Bettina E. B. Jensen

Poly(Vinyl Alcohol) Physical Hydrogels: New Vista on a Long Serving Biomaterial

Poly(vinyl alcohol), PVA, and physical hydrogels derived thereof have an excellent safety profile and a successful history of biomedical applications. However, these materials are hardly in the focus of biomedical research, largely due to poor opportunities in nano- and micro-scale design associated with PVA hydrogels in their current form. In this review we aim to demonstrate that with PVA, a (sub)molecular control over polymer chemistry translates into fine-tuned supramolecular association of chains and this, in turn, defines macroscopic properties of the material. This nano- to micro- to macro- translation of control is unique for PVA and can now be accomplished using modern tools of macromolecular design. We believe that this strategy affords functionalized PVA physical hydrogels which meet the demands of modern nanobiotechnology and have a potential to become an indispensable tool in the design of biomaterials.

Liposomes as Drug Deposits in Multilayered Polymer Films

The ex vivo growth of implantable hepatic or cardiac tissue remains a challenge and novel approaches are highly sought after. We report an approach to use liposomes embedded within multilayered films as drug deposits to deliver active cargo to adherent cells. We verify and characterize the assembly of poly(l-lysine) (PLL)/alginate, PLL/poly(l-glutamic acid), PLL/poly(methacrylic acid) (PMA), and PLL/cholesterol-modified PMA (PMAc) films, and assess the myoblast and hepatocyte adhesion to these coatings using different numbers of polyelectrolyte layers. The assembly of liposome-containing multilayered coatings is monitored by QCM-D, and the films are visualized using microscopy. The myoblast and hepatocyte adhesion to these films using PLL/PMAc or poly(styrenesulfonate) (PSS)/poly(allyl amine hydrochloride) (PAH) as capping layers is evaluated. Finally, the uptake of fluorescent lipids from the surface by these cells is demonstrated and compared. The activity of this liposome-containing coating is confirmed for both cell lines by trapping the small cytotoxic compound thiocoraline within the liposomes. It is shown that the biological response depends on the number of capping layers, and is different for the two cell lines when the compound is delivered from the surface, while it is similar when administered from solution. Taken together, we demonstrate the potential of liposomes as drug deposits in multilayered films for surface-mediated drug delivery.

Surface-Adhered Composite Poly(Vinyl Alcohol) Physical Hydrogels: Polymersome-Aided Delivery of Therapeutic Small Molecules

Surface-mediated drug delivery (SMDD) is an approach which aims to administer therapeutics to adhering or suspension cells from implantable devices or from tissue engineering scaffolds. Herein is reported the proof of concept for polymersome-aided trapping of the cytotoxic hydrophobic compound thiocoraline in a physical poly(vinyl alcohol) (PVA) hydrogel matrix and the subsequent viability of myoblast cells cultured on this surfaceadhered composite hydrogel. The ABA triblock copolymer, poly( N -acryloyl morpholine)block -poly(cholesteryl acrylate)block -poly( N -acryloyl morpholine) (PNAMb -PChAb -PNAM), is synthesized, and the self-assembly of this polymer into polymersomes is shown. The polymersomes are loaded with the model cargo fl uorescein in order to visualize the entrapping of the small payload in this composite hydrogel. To render the PVA hydrogels cell adhesive, a poly(dopamine) (PDA) coating is successfully applied to the biointerface. Finally, to demonstrate the feasibility that active cargo can be entrapped and yield a cell response, the composite hydrogels are loaded with the cytotoxic depsipeptide thiocoraline and used in SMDD. The cargo activity is ascertained via monitoring the viability of myoblast cells adhering to the composite hydrogels ( Scheme 1 ). Taken together, the presented results contribute signifi cantly to the development of methods in SMDD for various biomedical applications. Among the most well-known examples in SMDD to date are hormone-eluting contraceptive coils, [ 1 ] and drug-eluting stents, which have demonstrated their clinical relevance by their unparalleled success in preventing restenosis. [ 2 ] Implantable drugeluting devices [ 3 ] are of great commercial interest, [ 4 ] and there is a need for sophisticated coatings when it comes to delivery of a broad range of different cargo. Further, there is a considerable demand for hydrogel-based drug-eluting [ 5 ] micro-structured [ 6 ]

Assembly of Poly(dopamine)/Poly(N-isopropylacrylamide) Mixed Films and Their Temperature-Dependent Interaction with Proteins, Liposomes, and Cells

Many biomedical applications benefit from responsive polymer coatings. The properties of poly(dopamine) (PDA) films can be affected by codepositing dopamine (DA) with the temperature-responsive polymer poly(N-isopropylacrylamide) (pNiPAAm). We characterize the film assembly at 24 and 39 °C using DA and aminated or carboxylated pNiPAAm by a quartz crystal microbalance with dissipation monitoring (QCM-D), X-ray photoelectron spectroscopy, UV-vis, ellipsometry, and atomic force microscopy. It was found that pNiPAAm with both types of end groups are incorporated into the films. We then identified a temperature-dependent adsorption behavior of proteins and liposomes to these PDA and pNiPAAm containing coatings by QCM-D and optical microscopy. Finally, a difference in myoblast cell response was found when these cells were allowed to adhere to these coatings. Taken together, these fundamental findings considerably broaden the potential biomedical applications of PDA films due to the added temperature responsiveness.

Bioresorbable Surface-Adhered Enzymatic Microreactors Based on Physical Hydrogels of Poly(vinyl alcohol)

Hydrogel biomaterials based on poly(vinyl alcohol), PVA, have an extensive history of biomedical applications, yet in their current form suffer from significant shortcomings, such as a lack of mechanism of biodegradation and poor opportunities in controlled drug release. We investigate physical hydrogels of PVA as surface-adhered materials and present biodegradable matrices equipped with innovative tools in substrate-mediated drug release. Toward the final goal, PVA chains with narrow polydispersities (1.1-1.2) and molecular weights of 5, 10, and 28 kDa are synthesized via controlled radical polymerization (RAFT). These molecular weights are shown to be suitably high to afford robust hydrogel matrices and at the same time suitably low to allow gradual erosion of the hydrogels with kinetics of degradation controlled via polymer macromolecular characteristics. For opportunities in controlled drug release, hydrogels are equipped with enzymatic cargo to achieve an in situ conversion of externally added prodrug into a final product, thus giving rise to surface-adhered enzymatic microreactors. Hydrogel-mediated enzymatic activity was investigated as a function of polymer molecular weight and concentration of solution taken for assembly of hydrogels. Taken together, we present, to the best of our knowledge, the first example of bioresorbable physical hydrogel based on PVA with engineered opportunities in substrate-mediated enzymatic activity and envisioned utility in surface-mediated drug delivery and tissue engineering.

Surface adhered poly(vinyl alcohol) physical hydrogels as tools for rational design of intelligent biointerfaces

We develop three independent non-cryogenic techniques for the production of physical hydrogels based on poly(vinyl alcohol), PVA, as surface adhered materials. Hydrogelation of the micro-structured PVA films was achieved using a coagulating salt (sodium sulfate), aqueous isopropanol, and poly(ethylene glycol), and each of the methods afforded robust hydrogels which remained stable under physiological conditions for at least 48 h. The choice of the stabilization method and polymer molecular weight was decisive in controlling hydrogel dimensions (swelling) and Youngs modulus, the latter varied from 20 to 2000 kPa. Immobilization and retention of model low molecular weight cargo were monitored using a custom-made PVA with terminal amine groups, conjugating a fluorescent probe to this polymer and co-gelation of the conjugate with matrix forming PVA. Polymer losses during stabilization and kinetics of cargo release in PBS over time differed depending on the hydrogel stabilization method employed making the latter an effective tool in the design of intelligent biointerfaces. For each stabilization method, we demonstrate retention of model protein cargo over at least 48 h of incubation in physiological buffer, a phenomenon which was used to render the non-adhesive pristine PVA gels well suited for cell adhesion and proliferation. Taken together, we present a flexible and highly adaptable platform for the design of intelligent biointerfaces towards their use in tissue engineering and surface mediated drug delivery.

Lipogels: surface-adherent composite hydrogels assembled from poly(vinyl alcohol) and liposomes.

Drug-eluting engineered surface coatings are of paramount importance for many biomedical applications from implantable devices to tissue engineering. Herein, we present the assembly of lipogels, composite physical hydrogels assembled from poly(vinyl alcohol) and liposomes using thiol-disulfide exchange between end group modified PVA and thiocholesterol containing liposomes, and the response of adhering cells to these coatings. We demonstrate the controlled loading of liposomes into the polymer matrix and the preserved mechanical properties of the lipogels. Furthermore, the lipogels are successfully rendered cell adhesive by incorporation of poly(l-lysine) into the PVA polymer matrix or by poly(dopamine) coating of the lipogels. The successful lipid uptake from the lipogels by macrophages, hepatocytes, and myoblasts was monitored by flow cytometry. Finally, the delivery of active cargo, paclitaxel, to adherent myoblasts is shown, thus illustrating the potential of the lipogels as a drug eluting interface for biomedical applications.

Surface grafted glycopolymer brushes to enhance selective adhesion of HepG2 cells

This work demonstrates the application of carbohydrate based methacrylate polymer brush, poly(2-lactobionamidoethyl methacrylate), for the purpose of cell adhesion studies. The first part of the work illustrates the effects of the structure of the aminosilane based ATRP initiator layer on the polymerization kinetics of 2-lactobionamidoethyl methacrylate) (LAMA) monomer on thermally oxidized silicon wafer. Both monolayer and multilayered aminosilane precursor layers have been prepared followed by reaction with 2-bromoisobutyrylbromide to form the ATRP initiator layer. It is inferred from the kinetic studies that the rate of termination is low on a multilayered initiator layer compared to a disordered monolayer structure. However both initiator types results in similar graft densities. Furthermore, it is shown that thick comb-like poly(LAMA) brushes can be constructed by initiating a second ATRP process on a previously formed poly(LAMA) brushes. The morphology of human hepatocellular carcinoma cancer cells (HepG2) on the comb-like poly(LAMA) brush layer has been studied. The fluorescent images of the HepG2 cells on the glycopolymer brush surface display distinct protrusions that extend outside of the cell periphery. On the other hand the cells on bare glass substrate display spheroid morphology. Further analysis using ToF-SIMS imaging shows that the HepG2 cells on glycopolymer surfaces is enriched with protein fragment along the cell periphery which is absent in the case of cells on bare glass substrate. It is suggested that the interaction of the galactose units of the polymer brush with the asialoglycoprotein receptor (ASGPR) of HepG2 cells has resulted in the protein enrichment along the cell periphery.

Macromolecular (pro)drugs with concurrent direct activity against the hepatitis C virus and inflammation.

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.

Poly(vinyl alcohol) Physical Hydrogels: Matrix-Mediated Drug Delivery Using Spontaneously Eroding Substrate.

Poly(vinyl alcohol) hydrogels have a long and successful history of applications in biomedicine. Historically, these matrices were developed to be nondegradable—limiting their utility to applications as permanent implants. For tissue engineering and drug delivery, herein we develop spontaneously eroding physical hydrogels based on PVA. We characterize in detail a mild, noncryogenic method of producing PVA physical hydrogels using poly(ethylene glycol) as a gelating agent, and investigate PVA molar mass as a means to define the kinetics of erosion of these biomaterials. PVA hydrogels are characterized for associated inflammatory response in adhering macrophages, antiproliferative effects mediated through delivery of cytotoxic drugs to myoblasts, and pro-proliferative activity achieved via presentation of conjugated growth factors to endothelial cells. Together, these data present a multiangle characterization of these novel multifunctional matrices for applications in tissue engineering and drug delivery mediated by implantable biomaterials.