over 850-fold more efficient than the physically mixed parent PEIs. The relative efficiencies of these polymers were even up to over twice as high as that of the linear 22-kDa PEI, considered the "gold standard" for in vitro and systemic gene delivery. The potent cross-linked polycations identified were also less toxic than the 22-kDa PEI. The optimal vector in vivo was the mixed PEI cross-linked with propylene glycol glycerolate diacrylate (diacrylate 7); it mediated the highest gene expression in the lungs, followed by the spleen, with the expression in the former being 53-fold higher compared to the latter. In contrast, the parent PEIs mediated no gene expression at all under similar conditions, and injection of the polyplexes of the 22-kDa PEI at its optimal N/P of 10 prepared under identical conditions killed half of the mice injected. CONCLUSIONS: High-throughput synthesis and transfection assay of a cross-linked library of biodegradable PEIs was proven effective in identifying highly transfecting vectors. The identified vectors exhibited dramatically superior efficiency compared to their parents both in vitro and in an in vivo systemic gene delivery model. The majority of these vectors mediated preferential gene delivery to the lung, and their in vivo toxicity paralleled that in vitro.

Polymeric coatings that inactivate both influenza virus and pathogenic bacteria.

Painting a glass slide with branched or linear N,N-dodecyl methyl-polyethylenimines (PEIs) and certain other hydrophobic PEI derivatives enables it to kill influenza virus with essentially a 100% efficiency (at least a 4-log reduction in the viral titer) within minutes, as well as the airborne human pathogenic bacteria Escherichia coli and Staphylococcus aureus. For most of the coating polyions, this virucidal action is shown to be on contact, i.e., solely by the polymeric chains anchored to the slide surface; for others, a contribution of the polyion leaching from the painted surface cannot be ruled out. A relationship between the structure of the derivatized PEI and the resultant virucidal activity of the painted surface has been elucidated.

Full deacylation of polyethylenimine dramatically boosts its gene delivery efficiency and specificity to mouse lung.

High-molecular-mass polyethylenimines (PEIs) are widely used vectors for nucleic acid delivery. We found that removal of the residual N-acyl moieties from commercial linear 25-kDa PEI enhanced its plasmid DNA delivery efficiency 21 times in vitro, as well as 10,000 times in mice with a concomitant 1,500-fold enhancement in lung specificity. Several additional linear PEIs were synthesized by acid-catalyzed hydrolysis of poly(2-ethyl-2-oxazoline), yielding the pure polycations. PEI87 and PEI217 exhibited the highest efficiency in vitro: 115-fold and 6-fold above those of the commercial and deacylated PEI25s, respectively; moreover, PEI87 delivered DNA to mouse lung as efficiently as the pure PEI25 but at a lower concentration and with a 200-fold lung specificity. These improvements stem from an increase in the number of protonatable nitrogens, which presumably results in a tighter condensation of plasmid DNA and a better endosomal escape of the PEI/DNA complexes. As a validation of the potential of such linear, fully deacylated PEIs in gene therapy for lung diseases, systemic delivery in mice of the complexes of a short interfering RNA (siRNA) against a model gene, firefly luciferase, and PEI25 or PEI87 afforded a 77% and 93% suppression of the gene expression in the lungs, respectively. Furthermore, a polyplex of a siRNA against the influenza viral nucleocapsid protein gene and PEI87 resulted in a 94% drop of virus titers in the lungs of influenza-infected animals.

Cross-linked small polyethylenimines: while still nontoxic, deliver DNA efficiently to mammalian cells in vitro and in vivo.

PURPOSE: Polyethylenimine (PEI) is among the most efficient nonviral gene delivery vectors. Its efficiency and cytotoxicity depend on molecular weight, with the 25-kDa PEI being most efficient but cytotoxic. Smaller PEIs are noncytotoxic but less efficient. Enhancement in gene delivery efficiency with minimal cytotoxicity by cross-linking of small PEIs via potentially biodegradable linkages was explored herein. The hypothesis was that cross-linking would raise the polycation's effective molecular weight and hence the transfection efficiency, while biodegradable linkages would undergo the intracellular breakdown after DNA delivery and hence not lead to cytotoxicity. Toward this goal, we carried out cross-linking of branched 2-kDa PEI and its 1:1 (w/w) mixture with a linear 423-Da PEI via ester- and/or amide-bearing linkages; the in vitro and in vivo gene delivery efficiency, as well as toxicity to mammalian cells, of the resultant cross-linked polycations were investigated. METHODS: The efficiency of the cross-linked PEIs in delivering in vitro a plasmid containing beta-galactosidase gene and their cytotoxicity were investigated in monkey kidney cells (COS-7). Dynamic light scattering was used to compare the relative DNA condensation efficiency of the unmodified and cross-linked PEIs. In vivo gene delivery efficiency was evaluated by intratracheal delivery in mice of the complexes of a luciferase-encoding plasmid and the PEIs and estimating the luciferase expression in the lungs. RESULTS: Cross-linking boosted the gene delivery efficiency of the small PEIs by 40- to 550-fold in vitro; the efficiency of the most potent conjugates even exceeded by an order of magnitude that of the branched 25-kDa PEI. Effective condensation of DNA was evident from the fact that the mean diameter of the complexes of the cross-linked PEIs was some 300 nm with a narrow size distribution, while the complexes of the unmodified small PEIs exhibited a mean size of >700 nm with a very broad size distribution. At concentrations where the 25-kDa PEI resulted in >95% cell death, the conjugates afforded nearly full cell viability. The cross-linked PEIs were 17 to 80 times more efficient than the unmodified ones in vivo; furthermore, their efficiencies were up to twice that of the 25-kDa PEI. CONCLUSIONS: Cross-linking of small PEIs with judiciously designed amide- and ester-bearing linkers boosts their gene delivery efficiency both in vitro and in vivo without increasing the cytotoxicity. The high efficiency is dependent on the nature of the linkages and the PEIs used.

Inhibition of influenza virus production in virus-infected mice by RNA interference.

Influenza A virus infection is a major source of morbidity and mortality worldwide. Because the effectiveness of existing vaccines and antiviral drugs is limited, development of new treatment modalities is needed. Here, we show that short interfering RNAs (siRNAs) specific for conserved regions of influenza virus genes can prevent and treat influenza virus infection in mice. Virus production in lungs of infected mice is reduced by siRNAs given either before or after initiating virus infection, by using slow i.v. administration of small volumes containing siRNAs in complexes with a polycation carrier. Similar effects also are observed when mice are given DNA vectors i.v. or intranasally, from which siRNA precursors can be transcribed. Development of delivery systems that may be compatible with human use demonstrates the potential utility of siRNAs for prophylaxis and therapy of influenza virus infections in humans.

Poly-beta amino ester-containing microparticles enhance the activity of nonviral genetic vaccines.

Current nonviral genetic vaccine systems are less effective than viral vaccines, particularly in cancer systems where epitopes can be weakly immunogenic and antigen-presenting cell processing and presentation to T cells is down-regulated. A promising nonviral delivery method for genetic vaccines involves microencapsulation of antigen-encoding DNA, because such particles protect plasmid payloads and target them to phagocytic antigen-presenting cells. However, conventional microparticle formulations composed of poly lactic-co-glycolic acid take too long to release encapsulated payload and fail to induce high levels of target gene expression. Here, we describe a microparticle-based DNA delivery system composed of a degradable, pH-sensitive poly-beta amino ester and poly lactic-co-glycolic acid. These formulations generate an increase of 3-5 orders of magnitude in transfection efficiency and are potent activators of dendritic cells in vitro. When used as vaccines in vivo, these microparticle formulations, unlike conventional formulations, induce antigen-specific rejection of transplanted syngenic tumor cells.