1. Preparation and Characterization of Protective Silk-Based Composite Materials

2. Assessment of Stability of Medications in the Protective Materials

3. Mechanisms of Stabilization

Experimental Approaches -

Year 2

We focused the Year 2 on silk films loaded with drugs. For the study, two drugs were selected: Ampicillin (known to be stable to radiation), and Clavulanate (known to be unstable to radiation). Silk films were irradiated under different conditions: X-rays (0.1 and 1 kGy), Protons (1.0 and 200 kGy), Solar Particles Event (SPE) simulation (SPE, 1.0 Gy), and Galactic Cosmic Radiation (GCR, 0.5 Gy). X-ray and proton radiation exposures were conducted at Columbia University (NY) with the help of Dr. Guy Garty and Dr. David Brenner, SPE and GCR were run at the NASA national radiation lab (Brookhaven, NY) with the help of Dr. Afshin Beheshti. High Pressure Liquid Chromatography (HPLC) is being used to quantify the recovery and to analyze the stability of the medications being studied, while Fourier Transform Infrared Spectroscopy (FTIR) is used to evaluate changes in the silk structure. Films were modified with the addition of inorganic or organic compounds to further enhance their protective properties, such as to increase mechanical toughness and free radical scavenging capabilities. The radiation had a minimal effect on silk structures at the exposures utilized in the experiments, demonstrating suitable stability of this protein matrix. With the addition of SiO2, the dissolution of silk to enable the recovery of the sequestered drugs, and the recovery of the drugs, were minimal, demonstrating the need to further study the interactions of the drugs with SiO2. Finally, it was also observed that the most significant environmental impact on drug recovery and silk dissolution was the humidity. The crystallinity of the silk increased at the higher humidity, due to chain dynamics and thus beta sheet formation (by FTIR), preventing the dissolution of the films in aqueous systems, hence the poor recovery of drug. Under low humidity, 100% of the ampicillin was recovered when embedded in silk films, and increased recovery of clavulanate was also obtained compared to the free powder (control) with GCR exposure. These results demonstrate the efficiency of silk as a stabilizing matrix to some space-related radiation.

Modeling Approaches -

Year 2

Atomistic modeling of the effect of silk proteins on the reactivity of several commonly-used drug molecules was performed using the combination of molecular dynamics (MD) and density-functional theory (DFT) calculations. More specifically, equilibrated configurations of selected drug molecules (ibuprofen, ampicillin, and clavulanate) binding to silk proteins were identified from MD results and then fed as input to the conceptual DFT calculations of their reactivity descriptors. The comparison between the reactivity descriptors of (i) free-standing drug molecules and (ii) drug molecules bound to the residues in silk proteins can help us understand how the presence of silk materials affects the reactivity of drugs, as well as their susceptibility to space radiation.

Next Steps -

Future work would include the study of the interaction between SiO2 and the drug to elucidate the mechanisms involved. Additional beam time (GCR and SPE) would be helpful to obtain better insight into the effect of radiation on drugs and silk. All the samples would be prepared under controlled humidity to evaluate the range of stability or crystallization outcomes. Finally, we would like to study edible patches such as their dissolution in the gastrointestinal track for future uses, where even with the humidity-induced crystallization and thus poor drug recovery from the silk matrix, the systems would still be useful as edible reservoirs for the drugs. The design of alternative silk sequences that interact (and protect) specially with the reactive areas of the sensitive regions of the structures of the drugs would be another direction to pursue. In work that we could not finish, we planned to perform a parameter study based on the existing modeling approach to find the density of silk fibroin and the threshold spacing between silk and drugs to make the drugs less reactive in the presence of silk materials. We will also implement the atomistic model to investigate the interaction between drugs and inorganic particles such as SiO2.

The medications needed to keep a crew healthy during a deep space exploration missions must remain effective while enduring more than a year in deep space, which includes exposure to space radiation. Medications must be stored in containers that are resilient to such conditions, which is the goal of our project. The objective is to utilize silk protein composite materials to protect these compounds. Silk protein is a biomaterial that has previously been approved by the US Food and Drug Administration for various medical products. In the study for NASA, formulations including silk proteins will be used with inorganic particles and additives to shield a variety of drugs from exposure to environmental extremes. The protective ability of the materials and mechanisms underlying this protection will be explored and optimized through a combination of experimental testing and molecular modeling. The specific objectives are: 1. Preparation and Characterization of Protective Silk-Based Composite Materials; 2. Assessment of Stability of Medications in the Protective Materials; 3. Mechanisms of Stabilization Past Work Related to the Project.

Previously, silk materials have been shown to be effective at stabilizing a number of drugs and complex antibodies, as well as providing protective functions for otherwise labile biological materials (e.g., foods). Further, in neat forms and as silica composites, silk films were flown for 18 months on the International Space Station (MISSE-6) to assess the impact of space radiation on structure and function. Of the material formulations tested, it was observed that silk composites (silk-silica) remained mainly intact in morphology and size. Based on these finding, we are working on the design of formulations including silk proteins with inorganic particles to shield a variety of drugs from exposure to environmental extremes.

Experimental Approaches -- We designed two different silk formulations for our experiments; the first being silk films with medications embedded inside the films, while in the second format, the medication is loaded as powder or tablet inside silk pouches. The films and pouches were subjected to incubation at elevated temperature and humidity as accelerated aging experimental conditions (e.g., 40oC and 75% relative humidity). High Pressure Liquid Chromatography (HPLC) is being used to quantify the recovery and to analyze the stability of the medications being studied, while several other instrumental analyses such as Fourier Transform Infrared Spectroscopy (FTIR) and Scanning Electron Microscopy (SEM) are used to evaluate changes in the silk structure and morphology, respectively. Films are being modified with the addition of inorganic or organic compounds to further enhance their protective properties, such as to increase mechanical toughness and free radical scavenging capabilities. Examples of drugs being used initially include ciprofloxacin, tetracycline, and ampicillin. Other drugs known to be susceptible to space radiation, such as clavulanate and levothyroxine, will be included after designing suitable silk formulations. The type of radiation that will be initially tested includes gamma rays and X-rays, while we will also subject the samples and the drugs to different ions and energy at the Brookhaven National Lab to simulate Galactic Cosmic Radiation, more relevant to deep space radiations.

Modeling Approaches -- Density Functional Theory (DFT) calculations of commonly-used drug molecules, the amino acids and sequences in silk with different lengths are being performed. We used the energy and electron density obtained from the DFT results to calculate conceptual DFT reactivity descriptors, which allow us to evaluate the tendency of each molecule to transfer electrons and to identify the locations of reactive sites in the chemical structures.

Next Steps -- For the next year, we will with the two device designs, films and pockets, focusing on improvements in production efficiency, mass balance assessments, and additions of inorganic particles such as silica and antioxidants such as Vitamin C and Trolox to enhance protective capabilities. We plan to irradiate samples with gamma rays, x-rays, and heavy ions to simulate Galactic Cosmic Radiations (GCR). All-atom and/or coarse-grained molecular dynamics simulations will be used to investigate the mechanisms of radiation effects on the silk films at the molecular level.