Macromolecular prodrugs are developed as multiarmed agents against diverse viral pathogens. Lead candidates inhibit infectivity and replication of HIV, Ebola, influenza, measles, RSV, etc-thus being broad-spectrum antiviral agents.

Layer-by-layer (LbL) assembly is the process of building functional multilayered thin films. Owing to its highly modular and versatile nature, it has been used to coat a wide variety of different surfaces, including inorganic substrates, membranes, implants, nanoparticles and even living cells. It provides ways to induce responsiveness through both the chemically engineered macromolecular components, and the way the multilayers are built up. For example, assembly degradability can be adjusted by using degradable polymers or crosslinkers, while physical properties can be altered through the use of additives, or by the assembly method. This chapter is dedicated to LbL fabrication-specific responsiveness, and to recent developments in multilayers composed of specifically tailored polymers. It further focuses on chemically and biologically responsive LbL systems, with main applications in the biomedical field. The introduction covers general aspects of LbL assembly and physicochemical aspects of the assemblies. The second part describes physicochemical aspects in more detail with examples on how variation in deposition conditions, e.g. pH and ionic strength, as well as specific additives, induce responsiveness to the resulting multilayers. It also highlights several reports on compartmentalized multilayered coating fabrication for tunable disassembly or release of incorporated materials. The third part describes multilayers fabricated with chemically tailored biomaterials for different chemical and biological responsiveness. More specifically, multilayer disassembly can be triggered by the inherent responsiveness of one of the multilayer components, through incorporation of labile bonds that respond to specific external triggers, or through disruption of the interlayer interaction between two or more multilayer components.

Layer-by-layer (LbL) assembly has been extensively researched as a versatile method to produce smart coatings and capsules with a distinct multilayered structure. This specific fabrication technique provides ways to induce responsiveness not only through the chemically engineered macromolecular components, but also through the way the multilayers are built up. This chapter is dedicated to LbL fabrication-specific responsiveness, as well as to the recent developments in multilayers composed of specifically tailored polymers. This chapter further focuses on chemically- and biologically-responsive LbL systems, with main applications in the biomedical field. In the introduction, general aspects of LbL assembly as a fabrication technique are described, along with physicochemical aspects of the assemblies. The second part describes the physicochemical aspects in more detail with examples on how variation in deposition conditions, such as pH and ionic strength, can be used to induce responsiveness upon the resulting multilayers. This section also highlights several reports on the fabrication of compartmentalized multilayered coatings for tunable disassembly or release of incorporated materials. The third part describes recent examples of multilayers fabricated with chemically tailored biomaterials for various chemical and biological responsiveness. More specifically, the multilayer disassembly can be triggered through inherent responsiveness of one of the multilayer components, through chemically- or biologically-triggered degradation of one of the multilayer components, or through disruption of the interlayer interaction between two or more components of the multilayers.

We report the first successful implementation of transfection agents to facilitate the delivery of non-nucleic acid based anti-inflammatory and anti-viral drugs. In doing so, we illustrate a new paradigm in the intracellular delivery of polyanionic drugs and also extend the scope and utility of successful tools of gene transfer into a new area of biomedical research.

Efficacious, potent, and at the same time nontoxic macromolecular prodrugs of ribavirin are designed taking advantage over prodrug activation by the intracellular milieu. Activity of these prodrugs is illustrated in the cells hosting hepatitis C virus replication and also in the cells implicated in the inflammatory response to the viral infection.

Ribavirin (RBV)-containing polymers are synthesized based on poly(N-vinylpyrrolidone) and poly(acrylic acid), two polymers with extensive characterization in biomedicine. The copolymers are shown to exhibit a minor to negligible degree of association with erythrocytes, thus effectively eliminating the origin of the main side effects of RBV. The therapeutic benefit of macromolecular RBV prodrugs is illustrated by matched efficacy in suppressing production of nitric oxide by stimulated cultured macrophages as compared to pristine RBV with no associated cytotoxicity, which is in stark contrast to an RBV-based treatment which results in a significant decrease in cell viability. These results contribute to the development of antiviral polymer therapeutics and delivery of RBV in particular.

Polymers in tune. Automated parallel polymer synthesis is developed to obtain libraries of macromolecular prodrugs of ribavirin, a broad-spectrum antiviral agent. As many as 10 identified lead polymer conjugates exhibit therapeutic efficacy matching that of the pristine drug and at the same time suppressed the origin of the main side effect of ribavirin.

Front Cover: Macromolecular prodrugs of ribavirin are synthesized by Alexander N. Zelikin and colleagues to overcome the origin of the main side effect of this drug, namely, accumulation in the red blood cells. On page 173, polymeric prodrugs are successful in accomplishing set goal, deliver their payload into the cells with hepatic relevance, and elicit a therapeutic response comparable to the parent drug. Together, these results constitute a major step forward towards safer delivery of ribavirin, a broad spectrum antiviral agent.

Macromolecular prodrugs (MPs) are a powerful tool to alleviate side-effects and improve the efficacy of the broad-spectrum antiviral agent ribavirin. In this work, we sought an understanding of what makes an optimal formulation within the macromolecular parameter space--nature of the polymer carrier, average molar mass, drug loading, or a good combination thereof. A panel of MPs based on biocompatible synthetic vinylic and (meth)acrylic polymers was tested in an anti-inflammatory assay with relevance to alleviating inflammation in the liver during hepatitis C infection. Pristine polymer carriers proved to have a pronounced anti-inflammatory activity, a notion which may prove significant in developing MPs for antiviral and anticancer treatments. With conjugated ribavirin, MPs revealed enhanced activity but also higher toxicity. Therapeutic windows and therapeutic indices were determined and discussed to reveal the most potent formulation and those with optimized safety. Polymers were also tested as inhibitors of replication of the hepatitis C viral RNA using a subgenomic viral replicon system. For the first time, negatively charged polymers are revealed to have an intracellular activity against hepatitis C virus replication. Concerted activity of the polymer and ribavirin afforded MPs which significantly increased the therapeutic index of ribavirin-based treatment. Taken together, the systematic investigation of the macromolecular space identified lead candidates with high efficacy and concurrent direct activity against the hepatitis C virus and inflammation.

Human immunodeficiency virus (HIV) and hepatitis C virus (HCV) represent tremendous healthcare burdens with a large proportion of patients hosting the two viruses at the same time. An altered hepatic function and immunity as well as cross-interference of drugs make treatment of co-infection increasingly challenging. Herein we report the first design of macromolecular prodrugs (MP) with concurrent success in fighting HIV and alleviating hepatitis (liver inflammation). To achieve this, polymer compositions were systematically screened in a broad range of molar mass and content of ribavirin - a broad spectrum antiviral agent. For the first time, we report that ribavirin is efficacious in fighting HIV and in the form of MP, the treatment is safe, both in terms of lack of association of ribavirin with red blood cells and lack of toxicity upon cellular internalization. The lead polymer compositions were also potent in anti-inflammatory assays with relevance to viral hepatitis - thus making up formulations with potential for treatment of co-infection with HIV and HCV.

Macromolecular (pro) drugs are a sub-discipline of medicinal and polymer chemistries aiming to optimize the delivery of drugs to their site of action. In recent decades, this field of science has undergone a tremendous development, with the soundest achievements registered in the delivery of anticancer drugs. Surprisingly, the development of these tools for applications in antiviral treatment lags significantly behind -despite the fact that the first in vivo successes of polymers in fighting viruses were reported half a century ago. Furthermore, the unique scope and utility of polymers in antiviral research is that macromolecules themselves exhibit highly potent activity against diverse viruses. Herein, in an attempt to revive the research interest in this field, we aim to provide an overview of successes (and failures) of polymers as antiviral agents and macromolecular prodrugs. Specifically, we discuss inhibition of the entry of the virus into mammalian cells by polymers, give an overview of the synthetic schemes applied for the conjugation of drugs to carrier polymers, and also present guidance with regard to potential reporter systems which can be used for the characterization of novel drug delivery systems in virus-free cell cultures.

Automated parallel synthesis is applied by A. Postma, A. Zelikin, and team on page 1404 to obtain libraries of macromolecular prodrugs of ribavirin. Polymers differ in their average molar mass and drug content, the two parameters independently vary in broad respective ranges. Screening the libraries for intracellular activity identifies at least 10 formulations with efficacy matching that of the drug and at the same time significantly reducing toxicity.

Macromolecular (pro)drugs are a sub-discipline of medicinal and polymer chemistries aiming to optimize the delivery of drugs to their site of action. In recent decades, this field of science has undergone a tremendous development, with the soundest achievements registered in the delivery of anticancer drugs. Surprisingly, the development of these tools for applications in antiviral treatment lags significantly behind – despite the fact that the first in vivo successes of polymers in fighting viruses were reported half a century ago. Furthermore, the unique scope and utility of polymers in antiviral research is that macromolecules themselves exhibit highly potent activity against diverse viruses. Herein, in an attempt to revive the research interest in this field, we aim to provide an overview of successes (and failures) of polymers as antiviral agents and macromolecular prodrugs. Specifically, we discuss inhibition of the entry of the virus into mammalian cells by polymers, give an overview of the synthetic schemes applied for the conjugation of drugs to carrier polymers, and also present guidance with regard to potential reporter systems which can be used for the characterization of novel drug delivery systems in virus-free cell cultures.

The release of azidothymidine from macromolecular prodrugs was designed to respond to the intracellular disulfide reshuffling. This drug has no thiol groups, and a response to this trigger was engineered using a self-immolative linker. The resulting formulations were fast-acting, efficacious, and highly potent with regards to suppressing the infectivity of the virus.

Hydrogel nanoparticles (HNP) are an emerging tool of biomedicine with unique materials characteristics, scope, and utility. These hydrated, soft colloidal carriers can penetrate through voids with dimensions narrower than the size of the particle, provide stabilization for fragile biological cargo and allow diffusion and exchange of solutes with external phase. However, techniques to assemble HNP are few; solitary examples exist of biocompatible polymers being formulated into HNP; and knowledge on the biomedical properties of HNP remains rather cursory. In this work, we investigate assembly of HNP based on a polymer with decades of prominence in the biomedical field, poly(vinyl alcohol), PVA. We develop a novel method for production of PVA HNP through nanoprecipitation-based assembly of polymer nanoparticles and subsequent physical hydrogelation of the polymer. Polymer nanoparticles and HNP were visualized using scanning electron microscopy and fluorescence imaging, and characterized using dynamic light scattering and zeta potential measurements. Interaction of PVA HNP with mammalian cells was investigated using flow cytometry, viability screening, and measurements of nitric oxide production by cultured macrophages. The latter analyses revealed that PVA administered as a polymer solution or in the form of HNP resulted in no measurable increase in production of the inflammation marker. Unexpectedly, PVA HNP exerted a pronounced inhibition of NO synthesis by stimulated macrophages, that is, had an anti-inflammatory activity. This effect was accomplished with a negligible change in the cell viability and was not observed when PVA was administered as a polymer solution. To the best of our knowledge, this is the first observation of inhibition of NO synthesis in macrophages by administered nanoparticles and specifically hydrogel nanoparticles. Taken together, our results present PVA HNP as promising colloidal hydrogel nanocarriers for biomedical applications, specifically drug delivery and assembly of intracellular biosensors.

Chemi-enzymatic synthesis of ribavirin acrylate and subsequent RAFT co-polymerization with acrylic acid afforded a formulation of a broad spectrum antiviral drug which avoids accumulation in erythrocytes, the origin of the main side effect of ribavirin. In cultured macrophages the macromolecular prodrugs exhibited decreased toxicity while maintaining the anti-inflammatory action of ribavirin.

Ribavirin (RBV), a broad-spectrum antiviral agent, is a standard medication against hepatitis C virus (HCV). However, despite the decades of clinical success, the mechanism of action of this drug against HCV remains a subject of debate. Furthermore, the appeal of this therapeutic agent is considerably lessened by unfavorable pharmacokinetics. This interdisciplinary study contributes to the understanding of intracellular effects exerted by RBV and presents a successful design of macromolecular prodrugs of RBV to achieve a safer treatment. Specifically, we demonstrate that RBV exhibits a pronounced anti-inflammatory activity in cultured macrophages as is evidenced by a 2-fold decrease in the levels of produced nitric oxide achieved using a clinically relevant concentration of this drug. However, this effect was characterized by a rather narrow therapeutic window with experimental values of EC50 and IC50 being 7 and 19 μM, respectively. Macromolecular prodrugs were obtained using an acrylate derivative of RBV, RAFT polymerization technique, and N-vinyl pyrrolidone as a partner monomer. The synthesized polymers were characterized with uniform molecular weights, relatively narrow polydispersities, and gradually increasing content of RBV. The resulting polymer therapeutics were effective in delivering their payload to the cultured macrophages and afforded a significantly wider therapeutic window, as much as >1000 μM (18-fold in relative values). Taken together, this work contributes significantly to the development of safer methods for delivery of RBV, as well as understanding the mechanism of action and origins of the side effects of this broad-spectrum antiviral agent.

Due to its versatility and ease of use the layer-by-layer (LbL) assembly technique has been under intensive investigation for drug and gene delivery applications. Especially the development of responsive LbL materials has advanced significantly in recent years. Responsiveness plays an important role in many delivery applications, either for loading of therapeutics or controlled and triggered release. In general four basic mechanisms within responsive LbL films have been identified: disruption of layer interactions, degradation of the LbL film, multilayer destruction via physical stimuli, and phase transitions or polymer rearrangements within the LbL film. This review will outline these different mechanisms and highlight recent advances in these fields.