Folate-Targeted Monodisperse PEG-Based Conjugates Made by Chemo-Enzymatic Methods for Cancer Diagnosis and Treatment.

This paper focuses on preliminary in vitro and in vivo testing of new bivalent folate-targeted PEGylated doxorubicin (DOX) made by modular chemo-enzymatic processes (FA2-dPEG-DOX2). A unique feature is the use of monodisperse PEG (dPEG). The modular approach with enzyme catalysis ensures exclusive γ-conjugation of folic acid, full conversion and selectivity, and no metal catalyst residues. Flow cytometry analysis showed that at 10 µM concentration, both free DOX and FA2-dPEG-DOX2 would be taken up by 99.9% of triple-negative breast cancer cells in 2 h. Intratumoral injection to mice seemed to delay tumor growth more than intravenous delivery. The mouse health status, food, water consumption, and behavior remained unchanged during the observation.

Polyisobutylene-New Opportunities for Medical Applications.

This paper presents the results of the first part of testing a novel electrospun fiber mat based on a unique macromolecule: polyisobutylene (PIB). A PIB-based compound containing zinc oxide (ZnO) was electrospun into self-supporting mats of 203.75 and 295.5 g/m2 that were investigated using a variety of techniques. The results show that the hydrophobic mats are not cytotoxic, resist fibroblast cell adhesion and biofilm formation and are comfortable and easy to breathe through for use as a mask. The mats show great promise for personal protective equipment and other applications.

Screening of Polymer-Based Drug Delivery Vehicles Targeting Folate Receptors in Triple-Negative Breast Cancer.

PURPOSE: To compare cellular uptake and cytotoxicity of fluorescein (FL)-labeled polyethylene glycols (PEGs) carrying 2 folate groups (targeted delivery vehicles [TDVs]) to non-PEGylated molecules with 1 or 2 folate groups. MATERIALS AND METHODS: Three PEGylated TDVs and 2 non-PEGylated folic acid (FA)-fluorescein (FL) conjugates (FA-FL and FA-FL-FA) were synthesized. Two triple-negative breast cancer cell lines (MDA-MB-231and MDA-MB-468) were cultured to 70% confluency and incubated for 2 h in a folate-depleted medium. Folate receptor (FR) expression was confirmed by immunocytochemistry. Cellular uptake and cytotoxicity of compounds were measured by flow cytometry. Intracellular localization was confirmed using confocal microscopy. RESULTS: MDA-MB-231 demonstrated 40% more FR staining than MD-MB-468. Intracellular localization of the 2 non-PEGylated molecules (FA-FL and FA-FL-FA) and the 3 PEGylated TDVs was confirmed with confocal microscopy. Cellular uptake was independent of concentration for FA-FL, but there was 26.8% more cytotoxicity at 30 µg/mL compared with no treatment (P ≤ .05). Uptake was > 90% for FA-FL-FA at 10 µg/mL and 30 µg/mL without significant cytotoxicity (P ≤ .005). Cellular uptake was > 80% for all TDVs. The molecule containing monodispersed PEG with Mn = 1,000 g/mol had the highest uptake in both cell lines without cytotoxicity. Maximum toxicity was demonstrated by the molecule containing PEG2,000 only at the highest dose of 30 µg/mL (8.66% ± 3.94% cytotoxicity; cut-off was 20%). CONCLUSIONS: The molecule containing monodispersed PEG with Mn = 1,000 g/mol and 2 FA targeting groups demonstrated better targetability and cellular uptake as a TDV.

PEGylation of Fluorescein by Enzyme-Catalyzed "Click" Michael Addition.

This paper reports the first "Click" Michael addition catalyzed by Candida antarctica lipase B (CALB) between fluorescein o-acrylate and thiol-functionalized poly(ethylene glycol)s (HS-PEG-SH, Mn = 1200 g mol-1 , D = 1.14, and Mn = 2200 g mol-1 , D = 1.09). The progress of the reactions is monitored with 1 H-NMR spectroscopy. In the absence of CALB, the reaction does not go to completion even after 18 h but completes in less than 2 min when CALB is added. Similarly, the reaction with HS-PEG-SH having Mn = 2200 g mol-1 and D = 1.09 completes in less than 2 min by CALB catalysis. The structures of the products are also confirmed by 13 C-NMR. This enzyme-catalyzed "Click" Michael addition is found to be a powerful tool to synthesize fluorescein-based polymeric conjugates for a wide variety of applications.

Green polymer chemistry: enzyme catalysis for polymer functionalization.

Enzyme catalyzed reactions are green alternative approaches to functionalize polymers compared to conventional methods. This technique is especially advantageous due to the high selectivity, high efficiency, milder reaction conditions, and recyclability of enzymes. Selected reactions can be conducted under solventless conditions without the application of metal catalysts. Hence this process is becoming more recognized in the arena of biomedical applications, as the toxicity created by solvents and metal catalyst residues can be completely avoided. In this review we will discuss fundamental aspects of chemical reactions biocatalyzed by Candida antarctica lipase B, and their application to create new functionalized polymers, including the regio- and chemoselectivity of the reactions.

Green polymer chemistry: investigating the mechanism of radical ring-opening redox polymerization (R3P) of 3,6-dioxa-1,8-octanedithiol (DODT).

The mechanism of the new Radical Ring-opening Redox Polymerization (R3P) of 3,6-dioxa-1,8-octanedithiol (DODT) by triethylamine (TEA) and dilute H2O2 was investigated. Scouting studies showed that the formation of high molecular weight polymers required a 1:2 molar ratio of DODT to TEA and of DODT to H2O2. Further investigation into the chemical composition of the organic and aqueous phases by 1H-NMR spectroscopy and mass spectrometry demonstrated that DODT is ionized by two TEA molecules (one for each thiol group) and thus transferred into the aqueous phase. The organic phase was found to have cyclic disulfide dimers, trimers and tetramers. Dissolving DODT and TEA in water before the addition of H2O2 yielded a polymer with Mn = 55,000 g/mol, in comparison with Mn = 92,000 g/mol when aqueous H2O2 was added to a DODT/TEA mixture. After polymer removal, MALDI-ToF MS analysis of the residual reaction mixtures showed only cyclic oligomers remaining. Below the LCST for TEA in water, 18.7 °C, the system yielded a stable emulsion, and only cyclic oligomers were found. Below DODT/TEA and H2O2 1:2 molar ratio mostly linear oligomers were formed, with <20% cyclic oligomers. The findings support the proposed mechanism of R3P.

In Vivo Release of Zafirlukast from Electrospun Polyisobutylene-Based Fiber Mats to Reduce Capsular Contracture of Silicone Breast Prostheses.

Silicone rubber tissue expanders and breast implants are associated with chronic inflammation, leading to the formation of fibrous capsules. If the inflammation is left untreated, the fibrous capsules can become hard and brittle and lead to formation of capsular contracture. When capsular contracture occurs, implant failure and reoperation is unavoidable. Fibrous capsule formation to medical grade silicone rubber breast implants and polyisobutylene-based electrospun fiber mats attached to silicone rubber with and without an anti-inflammatory therapeutic were compared. A linear polyisobutylene (PIB)-based thermoplastic elastomer is currently applied as a polymer coating for drug release on coronary stents to reduce restenosis. Recent work has created a drug releasing electrospun fiber mat from PIB-based materials. Important to this study, poly(alloocimene-b-isobutylene-b-alloocimene) (AIBA) was electrospun with zafirlukast (ZAF). ZAF is an anti-inflammatory drug that is able to reduce capsule formation and complications to silicone breast implants. Fiber mats are advantageous for local drug delivery because of their high porosity and surface area for drug release. The chief hypothesis was that local release of ZAF from AIBA would lower inflammatory signaling and resulting capsular formation after 90 days in vivo. Electrospun AIBA mats locally released ZAF, lowering inflammation and fibrous capsule development compared to medical grade silicone rubber. Locally and orally released ZAF led to similar results, but the former had much lower concentration that highlights local delivery's therapeutic potential. Released ZAF from AIBA fiber mats mitigated inflammation and serves as an alternative to existing clinical approaches.

Preparation of surgical meshes using self-regulating technology based on reaction-diffusion processes.

While reaction-diffusion processes are utilized in multiple scientific fields, these phenomena have seen limited practical application in the polymer industry. Although self-regulating processes driven by parallel reaction and diffusion can lead to patterned structures, most polymeric products with repeating subunits are still prepared by methods that require complex and expensive instrumentation. A notable, high-added-value example is surgical mesh, which is often manufactured by weaving or knitting. In our present work, we demonstrate how the polymer and the biomedical industry can benefit from the pattern-forming capabilities of reaction-diffusion. We would like to propose a self-regulating method that facilitates the creation of surgical meshes from biocompatible polymers. Since the control of the process assumes a thorough understanding of the underlying phenomena, the theoretical background, as well as a mathematical model that can accurately describe the empirical data, is also introduced and explained. Our method offers the benefits of conventional techniques while introducing additional advantages not attainable with them. Most importantly, the method proposed in this paper enables the rapid creation of meshes with an average pore size that can be adjusted easily and tailored to fit the intended area of application.

Natural rubber - Increasing diversity of an irreplaceable renewable resource.

This paper discusses the importance of introducing domestic natural rubber production and presents the rediscovery of a rubber-producing species, Scorzonera tau-saghyz or "mountain gum", originally discovered in 1929 on the Karatau mountains in Kazakhstan. This plant could potentially also be cultivated in the U.S. In this exploratory work, roots (2-5 years old) were harvested on June 16, 2021 from wild strands in the Karatau mountains, Kumantas ridge, and Saraba, Kazakhstan, and processed at the Ohio State University. The rubber extraction method was based on an indigenous method in Kazakhstan to make natural chewing gum. Water extraction followed by purification yielded 16.2 wt% rubber from the dry roots, in comparison with 4-8 wt% from most rubber dandelion (Taraxacum kok-saghyz) plants, also a potential domestic rubber producing plant. High-resolution size exclusion chromatography was used to analyze rubber samples. The molecular weights and gel and oligomer contents were very similar to the rubber from Hevea brasiliensis, the current commercial source of natural rubber. More detailed investigations of this very interesting rubber-producing plant are in progress.

Green polymer chemistry VIII: synthesis of halo-ester-functionalized poly(ethylene glycol)s via enzymatic catalysis.

Halo-ester-functionalized poly(ethylene glycol)s (PEGs) are successfully prepared by the transesterification of alkyl halo-esters with PEGs using Candida antarctica lipase B (CALB) as a biocatalyst under the solventless conditions. Transesterifications of chlorine, bromine, and iodine esters with tetraethylene glycol monobenzyl ether (BzTEG) are quantitative in less than 2.5 h. The transesterification of halo-esters with PEGs are complete in 4 h. (1) H and (13) C NMR spectroscopy with MALDI-ToF and ESI mass spectrometry confirm the structure and purity of the products. This method provides a convenient and "green" process to effectively produce halo-ester PEGs.

Green polymer chemistry: living dithiol polymerization via cyclic intermediates.

This paper reports the synthesis and characterization of disulfide polymers obtained by oxidation of 2-[2-(2-sulfanylethoxy)ethoxy]ethanethiol (DODT) using a benign, synergistic system comprised of air, dilute hydrogen peroxide and triethylamine as a catalyst that can be recycled. The dn/dc value of the polymer in THF was determined to obtain absolute molecular weight measurements. High molecular weight disulfide polymers (up to M(n) = 250000 g/mol) with polydispersity indices as low as M(w)/M(n) = 1.15 were obtained. Thermal analysis by DSC and TGA demonstrated that the rubbery polymers had a T(g) of -50 °C and began to degrade at 250 °C. Dithiothreitol reduced the polymers back to the original monomeric units in 33 h. MALDI-ToF showed the involvement of oligodisulfide rings (2-14 mers) in the polymerization that displayed the characteristics of a living/controlled polymerization; poly(DODT) was readily chain extended with 1,2-ethanedithiol. The chain extension indicates a class of living polymerization which is governed by radical recombination.

Multifunctional PEG Carrier by Chemoenzymatic Synthesis for Drug Delivery Systems: In Memory of Professor Andrzej Dworak.

This paper describes the synthesis and characterization of new bivalent folate-targeted PEGylated doxorubicin (FA2-dPEG-DOX2) made by modular chemo-enzymatic processes using Candida antarctica lipase B (CALB) as a biocatalyst. Unique features are the use of monodisperse PEG (dPEG) and the synthesis of thiol-functionalized folic acid yielding exclusive γ-conjugation of folic acid (FA) to dPEG. The polymer-based drug conjugate is built up by a series of transesterification and Michael addition reactions all catalyzed be CALB. In comparison with other methods in the literature, the modular approach with enzyme catalysis leads to selectivity, full conversion and high yield, and no transition metal catalyst residues. The intermediate product with four acrylate groups is an excellent platform for Michael-addition-type reactions for a wide variety of biologically active molecules. The chemical structures were confirmed by nuclear magnetic resonance spectroscopy (NMR). Flow cytometry analysis showed that, at 10 µM concentration, both free DOX and FA2-dPEG-DOX2 were taken up by 99.9% of triple-negative breast cancer cells in 2 h. Fluorescence was detected for 5 days after injecting compound IV into mice. Preliminary results showed that intra-tumoral injection seemed to delay tumor growth more than intravenous delivery.

Highly hydrophobic electrospun fiber mats from polyisobutylene-based thermoplastic elastomers.

This paper is the first report of electrospinning neat polyisobutylene-based thermoplastic elastomers. Two generations of these materials are investigated: a linear poly(styrene-b-isobutylene-b-styrene) (L_SIBS) triblock copolymer and a dendritic poly(isobutylene-b-p-methylstyrene) (D_IB-MS), also a candidate for biomedical applications. Cross-polarized optical microscopy shows birefringence, indicating orientation in the electrospun fibers, which undergo large elongation and shear during electrospinning. In contrast to the circular cross section of L_SIBS fibers, D_IB-MS yields dumbbell-shaped fiber cross sections for the combination of processing conditions, molecular weight, and architecture. Hydrophobic surfaces with a water contact angle as high as 146 ± 3° were obtained with D_IB-MS that had the noncircular fiber cross section and a hierarchical arrangement of nano- to micrometer-sized fibers in the mat. These highly water repellent fiber mats were found to serve as an excellent scaffold for bovine chondrocytes to produce cartilage tissue.

Investigation of the Cytotoxicity of Electrospun Polysuccinimide-Based Fiber Mats.

This study investigated cell viability in the presence of allylamine-modified and plasma-treated electrospun polysuccinimide fiber mats (PSI-AAmp). Low pressure non-equilibrium plasma was used for crosslinking the PSI-AAm. Comparison of FTIR and XPS analyses demonstrated that crosslinking occurred on the surface of the samples. Cell viability was investigated using the MG-63 osteosarcoma cell line and WST-1 viability reagent. Since PSI hydrolyzes to poly(aspartic acid) (PASP), PASP was used in addition to the regular controls (cells only). Phase contrast showed normal morphology in all cases at 24 h; however, in the presence of PSI-AAmp at 72 h, some rounded, dead cells could also be seen, and proliferation was inhibited. Since proliferation in the presence of PASP alone was not inhibited, the cause of inhibition was not the final product of the hydrolysis. Further investigations will be carried out to pinpoint the cause.

Architectured helically coiled scaffolds from elastomeric poly(butylene succinate) (PBS) copolyester via wet electrospinning.

Electrospinning is one of the most investigated methods used to produce polymeric fiber scaffolds that mimic the morphology of native extracellular matrix. These structures have been extensively studied in the context of scaffolds for tissue regeneration. However, the compactness of materials obtained by traditional electrospinning, collected as two-dimensional non-woven scaffolds, can limit cell infiltration and tissue ingrowth. In addition, for applications in smooth muscle tissue engineering, highly elastic scaffolds capable of withstanding cyclic mechanical strains without suffering significant permanent deformations are preferred. In order to address these challenges, we report the fabrication of microscale 3D helically coiled scaffolds (referred as 3D-HCS) by wet-electrospinning method, a modification of the traditional electrospinning process in which a coagulation bath (non-solvent system for the electrospun material) is used as the collector. The present study, for the first time, successfully demonstrates the feasibility of using this method to produce various architectures of 3D helically coiled scaffolds (HCS) from segmented copolyester of poly (butylene succinate-co-dilinoleic succinate) (PBS-DLS), a thermoplastic elastomer. We examined the role of process parameters and propose a mechanism for the HCS formation. Fabricated 3D-HCS showed high specific surface area, high porosity, and good elasticity. Further, the marked increase in cell proliferation on 3D-HCS confirmed the suitability of these materials as scaffolds for soft tissue engineering.

Lean on Your Land Grant: One University's Approach to Address the Food Supply Chain Workforce during the COVID-19 Pandemic.

During the spring 2020 COVID-19 outbreak, faculty and staff within Ohio State University's College of Food, Agricultural and Environmental Sciences came together from multiple disciplines to support essential agricultural workers. Concerted leadership from administration provided a framework for this interaction to occur while faculty worked off-campus to address the many issues identified by the agricultural community, the industry sector, and other state agencies. During the onset period, much of our work was reactive; our efforts to address worker safety and health involved three primary areas within: 1) production agricultural workers, 2) produce growers and direct marketing enterprises, and 3) meat supply chain workers. Communication to target audiences relied upon our ability to convert face-to-face programming into virtual webinars, social media, and digital publications. A Food System Task Force mobilized specialists to address emerging issues, with one specific topic related to Personal Protective Equipment (PPE). As we continue to face new seasons in agriculture production, and pockets of COVID-19 outbreaks within our state, we will continue to address the dynamic needs of our food supply systems. There are implications for how we will teach the agricultural workforce within a virtual platform, including the evaluation of the effectiveness of those training programs. There are renewed opportunities to integrate health and safety content into other Extension teams who conventionally focused on production practices and farm management topics. Several research themes emerged during subgroup dialog to pursue new knowledge in workers' cultural attitude and barriers, PPE design, PPE access, and overall attitude toward COVID-19 health practices.

Synthesis of POSS-Functionalized Polyisobutylene via Direct Initiation.

POSS-functionalized polyisobutylenes (PIBs) were synthesized by carbocationic polymerization using an epoxy-POSS/TiCl(4) initiating system in hexane/methyl chloride (60:40 v/v) solvent mixture at -80 °C. (1) H NMR spectroscopy verified the incorporation of one epoxy-POSS per polymer chain. Light scattering and TEM analysis demonstrated the formation of 50-100 nm sized aggregates and micron-sized clusters.

Folic acid conjugated polymeric drug delivery vehicle for targeted cancer detection in hepatocellular carcinoma.

Targeted therapies provide increased efficiency for the detection and treatment of cancer with reduced side effects. Folate receptor (alpha subunit) is overexpressed in multiple tumors including liver cancer. In this study, we evaluated the specificity and toxicity of a folic acid-containing drug delivery vehicle (DDV) in a hepatocellular carcinoma (HCC) model. The DDV was prepared with two units each of folic acid (FA) and fluorescein isothiocyanate (FITC) molecules and conjugated to a central poly (ethylene glycol) (PEG) core via a modified chemo-enzymatic synthetic process. Rat hepatoma (N1S1) and human monocytic (U937) cell lines were used for cell culture-based assays and tested for DDV uptake and toxicity. Folate receptor expressions in liver tissues and cell lines were verified using standard immunohistochemistry techniques. Rat HCC model was used for in vivo assessment. The DDV was injected via intra-arterial or intravenous methods and imaged with IVIS spectrum in vivo imaging system. Strong signals of FITC in the liver tumor region correlated to targeted DDV uptake. The use of PEG enhanced water-solubility and provided flexibility for the interaction of FA ligands with multiple cell surface folate receptors that resulted in increased specific uptake. Our study suggested that PEG incorporation and folate targeting via intra-arterial approach is an efficient strategy for targeted delivery in HCC therapy.

Enzymatic Degradation of Poly(butylene succinate) Copolyesters Synthesized with the Use of Candida antarctica Lipase B.

Biodegradable polymers are an active area of investigation, particularly ones that can be produced from sustainable, biobased monomers, such as copolymers of poly(butylene succinate) (PBS). In this study, we examine the enzymatic degradation of poly(butylene succinate-dilinoleic succinate) (PBS-DLS) copolymers obtained by "green" enzymatic synthesis using lipase B from Candida antarctica (CALB). The copolymers differed in their hard to soft segments ratio, from 70:30 to 50:50 wt %. Enzymatic degradation was carried out on electrospun membranes (scaffolds) and compression-moulded films using lipase from Pseudomomas cepacia. Poly(ε-caprolactone) (PCL) was used as a reference aliphatic polyester. The degradation process was monitored gravimetrically via water uptake and mass loss. After 24 days, approx. 40% mass loss was observed for fibrous materials prepared from the PBS-DLS 70:30 copolymer, as compared to approx. 10% mass loss for PBS-DLS 50:50. Infrared spectroscopy (FTIR) and size exclusion chromatography (SEC) analysis were used to examine changes in chemical structure. Differential scanning calorimetry (DSC) and scanning light microscopy (LSM) revealed changes in degree of crystallinity, and changes in surface morphology, consistent with a surface erosion mechanism. We conclude that the obtained copolymers are suitable for tissue engineering applications thanks to tuneable degradation and lack of acidification during breakdown.

Biocompatibility evaluation of a thermoplastic rubber for wireless telemetric intracranial pressure sensor coating.

This study investigated the biocompatibility of the experimental thermoplastic rubber Arbomatrix(™) that will be used as the protective coating on a novel intracranial pressure (ICP) sensor silicon chip. Arbomatrix(™) was benchmarked against biocompatible commercial silicone rubber shunt tubing in the brain via a rat model with 60-day implant duration. A bare silicon chip was also implanted. The results showed similar cellular distribution in the brain-implant boundary and surrounding tissues. Quantitative analysis of neuron and glia density did not show significant difference between implants. Through histological and immunohistochemical evaluation we conclude that Arbomatrix(™) is well tolerated by the brain. Due to its exceptional barrier properties Arbomatrix(™) has already been shown to be an excellent protective coating for new ICP monitoring chip.