Silk Fibroin Microspheres: Improving Drug Delivery to the Lungs for the Treatment of Neonatal Respiratory Distress Syndrome (NRDS)
Advisor: Dr. Fisher
Internship Location: Georgetown Nanotech and Biomedical Device Lab
Onsite Mentor: Professor Makarand Paranjape
Neonatal Respiratory Distress Syndrome (NRDS) is a breathing disorder that is the leading cause of death in premature infants. It occurs when an infant is unable to produce enough lung surfactant, causing insufficient oxygen exchange and lung collapse. The prevailing treatment for NRDS is delivering natural animal pulmonary surfactant to the lungs to affect oxygenation and lung compliance. Known as INtubation-SURfactant Extubation (INSURE), this procedure unfortunately involves an unstandardized protocol and is often inefficient and ineffective due to its inability to maximize the delivery of lung surfactant to the alveoli. However, emerging studies have suggested that a more effective approach to drug delivery is administration through microspheres, as aerosolized microspheres containing the drug can maximize the surface area of the lung that is exposed to the surfactant. Specifically, Silk Fibroin (SF) best facilitates microsphere drug delivery, as SF is biocompatible and requires little resources to fabricate. In my research at the Georgetown Nanotech and Biomedical Device Lab, I hope to introduce a protocol for designing SF microspheres containing CUROSURF®, a popular pulmonary surfactant, through the simple yet effective process of phase separation between SF and Polyvinyl alcohol (PVA). I will optimize my protocol by iterating the procedure based on observations and characterizations of the resulting microspheres. With this research, I hope to offer insight into a new method of drug delivery for treating NRDS in infants that addresses the long-standing issues of lung surfactant delivery experienced in today’s intensive care nurseries.
Project Blog
Post 1: Introduction
Hi everyone, I'm Chelsea and welcome to my Senior Project blog!
Through my project, I hope to use Silk Fibroin (SF), a protein from the Bombyx mori silkworm, to create drug-loaded microsphere carriers. My project is specifically focused on the treatment of Neonatal Respiratory Distress Syndrome (NRDS), so the goal of my research is to fabricate SF microspheres loaded with lung surfactant, which is critical for improving oxygenation in newborns. As I mentioned in my abstract, the prevailing treatment for NRDS lung surfactant delivery is through the INtubation-SURfactant Extubation (INSURE) method. Unfortunately, this method is inefficient, dangerous, and poses increased risks for pulmonary injuries in newborns. My research intervenes here and proposes a safer, innovative approach by delivering lung surfactant through biomaterials, specifically SF surfactant-loaded microspheres. Over the next few weeks, I'll focus on devising a method to create these microspheres and then examine their fabrication and characteristics.
Coming up for the first week of my Senior Project, I will be focusing on a very crucial part of all research: literature review! I plan on looking closely at past research that has created protocols for creating SF microspheres and what steps and materials will be necessary for carrying out the methodology for microsphere fabrication.
I am super excited to work on this project as it could contribute substantial advancement in neonatal intensive care units, offering a more reliable and safer treatment for NRDS. I am looking forward to sharing my experiences and trials (and errors) with you all!
Week 1: Literature Review
Hi everyone! This was my first week working on my senior project research! Like I mentioned in last week’s blog, in this first week I spent a lot of time doing literature review. I used Google Scholar to look at papers that were relevant to Silk Fibroin material (microsphere) fabrication and using SF for drug delivery. From there, I looked at the citations of papers which lead me to other relevant papers and resources.
There are a couple of questions you might have that I want to first address. You might be wondering why I chose to research using Silk Fibroin (SF) as the specific biomaterial to create microspheres with. Well from the research that I’ve seen, SF has several really important characteristics that make it good for biomedical applications. For example, SF is biocompatible (in that it is compatible with living tissue), slowly biodegradable, and is endowed with excellent mechanical properties and processability. In particular, the structure of silk fibroin and its tightly-packed amino acids, along with other characteristics such as its both hydrophobic and hydrophilic amino acid chains, allow for the product to have a versatile array of uses in many fields (Wang et al, 2010).
In the context of drug delivery, it has been demonstrated that Silk Fibroin matrices are able to successfully deliver protein drugs and preserve their potency. SF exhibits high encapsulation efficiency and controllable drug release kinetics due to the control of crystalline beta-sheet formation (Kim et al, 2015). I thought this would apply perfectly to treating Neonatal Respiratory Distress Syndrome (NRDS) because for treatment of NRDS, it is crucial for the pulmonary surfactant to be continuously delivered to the lungs as soon as possible. Due to its controlled degradation rate, SF would be able to facilitate this critical function.
But aside from its material properties, another reason initially I was really interested in silk and looking at silk proteins was because I actually used to raise silkworms when I was younger! Those silkworms I raised are actually the same exact breed, the Bombyx mori silkworm, as the ones that are commonly used in these materials science lab settings. I thought this was such a cool crossover from my personal life with this research!
(These are pictures of the silkworms that I raised when I was younger!)
Stay tuned for next week's update, where I plan to have my initial methodology drafted, reviewed, and discussed with my mentor!
References:
Wang, X., Yucel, T., Lu, Q., Hu, X., & Kaplan, D. L. (2010). Silk nanospheres and microspheres from silk/PVA blend films for Drug Delivery. Biomaterials, 31(6), 1025–1035. https://doi.org/10.1016/j.biomaterials.2009.11.002
Kim, S. Y., Naskar, D., Kundu, S. C., Bishop, D. P., Doble, P. A., Boddy, A. V., Chan, H.-K., Wall, I. B., & Chrzanowski, W. (2015). Formulation of biologically-inspired silk-based drug carriers for pulmonary delivery targeted for Lung Cancer. Scientific Reports, 5(1). https://doi.org/10.1038/srep11878
Week 2: Methodology
Welcome to week 2 of my senior project research! This week I created my initial protocol for fabricating the Silk Fibroin (SF) microspheres loaded with lung surfactant. This methodology is not finalized but will be adjusted and optimized through iterations of my trials in order to improve the quality and characteristics of the produced SF microspheres.
In regards to my methodology, from my literature review, I determined that these lung surfactant loaded SF microspheres would be created best through a process of phase separation of SF with Polyvinyl alcohol (PVA).
This is because both natural and synthetic materials can be used to fabricate microspheres for delivery systems, but the use of only natural polymers (like Silk Fibroin) for drug delivery applications has been limited due to its inconsistent batch qualities and uncontrollable degradation rates. However, when combined with polymers such as Polyvinyl alcohol (PVA), the characteristics (drug loading and release properties) of the microspheres become suitable for drug delivery. Past research has demonstrated that the phase separation between PVA and SF is especially conducive in creating microspheres because of its simplicity and denaturing of potentially bio-incompatible organic solvents. In fact, microspheres formed with PVA are found to be more uniform in size (Feng et al., 2001). This process of phase separation between polyvinyl alcohol (PVA) and silk occurs spontaneously when the two polymer solutions are mixed and subsequently cast into films.
The protocol follows the steps that are illustrated below.
Rockwood, D., Preda, R., Yücel, T. et al. Materials fabrication from Bombyx mori silk fibroin. Nat Protoc 6, 1612–1631 (2011). https://doi.org/10.1038/nprot.2011.379
Now that I have created and reviewed my proposed methodology with my mentor, in the next few weeks I will begin to carry out my initial trials!
References:
Feng, S.-shen, & Huang, G. (2001). Effects of emulsifiers on the controlled release of Paclitaxel (Taxol®) from nanospheres of biodegradable polymers. Journal of Controlled Release, 71(1), 53–69. https://doi.org/10.1016/s0168-3659(00)00364-3
Weeks 3-4: Initial Microsphere Fabrication
Hi everyone and welcome back to my blog! I have officially entered into the more hands-on portion of my senior project research. These past 2 weeks I focused the majority of my time in the lab on carrying out the methodology I created earlier.
The first step to create the microspheres through Silk Fibroin (SF) and Polyvinyl alcohol (PVA) phase separation was to make separate SF and PVA solutions. The SF solution was made by combining deionized water and lyophilized SF powder, and the PVA solution was created similarly by combining PVA powder and deionized water. The first issue I ran into this week was with creating the PVA solution. After a long time mixing the PVA solution (using a magnetic stir bar), I noticed that the PVA would not dissolve and would rather clump up. This was a problem that was really important to address because agglomerations in the solution could result in having irregularly formed and shaped microspheres. After researching this, I realized that the problem with the PVA dissolution was coming from the molecular weight of the PVA as its solubility is dependent on its molecular weight. After discussing this with my mentor, I was able to order new PVA with a lower molecular weight that resulted in much better dissolution.
After the solutions were created, they were mixed, poured into petri dishes, and dried to create films. These films were then dissolved into solutions and centrifuged, then sonicated. See below for some photos of the whole process!
In the next weeks, I will be able to analyze and characterize the microspheres I have made from these initial trials of the methodology. Based on what I see, I will adjust the protocol accordingly. I will update you all next week!
Week 5: Initial Observations
Welcome back to my blog! This week I was able to observe and characterize the microspheres I made from my methodology.
After discussing with my mentor, we decided that the best techniques for microsphere characterization given the available equipment in the lab would be dynamic light scattering, scanning electron microscopy, and light microscopy. Dynamic light scattering (DLS) can help to determine the size distribution profile of the microspheres; scanning electron microscopy (SEM) can provide detailed images of the surfaces of the microspheres in order to assess their surface topography and composition; and light microscopy (LM) can visualize the fine details of the microspheres.
In order to carry out all these techniques, I had to receive training on how to use the different equipment. For this week, I was first trained on the SEM and cleanroom procedures (where the SEM was located). To image my microspheres, I added 100 μl of the silk microsphere suspension directly on top of conductive tape mounted on a SEM sample. The samples were dried overnight in air, sputtered with platinum, then imaged using the software in the SEM. Below are some of the images of SF microspheres I successfully fabricated that I took from the SEM! It was super exciting to actually be able to visualize the microspheres!
As you may notice in the images, these initial samples of the SF microspheres had wide-ranging sizes and were agglomerated and clustered together. Using the software from the SEM, I was able to quantifiably observe the microspheres. There were some microspheres as small as 0.857 μm in diameter, to being as large as 15.110 μm in diameter. Based on these observations as well as the ones I will get from doing DLS and LM, I will adjust some parts of the methodology to improve the sizing and spread of the microspheres.
Next week, I will take on doing the DLS and LM on these initial trials and begin to brainstorm changes I can make in the methodology. Thanks for reading!
Weeks 6-7: Initial Observations Continued
Hi everyone and welcome to weeks 6 and 7 of my blog! In these last weeks, I was trained on how to carry out dynamic light scattering (DLS) and light microscopy (LM) to help me characterize my microspheres. Based on what I see from these tests, I will make some edits to my protocol to improve and optimize it.
To do light microscopy on the sample, I placed 20 μl of the silk sphere suspension under an inverted light microscope and then I used the installed software to visualize the details of microspheres at a x40 magnification. From the images, I was able to see the general spread of the microspheres throughout the solution. As indicated by the SEM images I took from the prior week, the LM images showed that the microspheres were not evenly distributed throughout the supernatant and sometimes agglomerated. Agglomeration occurs when smaller microspheres are associated into a cluster composed of two or more particles. Agglomerates decrease the number of microparticles at a constant filler concentration. Therefore, the clustering of particles should be avoided as much as possible which helps to ensure a more even release of CUROSURF surfactant drug in the lungs of infants with NRDS.
The DLS characterizations helped to describe the size distribution profiles of the microspheres. This was done through a value called the polydispersity index. The polydispersity index (PDI) describes the width or spread of the particle size distribution; a PDI closer to zero means a more homogeneous sample of microspheres size, while a higher PDI (closer to one) indicates a more polydisperse or aggregate sample. In beginning trials, the DLS data showed that the initial microspheres samples had a PDI of 0.98.
Now that I have data and observations from this first initial trial of microspheres, I will make adjustments in the methodology to account for the changes that need to be made. Specifically I will focus on how I can improve the sizing and spread of the microspheres.
See you next week!
Week 8: Modifications + Moving Forward!
Hi everyone and welcome back to my senior project blog! It’s hard to believe that 8 weeks have gone by already!
This week I focused mainly on two tasks. First was updating my methodology based on the observations I collected from my initial trial of Silk Fibroin (SF) microspheres. Second was including the CUROSURF lung surfactant loading process into my methodology.
Reflecting on the data I collected from my initial batch of microspheres, my goals for my new methodology were for it to create microspheres that were more uniform in size and more evenly distributed throughout the solution. I first decided to create the initial PVA and SF solutions over hot plates to allow for greater uniformity in the solution and to ensure that all particles were dissolved. I also decided to adjust the time and amplitude that was used for sonication of the final SF microsphere solution. This is because, in the methodology, sonication was used to disperse microspheres in the solution so in order to help separate the microparticles, I increased the time and amplitude of the sonication 30 seconds at 30% amplitude to help decrease clustering and agglomeration of the microspheres. In the next few weeks I will be able to see how these changes will affect the qualities and characteristics of the new SF microspheres.
After I made those adjustments to my protocol, I also added in the steps to load the microspheres with CUROSURF. As I explained in earlier weeks, this CUROSURF surfactant needs to be delivered into the lungs of babies affected with Neonatal Respiratory Disease Syndrome (NRDS). Here are the basic steps I followed:
To load the natural lung surfactant CUROSURF into our silk fibroin microspheres, stock solutions of CUROSURF were prepared with a concentration of 5 mg/mL in the PBS buffer (with a pH of 7.4) and stored at -20°C. The PBS buffer helped maintain a constant pH and also prevented cells from rupturing or shriveling up due to osmosis.
Before preparing the silk microspheres, certain amounts of CUROSURF stock solution were added to the silk solutions using a micropipette to reach a weight ratio of 1:100 between the drug (CUROSURF) stock solution and aqueous SF. After mixing, the SF and CUROSURF solution was used to blend with the PVA solution following the steps described in the silk sphere preparation in order to actually load the SF microspheres with the surfactant.
That’s all for this week! Stay tuned for next week where I will observe these new microspheres!
Post 8: Final weeks: Final Results + Analysis
Welcome to my very last blog post of my senior project. These past weeks have truly gone by so fast and I’ve been able to learn and try so many new things in the lab! While there were some bumps along the way, I found that approaching problems from different angles and trying new things helped to push my project past setbacks. Now let's dive into the final results of my project!
Last week I made a couple changes in my methodology and also incorporated the lung surfactant (CUROSURF) loading steps into my Silk Fibroin (SF) microsphere fabrication. In these past weeks I was able to carry out all 3 methods of characterization again (SEM, DLS, and LM) on new samples of microspheres to see how they reflected the changes I made in the methodology.
Above are the scanning electron microscopy (SEM) images from the most recent sample of SF microspheres. As you can see, there is significantly less clustering and agglomeration compared to the previous trials and the microspheres are much closer in size with most microspheres falling in the range of 1.5 to 2.7 microns. This was really great news because past research has established that to achieve targeted pulmonary drug delivery to the deep lungs, the optimal particle size for inhalation is <5 μm, and particles smaller than 3 μm have a 50–60% chance of reaching the alveolar regions. Because these SF microspheres are intended for delivering surfactant to the lung alveoli, the sizing of an average diameter 2.5 micrometers was a sufficient size. This was super exciting to see because it reflected how the changes I made in my protocol in terms of dissolution procedure and sonication settings (time and amplitude) were able to improve the final microsphere sizes and distribution.
The light microscopy images also showed a similar trend where the microspheres were more evenly spread out and similar in sizing. The dynamic light scattering (DLS) data further confirmed the beneficial changes the updated methodology made on the microspheres. The polydispersity index (PDI) of this new sample was 0.168 which was a lot closer to 0 than the initial PDI of 0.98. As I mentioned in my past blogs, a PDI closer to 0 indicates a more homogeneous sample of microspheres size, while a PDI closer to 1 indicates a more polydisperse or aggregate sample, which is something we want to avoid.
Looking at this data, I was able to successfully fabricate SF microspheres that were loaded with CUROSURF and optimize its characteristics! These findings lend insight into the potential of a more effective and efficient solution of delivering pulmonary surfactant to the lungs of newborns being affected by Neonatal Respiratory Distress Syndrome. I am super excited about the potential of my project as it not only would this method have less adverse effects on neonates, but it would also be a more efficient way to treat NRDS through the delivery of surfactant-microspheres through aerosolization.
Thanks so much for reading!